58 results on '"Ivan Zlatev"'
Search Results
2. Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity
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Maja M. Janas, Mark K. Schlegel, Carole E. Harbison, Vedat O. Yilmaz, Yongfeng Jiang, Rubina Parmar, Ivan Zlatev, Adam Castoreno, Huilei Xu, Svetlana Shulga-Morskaya, Kallanthottathil G. Rajeev, Muthiah Manoharan, Natalie D. Keirstead, Martin A. Maier, and Vasant Jadhav
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Science - Abstract
A subset of chemically-modified siRNAs conjugated to trivalent GalNAc may fail during nonclinical development due to rat hepatotoxicity. Here, the authors show that hepatotoxicity may be accounted for by microRNA-like off-target effects of siRNA and can be mitigated by a thermally destabilizing modification in the siRNA seed region.
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- 2018
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3. Crystal structure of Middle East respiratory syndrome coronavirus helicase.
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Wei Hao, Justyna Aleksandra Wojdyla, Rong Zhao, Ruiyun Han, Rajat Das, Ivan Zlatev, Muthiah Manoharan, Meitian Wang, and Sheng Cui
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Middle East respiratory syndrome coronavirus (MERS-CoV) remains a threat to public health worldwide; however, effective vaccine or drug against CoVs remains unavailable. CoV helicase is one of the three evolutionary most conserved proteins in nidoviruses, thus making it an important target for drug development. We report here the first structure of full-length coronavirus helicase, MERS-CoV nsp13. MERS-CoV helicase has multiple domains, including an N-terminal Cys/His rich domain (CH) with three zinc atoms, a beta-barrel domain and a C-terminal SF1 helicase core with two RecA-like subdomains. Our structural analyses show that while the domain organization of nsp13 is conserved throughout nidoviruses, the individual domains of nsp13 are closely related to the equivalent eukaryotic domains of Upf1 helicases. The most distinctive feature differentiating CoV helicases from eukaryotic Upf1 helicases is the interaction between CH domain and helicase core.
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- 2017
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4. A Glu-urea-Lys Ligand-conjugated Lipid Nanoparticle/siRNA System Inhibits Androgen Receptor Expression In Vivo
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Justin B Lee, Kaixin Zhang, Yuen Yi C Tam, Joslyn Quick, Ying K Tam, Paulo JC Lin, Sam Chen, Yan Liu, Jayaprakash K Nair, Ivan Zlatev, Kallanthottathil G Rajeev, Muthiah Manoharan, Paul S Rennie, and Pieter R Cullis
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androgen receptor ,lipid nanoparticles ,liposomes ,prostate cancer ,prostate specific membrane antigen ,siRNA ,Therapeutics. Pharmacology ,RM1-950 - Abstract
The androgen receptor plays a critical role in the progression of prostate cancer. Here, we describe targeting the prostate-specific membrane antigen using a lipid nanoparticle formulation containing small interfering RNA designed to silence expression of the messenger RNA encoding the androgen receptor. Specifically, a Glu-urea-Lys PSMA-targeting ligand was incorporated into the lipid nanoparticle system formulated with a long alkyl chain polyethylene glycol-lipid to enhance accumulation at tumor sites and facilitate intracellular uptake into tumor cells following systemic administration. Through these features, and by using a structurally refined cationic lipid and an optimized small interfering RNA payload, a lipid nanoparticle system with improved potency and significant therapeutic potential against prostate cancer and potentially other solid tumors was developed. Decreases in serum prostate-specific antigen, tumor cellular proliferation, and androgen receptor levels were observed in a mouse xenograft model following intravenous injection. These results support the potential clinical utility of a prostate-specific membrane antigen–targeted lipid nanoparticle system to silence the androgen receptor in advanced prostate cancer.
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- 2016
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5. Blood pressure-independent renoprotective effects of small interference RNA targeting liver angiotensinogen in experimental diabetes
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Edwyn O. Cruz‐López, Liwei Ren, Estrellita Uijl, Marian C. Clahsen‐van Groningen, Richard van Veghel, Ingrid M. Garrelds, Oliver Domenig, Marko Poglitsch, Ivan Zlatev, Timothy Rooney, Anne Kasper, Paul Nioi, Don Foster, A. H. Jan Danser, Internal Medicine, and Pathology
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Pharmacology ,SDG 3 - Good Health and Well-being - Abstract
Background and Purpose Small interfering RNA (siRNA) targeting liver angiotensinogen lowers blood pressure, but its effects in hypertensive diabetes are unknown. Experimental Approach To address this, TGR (mRen2)27 rats (angiotensin II-dependent hypertension model) were made diabetic with streptozotocin over 18 weeks and treated with either vehicle, angiotensinogen siRNA, the AT(1) antagonist valsartan, the ACE inhibitor captopril, valsartan + siRNA or valsartan + captopril for the final 3 weeks. Mean arterial pressure (MAP) was measured via radiotelemetry. Key Results MAP before treatment was 153 +/- 2 mmHg. Diabetes resulted in albuminuria, accompanied by glomerulosclerosis and podocyte effacement, without a change in glomerular filtration rate. All treatments lowered MAP and cardiac hypertrophy, and the largest drop in MAP was observed with siRNA + valsartan. Treatment with siRNA lowered circulating angiotensinogen by >99%, and the lowest circulating angiotensin II and aldosterone levels occurred in the dual treatment groups. Angiotensinogen siRNA did not affect renal angiotensinogen mRNA expression, confirming its liver-specificity. Furthermore, only siRNA with or without valsartan lowered renal angiotensin I. All treatments lowered renal angiotensin II and the reduction was largest (>95%) in the siRNA + valsartan group. All treatments identically lowered albuminuria, whereas only siRNA with or without valsartan restored podocyte foot processes and reduced glomerulosclerosis. Conclusion and Implications Angiotensinogen siRNA exerts renoprotection in diabetic TGR (mRen2)27 rats and this relies, at least in part, on the suppression of renal angiotensin II formation from liver-derived angiotensinogen. Clinical trials should now address whether this is also beneficial in human diabetic kidney disease.
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- 2023
6. Small circular interfering RNAs (sciRNAs) as a potent therapeutic platform for gene-silencing
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Krishna Aluri, Swati Gupta, Sarah Leblanc, Martin Egli, Tim Racie, Muthiah Manoharan, Anna Bisbe, Peter Podbevšek, Vasant Jadhav, John Szeto, Christopher R. Brown, Hartmut Jahns, Ivan Zlatev, Dale C. Guenther, Martin Maier, Pawan Kumar, Adam Castoreno, Janez Plavec, and Rohan Degaonkar
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Small interfering RNA ,AcademicSubjects/SCI00010 ,Oligonucleotide ,RNA ,RNA, Circular ,Biology ,In vitro ,Cell biology ,Mice, Inbred C57BL ,Mice ,Chemical Biology and Nucleic Acid Chemistry ,Sense strand ,RNA interference ,Circular RNA ,Genetics ,Animals ,Gene silencing ,Female ,RNA Interference ,Gene Silencing ,RNA, Small Interfering - Abstract
In order to achieve efficient therapeutic post-transcriptional gene-silencing mediated by the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be chemically modified. Several supra-RNA structures, with the potential to stabilize siRNAs metabolically have been evaluated for their ability to induce gene silencing, but all have limitations or have not been explored in therapeutically relevant contexts. Covalently closed circular RNA transcripts are prevalent in eukaryotes and have potential as biomarkers and disease targets, and circular RNA mimics are being explored for use as therapies. Here we report the synthesis and evaluation of small circular interfering RNAs (sciRNAs). To synthesize sciRNAs, a sense strand functionalized with the trivalent N-acetylgalactosamine (GalNAc) ligand and cyclized using ‘click’ chemistry was annealed to an antisense strand. This strategy was used for synthesis of small circles, but could also be used for synthesis of larger circular RNA mimics. We evaluated various sciRNA designs in vitro and in vivo. We observed improved metabolic stability of the sense strand upon circularization and off-target effects were eliminated. The 5′-(E)-vinylphosphonate modification of the antisense strand resulted in GalNAc-sciRNAs that are potent in vivo at therapeutically relevant doses. Physicochemical studies and NMR-based structural analysis, together with molecular modeling studies, shed light on the interactions of this novel class of siRNAs, which have a partial duplex character, with the RNAi machinery.
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- 2021
7. A Chemical Approach to Introduce 2,6-Diaminopurine and 2-Aminoadenine Conjugates into Oligonucleotides without Need for Protecting Groups
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Mimouna Madaoui, Dhrubajyoti Datta, Kelly Wassarman, Ivan Zlatev, Martin Egli, Bruce S. Ross, and Muthiah Manoharan
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Adenine ,Organic Chemistry ,Oligonucleotides ,Physical and Theoretical Chemistry ,2-Aminopurine ,Biochemistry - Abstract
We report a simple, postsynthetic strategy for synthesis of oligonucleotides containing 2,6-diaminopurine nucleotides and 2-aminoadenine conjugates using 2-fluoro-6-amino-adenosine. The strategy allows introduction of 2,6-diaminopurine and other 2-amino group-containing ligands. The strongly electronegative 2-fluoro deactivates 6-NH
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- 2022
8. Chirality matters: stereo-defined phosphorothioate linkages at the termini of small interfering RNAs improve pharmacology in vivo
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Anna Bisbe, Martin Maier, Tuyen Nguyen, Martin Egli, Jennifer L. S. Willoughby, Muthiah Manoharan, Ivan Zlatev, Masaaki Akabane-Nakata, Matt Hettinger, Shigeo Matsuda, Hartmut Jahns, Christopher R. Brown, Nate Taneja, Rajar M. Manoharan, Kallanthottathil G. Rajeev, and Klaus Charisse
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0301 basic medicine ,Exonuclease ,Small interfering RNA ,Stereochemistry ,RNA Stability ,In silico ,010402 general chemistry ,01 natural sciences ,Mice ,03 medical and health sciences ,Isomerism ,RNA interference ,In vivo ,Genetics ,Animals ,RNA, Small Interfering ,RNA, Double-Stranded ,biology ,Diastereomer ,Argonaute ,Organophosphates ,0104 chemical sciences ,030104 developmental biology ,Phosphodiester bond ,biology.protein ,RNA Interference - Abstract
A critical challenge for the successful development of RNA interference-based therapeutics therapeutics has been the enhancement of their in vivo metabolic stability. In therapeutically relevant, fully chemically modified small interfering RNAs (siRNAs), modification of the two terminal phosphodiester linkages in each strand of the siRNA duplex with phosphorothioate (PS) is generally sufficient to protect against exonuclease degradation in vivo. Since PS linkages are chiral, we systematically studied the properties of siRNAs containing single chiral PS linkages at each strand terminus. We report an efficient and simple method to introduce chiral PS linkages and demonstrate that Rp diastereomers at the 5′ end and Sp diastereomers at the 3′ end of the antisense siRNA strand improved pharmacokinetic and pharmacodynamic properties in a mouse model. In silico modeling studies provide mechanistic insights into how the Rp isomer at the 5′ end and Sp isomer at the 3′ end of the antisense siRNA enhance Argonaute 2 (Ago2) loading and metabolic stability of siRNAs in a concerted manner.
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- 2021
9. The Nonclinical Disposition and Pharmacokinetic/Pharmacodynamic Properties of N-Acetylgalactosamine–Conjugated Small Interfering RNA Are Highly Predictable and Build Confidence in Translation to Human
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Krishna Aluri, Scott Waldron, Karyn Schmidt, Maja M. Janas, Elena Castellanos-Rizaldos, Xiumin Liu, Michael Arciprete, Diane Ramsden, Jing Li, Vasant Jadhav, Robin McDougall, Christopher R. Brown, Bahru A. Habtemariam, Dale C. Guenther, Gabriel J. Robbie, Saket Agarwal, Muthiah Manoharan, Akshay Vaishnaw, Ivan Zlatev, Jeffrey Kurz, Jayaprakash K. Nair, Martin Maier, Saeho Chong, Sagar Agarwal, Christopher S. Theile, Steven Liou, Muthusamy Jayaraman, Varun Goel, Kevin Fitzgerald, Christopher MacLauchlin, Klaus Charisse, Joseph A Cichocki, Ju Liu, Yuanxin Xu, Peter F Smith, Jing-Tao Wu, Xuemei Zhang, and Yongli Gu
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Pharmacology ,Small interfering RNA ,Pharmacokinetics ,Chemistry ,In vivo ,Pharmacodynamics ,Pharmaceutical Science ,Gene silencing ,Distribution (pharmacology) ,PK/PD models ,ADME - Abstract
Conjugation of oligonucleotide therapeutics, including small interfering ribonucleic acids (siRNAs) or antisense oligonucleotides (ASOs) to N-acetylgalactosamine (GalNAc) ligands has become the primary strategy for hepatocyte-targeted delivery, and with the recent approvals of GIVLAARI® (givosiran) for the treatment of acute hepatic porphyria, OXLUMOTM (lumasiran) for the treatment of primary hyperoxaluria, and Leqvio® (inclisiran) for the treatment of hypercholesterolemia, the technology has been well-validated clinically. While much knowledge has been gained over decades of development there is a paucity of published literature on the DMPK properties of GalNAc-siRNA. With this in mind the goals of this mini-review are to provide an aggregate analysis of these nonclinical ADME data to build confidence on the translation of these properties to human. Upon subcutaneous administration, GalNAc-conjugated siRNAs are quickly distributed to the liver, resulting in plasma pharmacokinetic (PK) properties that reflect rapid elimination through ASGPR-mediated uptake from circulation into hepatocytes. These studies confirm that liver PK, including half-life and, most importantly, siRNA levels in RNA-induced silencing complex (RISC) in hepatocytes are better predictors of pharmacodynamics (PD) than plasma PK. Several in vitro and in vivo nonclinical studies were conducted to characterize the absorption, distribution, metabolism and excretion (ADME) properties of GalNAc-conjugated siRNAs. These studies demonstrate that the PK/PD and ADME properties of GalNAc-conjugated siRNAs are highly conserved across species, largely predictable, and can be accurately scaled to human, allowing us to identify efficacious and safe clinical dosing regimens in the absence of human liver PK profiles. Significance Statement Several nonclinical ADME studies have been conducted in order to provide a comprehensive overview of the disposition and elimination of GalNAc-conjugated siRNAs and the PK/PD translation between species. These studies demonstrate that the ADME properties of GalNAc-conjugated siRNAs are well correlated and predictable across species building confidence in the ability to extrapolate to human.
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- 2021
10. Renoprotective Effects of Small Interfering RNA Targeting Liver Angiotensinogen in Experimental Chronic Kidney Disease
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Marian C. Clahsen-van Groningen, Ewout J. Hoorn, Jae B. Kim, Ingrid M. Garrelds, Oliver Domenig, Ivan Zlatev, Liwei Ren, Stephen Huang, Marko Poglitsch, Richard van Veghel, Estrellita Uijl, Lauren Melton, Xifeng Lu, A.H. Jan Danser, Dominique M Bovée, Don Foster, Internal Medicine, and Pathology
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Small interfering RNA ,Captopril ,Angiotensinogen ,030204 cardiovascular system & hematology ,Pharmacology ,Kidney ,Losartan ,Renin-Angiotensin System ,03 medical and health sciences ,0302 clinical medicine ,SDG 3 - Good Health and Well-being ,Renin–angiotensin system ,parasitic diseases ,Internal Medicine ,medicine ,Animals ,Arterial Pressure ,030212 general & internal medicine ,RNA, Small Interfering ,Renal Insufficiency, Chronic ,Antihypertensive Agents ,business.industry ,Angiotensin II ,medicine.disease ,Rats ,Disease Models, Animal ,Liver ,Cardiac hypertrophy ,business ,hormones, hormone substitutes, and hormone antagonists ,Kidney disease - Abstract
Small interfering RNA (siRNA) targeting liver angiotensinogen (AGT) lowers blood pressure, but its effectiveness in hypertensive chronic kidney disease is unknown. Considering that the kidney may generate its own AGT, we assessed the effectiveness of liver-targeted AGT siRNA in the 5/6th Nx (5/6th nephrectomy) rat—a hypertensive chronic kidney disease model. Five weeks after 5/6th Nx (baseline), rats were subjected to vehicle, AGT siRNA, AGT siRNA+losartan, losartan, or losartan+captopril. Baseline mean arterial pressure was 160±6 mm Hg. Over the course of 4 weeks, mean arterial pressure increased further by ≈15 mm Hg during vehicle treatment. This rise was prevented by AGT siRNA. Losartan reduced mean arterial pressure by 37±6 mm Hg and increased plasma Ang (angiotensin) II. Both AGT siRNA and captopril suppressed these effects of losartan, suggesting that its blood pressure–lowering effect relied on stimulation of vasodilator Ang II type 2 receptors by high Ang II levels. Proteinuria and cardiac hypertrophy increased with vehicle, and these increases were comparably abrogated by all treatments. No intervention improved glomerular filtration rate, while siRNA and losartan equally diminished glomerulosclerosis. AGT siRNA±losartan reduced plasma AGT by >95%, and this was accompanied by almost complete elimination of Ang II in kidney and heart, without decreasing renal AGT mRNA. Multiple linear regression confirmed both mean arterial pressure and renal Ang II as independent determinants of proteinuria. In conclusion, AGT siRNA exerts renoprotection in the 5/6th Nx model in a blood pressure–independent manner. This relies on the suppression of renal Ang II formation from liver-derived AGT. Consequently, AGT siRNA may prove beneficial in human chronic kidney disease.
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- 2021
11. Aminooxy Click Chemistry as a Tool for Bis-homo and Bis-hetero Ligand Conjugation to Nucleic Acids
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Dhrubajyoti Datta, Shohei Mori, Mimouna Madaoui, Kelly Wassarman, Ivan Zlatev, and Muthiah Manoharan
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Molecular Structure ,Nucleic Acids ,Organic Chemistry ,Oligonucleotides ,Click Chemistry ,Physical and Theoretical Chemistry ,Ligands ,Biochemistry - Abstract
An aminooxy click chemistry (AOCC) strategy was used to synthesize nucleoside building blocks for incorporation during solid-support synthesis of oligonucleotides to enable bis-homo and bis-hetero conjugation of various biologically relevant ligands. The bis-homo aminooxy conjugation leads to bivalent ligand presentation, whereas the bis-hetero conjugation allows the placement of different ligands with either the same or different chemical linkages. This facile synthetic methodology allows introduction of two different ligands with different biological functions simultaneously.
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- 2022
12. Blood pressure-independent renoprotection in diabetic rats treated with small interfering RNA targeting liver angiotensinogen
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Edwyn Omar Cruz Lopez, Liwei Ren, Estrellita Uijl, Marian C. Clahsen-Van Groningen, Richard Van Veghel, Ingrid M. Garrelds, Oliver Domenig, Marko Poglitsch, Ivan Zlatev, Timothy Rooney, Anne Kasper, Paul Nioi, Don Foster, A.H. Jan Danser, Internal Medicine, and Pathology
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SDG 3 - Good Health and Well-being ,Physiology ,Internal Medicine ,Cardiology and Cardiovascular Medicine - Abstract
OBJECTIVE: Small interfering RNA (siRNA) targeting liver angiotensinogen (AGT) exerts beneficial effects on blood pressure and kidney function, but its effects in diabetes are still unknown. DESIGN AND METHOD: To address this, TGR(mREN2)27 rats (a model of angiotensin II-dependent hypertension) were made diabetic with streptozotocin for 18 weeks and treated with vehicle, AGT siRNA, the angiotensin receptor blocker valsartan, the ACE inhibitor captopril, valsartan + siRNA, or valsartan + captopril for the final 3 weeks. Arterial pressure was measured via radiotelemetry. RESULTS: Baseline mean arterial pressure (MAP) was 164 ± 1 mm Hg. Diabetes resulted in albuminuria, accompanied by glomerulosclerosis and podocyte effacement, without a further rise in MAP or a change in glomerular filtration rate. All treatments lowered MAP (by ~50 mm Hg) and cardiac hypertrophy identically. Treatment with siRNA resulted in the largest reduction in AGT, while in combination with valsartan AGT virtually disappeared. Only the dual treatment groups and captopril lowered circulating angiotensin II and aldosterone. No treatment affected renal AGT mRNA expression, confirming the liver-specificity of the siRNA. Yet, siRNA with or without valsartan, and valsartan + captopril, but not valsartan alone, or captopril alone, reduced renal angiotensin I and II. All treatments lowered albuminuria and proteinuria, while only siRNA with or without valsartan improved glomerulosclerosis and podocyte dysfunction. Multiple linear regression confirmed both mean arterial pressure and renal angiotensin II as independent determinants of albuminuria. CONCLUSIONS: In conclusion, AGT siRNA exerts renoprotection in diabetic TGR(mREN2)27 rats, and this relies, at least in part, on the suppression of renal angiotensin II formation from liver-derived AGT. Consequently, AGT siRNA may prove beneficial in human diabetic kidney disease.
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- 2022
13. siRNAs containing 2′-fluorinated Northern-methanocarbacyclic (2′-F-NMC) nucleotides: in vitro and in vivo RNAi activity and inability of mitochondrial polymerases to incorporate 2′-F-NMC NTPs
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Mark K Schlegel, Derek K O'Flaherty, Maja M. Janas, Jason A. Gilbert, Namrata D. Erande, Ivan Zlatev, Shigeo Matsuda, Yongfeng Jiang, June Qin, Pawan Kumar, Rohan Degaonkar, Martha Mendez, Masaaki Akabane-Nakata, Muthiah Manoharan, Martin Egli, and Lauren Blair Woods
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Models, Molecular ,Small interfering RNA ,AcademicSubjects/SCI00010 ,DNA polymerase ,010402 general chemistry ,01 natural sciences ,Mitochondrial Proteins ,Mice ,03 medical and health sciences ,Organophosphorus Compounds ,Chemical Biology and Nucleic Acid Chemistry ,RNA interference ,Chlorocebus aethiops ,Genetics ,Animals ,Prealbumin ,Gene silencing ,Nucleotide ,RNA, Small Interfering ,Uridine ,Cells, Cultured ,Polymerase ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,Nucleotides ,Kinase ,RNA ,DNA-Directed RNA Polymerases ,DNA Polymerase gamma ,Mitochondria ,0104 chemical sciences ,Biochemistry ,chemistry ,Argonaute Proteins ,COS Cells ,biology.protein ,Pyrimidine Nucleotides ,RNA Interference - Abstract
We recently reported the synthesis of 2′-fluorinated Northern-methanocarbacyclic (2′-F-NMC) nucleotides, which are based on a bicyclo[3.1.0]hexane scaffold. Here, we analyzed RNAi-mediated gene silencing activity in cell culture and demonstrated that a single incorporation of 2′-F-NMC within the guide or passenger strand of the tri-N-acetylgalactosamine-conjugated siRNA targeting mouse Ttr was generally well tolerated. Exceptions were incorporation of 2′-F-NMC into the guide strand at positions 1 and 2, which resulted in a loss of the in vitro activity. Activity at position 1 was recovered when the guide strand was modified with a 5′ phosphate, suggesting that the 2′-F-NMC is a poor substrate for 5′ kinases. In mice, the 2′-F-NMC-modified siRNAs had comparable RNAi potencies to the parent siRNA. 2′-F-NMC residues in the guide seed region position 7 and at positions 10, 11 and 12 were well tolerated. Surprisingly, when the 5′-phosphate mimic 5′-(E)-vinylphosphonate was attached to the 2′-F-NMC at the position 1 of the guide strand, activity was considerably reduced. The steric constraints of the bicyclic 2′-F-NMC may impair formation of hydrogen-bonding interactions between the vinylphosphonate and the MID domain of Ago2. Molecular modeling studies explain the position- and conformation-dependent RNAi-mediated gene silencing activity of 2′-F-NMC. Finally, the 5′-triphosphate of 2′-F-NMC is not a substrate for mitochondrial RNA and DNA polymerases, indicating that metabolites should not be toxic.
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- 2021
14. No evidence for brain renin-angiotensin system activation during DOCA-salt hypertension
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Liwei Ren, Estrellita Uijl, Don Foster, Stephen Huang, Katrina M Mirabito Colafella, Jae B. Kim, Lauren Melton, Ingrid M. Garrelds, Richard van Veghel, Marko Poglitsch, A.H. Jan Danser, Ewout J. Hoorn, Oliver Domenig, Ivan Zlatev, and Internal Medicine
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Male ,medicine.medical_specialty ,medicine.drug_class ,Angiotensinogen ,Rats, Sprague-Dawley ,Renin-Angiotensin System ,Desoxycorticosterone Acetate ,chemistry.chemical_compound ,Mineralocorticoid receptor ,Internal medicine ,Renin ,Renin–angiotensin system ,medicine ,Animals ,cardiovascular diseases ,Sodium Chloride, Dietary ,Chemistry ,urogenital system ,Angiotensin II ,Brain ,General Medicine ,Receptor antagonist ,medicine.disease ,Hyperaldosteronism ,Blood pressure ,Endocrinology ,Valsartan ,Hypertension ,Spironolactone ,hormones, hormone substitutes, and hormone antagonists ,Brain Stem ,medicine.drug ,circulatory and respiratory physiology - Abstract
Brain renin–angiotensin system (RAS) activation is thought to mediate deoxycorticosterone acetate (DOCA)-salt hypertension, an animal model for human primary hyperaldosteronism. Here, we determined whether brainstem angiotensin II is generated from locally synthesized angiotensinogen and mediates DOCA-salt hypertension. To this end, chronic DOCA-salt-hypertensive rats were treated with liver-directed siRNA targeted to angiotensinogen, the angiotensin II type 1 receptor antagonist valsartan, or the mineralocorticoid receptor antagonist spironolactone (n = 6–8/group). We quantified circulating angiotensinogen and renin by enzyme-kinetic assay, tissue angiotensinogen by Western blotting, and angiotensin metabolites by LC-MS/MS. In rats without DOCA-salt, circulating angiotensin II was detected in all rats, whereas brainstem angiotensin II was detected in 5 out of 7 rats. DOCA-salt increased mean arterial pressure by 19 ± 1 mmHg and suppressed circulating renin and angiotensin II by >90%, while brainstem angiotensin II became undetectable in 5 out of 7 rats (
- Published
- 2021
15. Investigating the pharmacodynamic durability of GalNAc–siRNA conjugates
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Ryan Malone, Christopher S. Theile, Scott Lentini, Muthusamy Jayaraman, Mikyung Yu, Varun Goel, Rubina G. Parmar, Vasant Jadhav, Shigeo Matsuda, Mark K Schlegel, Guo He, Dennis Brown, June Qin, Swati Gupta, Timothy Racie, Svetlana Shulga-Morskaya, Anil V. Nair, Muthiah Manoharan, Jayaprakash K. Nair, Karyn Schmidt, Azar Shahraz, Ivan Zlatev, Christopher R. Brown, and Martin Maier
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Small interfering RNA ,Acetylgalactosamine ,Time Factors ,AcademicSubjects/SCI00010 ,NAR Breakthrough Article ,Asialoglycoprotein Receptor ,Biology ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Drug Stability ,RNA interference ,In vivo ,Genetics ,Animals ,Humans ,Prealbumin ,Gene silencing ,Gene Silencing ,RNA, Small Interfering ,030304 developmental biology ,Drug Carriers ,0303 health sciences ,Gene knockdown ,Biological Transport ,Biological activity ,Hydrogen-Ion Concentration ,Argonaute ,Cell biology ,Mice, Inbred C57BL ,Liver ,030220 oncology & carcinogenesis ,Argonaute Proteins ,Hepatocytes ,Nanoparticles ,Female ,Glycoconjugates ,Intracellular - Abstract
One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc–siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc–siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo.
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- 2020
16. Chimeric siRNAs with chemically modified pentofuranose and hexopyranose nucleotides: altritol-nucleotide (ANA) containing GalNAc–siRNA conjugates: in vitro and in vivo RNAi activity and resistance to 5′-exonuclease
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Pawan Kumar, Piet Herdewijn, Joel M. Harp, Ivan Zlatev, Muthiah Manoharan, Marie Capobianco, Martin Egli, Mark K Schlegel, Mikhail Abramov, Maja M. Janas, Charalambos Kaittanis, Yongfeng Jiang, Adam Castoreno, Rohan Degaonkar, Dale C. Guenther, and Guy Schepers
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musculoskeletal diseases ,Exonuclease ,Small interfering RNA ,Acetylgalactosamine ,AcademicSubjects/SCI00010 ,Carbohydrates ,Biology ,010402 general chemistry ,01 natural sciences ,Mice ,03 medical and health sciences ,Sugar Alcohols ,Chemical Biology and Nucleic Acid Chemistry ,RNA interference ,Chlorocebus aethiops ,Sense (molecular biology) ,Genetics ,Animals ,Prealbumin ,Nucleotide ,RNA, Small Interfering ,skin and connective tissue diseases ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Oligonucleotide ,RNA ,Ribonucleotides ,0104 chemical sciences ,stomatognathic diseases ,Biochemistry ,chemistry ,COS Cells ,Exoribonucleases ,Hepatocytes ,Nucleic acid ,biology.protein ,Nucleic Acid Conformation ,RNA Interference - Abstract
In this report, we investigated the hexopyranose chemical modification Altriol Nucleic Acid (ANA) within small interfering RNA (siRNA) duplexes that were otherwise fully modified with the 2'-deoxy-2'-fluoro and 2'-O-methyl pentofuranose chemical modifications. The siRNAs were designed to silence the transthyretin (Ttr) gene and were conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Sense and antisense strands of the parent duplex were synthesized with single ANA residues at each position on the strand, and the resulting siRNAs were evaluated for their ability to inhibit Ttr mRNA expression in vitro. Although ANA residues were detrimental at the 5' end of the antisense strand, the siRNAs with ANA at position 6 or 7 in the seed region had activity comparable to the parent. The siRNA with ANA at position 7 in the seed region was active in a mouse model. An Oligonucleotide with ANA at the 5' end was more stable in the presence of 5'-exonuclease than an oligonucleotide of the same sequence and chemical composition without the ANA modification. Modeling studies provide insight into the origins of regiospecific changes in potency of siRNAs and the increased protection against 5'-exonuclease degradation afforded by the ANA modification. ispartof: NUCLEIC ACIDS RESEARCH vol:48 issue:8 pages:4028-4040 ispartof: location:England status: published
- Published
- 2020
17. Discovery of a novel deaminated metabolite of a single-stranded oligonucleotide in vivo by mass spectrometry
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Krishna Aluri, Yuanxin Xu, Anna Bisbe, Jonathan O'Shea, Chris Tran, Jing Li, Ivan Zlatev, Ju Liu, Li-Hua Guan, Jennifer Enders, Diana Najarian, Michael Arciprete, and Klaus Charisse
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chemistry.chemical_classification ,0303 health sciences ,Oligonucleotide ,Metabolite ,010401 analytical chemistry ,Clinical Biochemistry ,Deamination ,General Medicine ,Mass spectrometry ,01 natural sciences ,0104 chemical sciences ,Analytical Chemistry ,03 medical and health sciences ,Medical Laboratory Technology ,chemistry.chemical_compound ,chemistry ,Biochemistry ,In vivo ,medicine ,Nucleotide ,Solid phase extraction ,General Pharmacology, Toxicology and Pharmaceutics ,Inosine ,030304 developmental biology ,medicine.drug - Abstract
Aim: A novel single-stranded deaminated oligonucleotide metabolite resulting from a REVERSIR™ oligonucleotide was discovered and identified in monkey liver after subcutaneous administration. Results & methodology: REVERSIR-A and its metabolites were extracted from biological matrices by solid phase extraction and analyzed using LC coupled with high-resolution MS under negative ionization mode. A novel 9-mer metabolite of REVERSIR-A, resulting from deamination of the 3′ terminal 2′- O-methyl-adenosine nucleotide to 2′- O-methyl-inosine, was discovered at significant levels in monkey liver. The metabolite's identity was confirmed by LC–MS/MS. Conclusion: This report describes the first observation of a long-chain deaminated metabolite of a single-stranded REVERSIR oligonucleotide in vivo in monkey liver.
- Published
- 2019
18. Expanding the Reach of RNAi Therapeutics with Next Generation Lipophilic siRNA Conjugates
- Author
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Maja M. Janas, Saeho Chong, Mark K Schlegel, Jason Gilbert, Saket Agarwal, Vasant Jadhav, Dale Gunther, Michelle Jung, Mark Keating, Kelly Wassarman, Shigeo Matsuda, Arlin Rogers, Yongfeng Jiang, Lei Johnson, Elena Castellanos-Rizaldos, Jessica E. Sutherland, Scott Lentini, Urjana Poreci, Klaus Charisse, Jing Li, Anne Kasper, Yesseinia Anglero-Rodriguez, Jeff Rollins, Jing-Tao Wu, Alex Kelin, Sarah Le Blanc Sarah Le Blanc, Diana Cha, Martin E. Maier, Adam Castoreno, Samantha Chigas, Tuyen Nguyen, Muthiah Manoharan, Wendell P Davis, Haiyan Peng, Jayaprakash K. Nair, John Petrulis, Christopher S. Theile, Tyler Chickering, Ivan Zlatev, Tim Racie, Kirk Brown, Christopher R. Brown, Stuart Milstein, Jessica Bombardier, Melissa Mobley, Jeffrey Allen, Raj Maganti, Donald Foster, Kevin Fitzgerald, Jeffrey Kurz, Lauren Blair Woods, and Robin McDougall
- Subjects
Chemistry ,Conjugate ,RNAi Therapeutics ,Cell biology - Abstract
RNA interference (RNAi) therapeutics are a new class of medicines that can address unmet medical needs by silencing disease-causing gene transcripts. While delivery of short interfering RNAs (siRNAs) to hepatocytes has yielded multiple drug approvals, novel delivery solutions are needed to expand the reach of RNAi therapeutics. Here we report that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables efficient silencing in the central nervous system (CNS), eye, and lung of multiple nonclinical species with broad cell type specificity. Intrathecally delivered C16-siRNAs are active across CNS regions and cell types, with sustained silencing for at least three months, which is an especially important outcome considering the challenging dosing route. Similarly, intravitreal and intranasal administration of C16-siRNAs resulted in potent and sustained knockdown in the eye and lung, respectively. Efficient delivery facilitated through C16 conjugation to optimized siRNA designs has enabled candidate selection for investigational human clinical trials assessing therapeutic silencing beyond the liver with infrequent (e.g. bi-annual) dosing.
- Published
- 2021
19. Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates
- Author
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Kirk M. Brown, Jayaprakash K. Nair, Maja M. Janas, Yesseinia I. Anglero-Rodriguez, Lan T. H. Dang, Haiyan Peng, Christopher S. Theile, Elena Castellanos-Rizaldos, Christopher Brown, Donald Foster, Jeffrey Kurz, Jeffrey Allen, Rajanikanth Maganti, Jing Li, Shigeo Matsuda, Matthew Stricos, Tyler Chickering, Michelle Jung, Kelly Wassarman, Jeff Rollins, Lauren Woods, Alex Kelin, Dale C. Guenther, Melissa W. Mobley, John Petrulis, Robin McDougall, Timothy Racie, Jessica Bombardier, Diana Cha, Saket Agarwal, Lei Johnson, Yongfeng Jiang, Scott Lentini, Jason Gilbert, Tuyen Nguyen, Samantha Chigas, Sarah LeBlanc, Urjana Poreci, Anne Kasper, Arlin B. Rogers, Saeho Chong, Wendell Davis, Jessica E. Sutherland, Adam Castoreno, Stuart Milstein, Mark K. Schlegel, Ivan Zlatev, Klaus Charisse, Mark Keating, Muthiah Manoharan, Kevin Fitzgerald, Jing-Tao Wu, Martin A. Maier, and Vasant Jadhav
- Subjects
Primates ,Amyloid beta-Protein Precursor ,Mice ,RNAi Therapeutics ,Biomedical Engineering ,Molecular Medicine ,Animals ,Bioengineering ,RNA Interference ,RNA, Small Interfering ,Applied Microbiology and Biotechnology ,Biotechnology - Abstract
Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.
- Published
- 2021
20. Safety evaluation of 2′-deoxy-2′-fluoro nucleotides in GalNAc-siRNA conjugates
- Author
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Krishna Aluri, Laura Sepp-Lorenzino, Christopher R. Brown, Kevin Fitzgerald, Akin Akinc, Martin Maier, Lauren Blair, Peter F Smith, Ju Liu, Jing Li, Ramesh Indrakanti, Biplab Das, Xiumin Liu, Ivan Zlatev, Scott A Barros, Kallanthottathil G. Rajeev, Yongfeng Jiang, Saket Agarwal, Akshay Vaishnaw, Chris Tran, Klaus Charisse, Jingxuan Liu, Muthiah Manoharan, Shigeo Matsuda, Jayaprakash K. Nair, Jessica E. Sutherland, Tracy Zimmermann, Yuanxin Xu, Jing-Tao Wu, Wendell P Davis, Xuemei Zhang, Maja M. Janas, Vasant Jadhav, and Mark K Schlegel
- Subjects
Male ,Small interfering RNA ,Acetylgalactosamine ,Deoxyribonucleotides ,Biology ,Rats, Sprague-Dawley ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Chemical Biology and Nucleic Acid Chemistry ,In vivo ,Genetics ,Animals ,Humans ,Nucleotide ,Viability assay ,RNA, Small Interfering ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Oligonucleotide ,RNA ,Fluorine ,Ligand (biochemistry) ,Rats ,chemistry ,Biochemistry ,Female ,030217 neurology & neurosurgery ,DNA - Abstract
For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2′-deoxy-2′-fluoro (2′-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2′-F-monomer metabolites was low and transient in rats and humans. In vitro, 2′-F-nucleoside 5′-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2′-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2′-F-monomers were observed after weekly administration of two GalNAc–siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2′-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc–siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.
- Published
- 2019
21. The Nonclinical Disposition and Pharmacokinetic/Pharmacodynamic Properties of
- Author
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Robin, McDougall, Diane, Ramsden, Sagar, Agarwal, Saket, Agarwal, Krishna, Aluri, Michael, Arciprete, Christopher, Brown, Elena, Castellanos-Rizaldos, Klaus, Charisse, Saeho, Chong, Joseph, Cichocki, Kevin, Fitzgerald, Varun, Goel, Yongli, Gu, Dale, Guenther, Bahru, Habtemariam, Vasant, Jadhav, Maja, Janas, Muthusamy, Jayaraman, Jeffrey, Kurz, Jing, Li, Ju, Liu, Xiumin, Liu, Steven, Liou, Chris, Maclauchlin, Martin, Maier, Muthiah, Manoharan, Jayaprakash K, Nair, Gabriel, Robbie, Karyn, Schmidt, Peter, Smith, Christopher, Theile, Akshay, Vaishnaw, Scott, Waldron, Yuanxin, Xu, Xuemei, Zhang, Ivan, Zlatev, and Jing-Tao, Wu
- Subjects
Acetylgalactosamine ,Hepatocytes ,Humans ,Asialoglycoprotein Receptor ,RNA, Small Interfering ,Porphyrias, Hepatic - Abstract
Conjugation of oligonucleotide therapeutics, including small interfering RNAs (siRNAs) or antisense oligonucleotides, to
- Published
- 2021
22. Synthesis, chirality-dependent conformational and biological properties of siRNAs containing 5'-(R)- and 5'-(S)-C-methyl-guanosine
- Author
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Namrata D. Erande, Mark K Schlegel, Martin Egli, Atsushi Mikami, Tyler Chickering, Ivan Zlatev, Lauren Blair Woods, Alexander V Kel'in, Shigeo Matsuda, Pradeep S. Pallan, and Muthiah Manoharan
- Subjects
Exonuclease ,Models, Molecular ,Stereochemistry ,AcademicSubjects/SCI00010 ,Endoribonuclease activity ,Guanosine ,Biology ,010402 general chemistry ,01 natural sciences ,03 medical and health sciences ,chemistry.chemical_compound ,Isomerism ,Chemical Biology and Nucleic Acid Chemistry ,Genetics ,Nucleotide ,Histone octamer ,RNA, Small Interfering ,Polymerase ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Oligonucleotide ,RNA ,0104 chemical sciences ,chemistry ,biology.protein ,Nucleic Acid Conformation - Abstract
Various chemical modifications have been identified that enhance potency of small interfering RNAs (siRNAs) and that reduce off-target effects, immune stimulation, and toxicities of metabolites of these therapeutic agents. We previously described 5′-C-methyl pyrimidine nucleotides also modified at the 2′ position of the sugar. Here, we describe the synthesis of 2′-position unmodified 5′-(R)- and 5′-(S)-C-methyl guanosine and evaluation of these nucleotides in the context of siRNA. The (R) isomer provided protection from 5′ exonuclease and the (S) isomer provided protection from 3′ exonuclease in the context of a terminally modified oligonucleotide. siRNA potency was maintained when these modifications were incorporated at the tested positions of sense and antisense strands. Moreover, the corresponding 5′ triphosphates were not substrates for mitochondrial DNA polymerase. Models generated based on crystal structures of 5′ and 3′ exonuclease oligonucleotide complexes with 5′-(R)- and 5′-(S)-C-methyl substituents attached to the 5′- and 3′-terminal nucleotides, respectively, provided insight into the origins of the observed protections. Structural properties of 5′-(R)-C-methyl guanosine incorporated into an RNA octamer were analysed by X-ray crystallography, and the structure explains the loss in duplex thermal stability for the (R) isomer compared with the (S) isomer. Finally, the effect of 5′-C-methylation on endoribonuclease activity has been explained.
- Published
- 2020
23. An efficient deprotection method for 5′-[O,O-bis(pivaloyloxymethyl)]-(E)-vinylphosphonate containing oligonucleotides
- Author
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Klaus Charisse, Christopher S. Theile, Ivan Zlatev, Jonathan O'Shea, Muthiah Manoharan, Rajat S. Das, Martin Maier, and I. Ramesh Babu
- Subjects
0301 basic medicine ,Diethylamine ,Aqueous solution ,Oligonucleotide ,Organic Chemistry ,Oligonucleotide synthesis ,010402 general chemistry ,Pivaloyloxymethyl ,01 natural sciences ,Biochemistry ,Combinatorial chemistry ,0104 chemical sciences ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,chemistry ,Drug Discovery ,Bioisostere - Abstract
5′-(E)-Vinylphosphonate (VP) is an effective bioisostere of the natural 5′-monophosphate in small interfering RNAs (siRNAs). Solid-phase synthesis of VP-siRNAs requires the use of appropriately protected VP-phosphoramidites in combination with optimal oligonucleotide deprotection conditions. Addition of 3% (v) neat diethylamine to the standard aqueous ammonia deprotection conditions allows clean and rapid one-step deprotection of 5′-[O,O-bis(pivaloyloxymethyl)] (POM)-protected VP oligonucleotides, minimizing side reactions and impurities, which broadly enhances the scope of VP oligonucleotide synthesis.
- Published
- 2018
24. Discovery of a novel deaminated metabolite of a single-stranded oligonucleotide
- Author
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Jing, Li, Ju, Liu, Jennifer, Enders, Michael, Arciprete, Chris, Tran, Krishna, Aluri, Li-Hua, Guan, Jonathan, O'Shea, Anna, Bisbe, Klaus, Charissé, Ivan, Zlatev, Diana, Najarian, and Yuanxin, Xu
- Subjects
Macaca fascicularis ,Liver ,Deamination ,Oligonucleotides ,Animals ,Inosine ,Mass Spectrometry - Published
- 2019
25. Oligonucleotide quantification and metabolite profiling by high-resolution and accurate mass spectrometry
- Author
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Saeho Chong, Krishna Aluri, Chris Tran, Li-Hua Guan, Ju Liu, Ivan Zlatev, Diana Najarian, Yuanxin Xu, Xuemei Zhang, Klaus Charisse, Valerie A. Clausen, Jing-Tao Wu, and Jing Li
- Subjects
Bioanalysis ,Small interfering RNA ,Clinical Biochemistry ,Oligonucleotides ,High resolution ,Mass spectrometry ,01 natural sciences ,Mass Spectrometry ,Analytical Chemistry ,03 medical and health sciences ,RNA interference ,Animals ,Metabolomics ,General Pharmacology, Toxicology and Pharmaceutics ,030304 developmental biology ,0303 health sciences ,Chromatography ,Base Sequence ,Oligonucleotide ,Chemistry ,010401 analytical chemistry ,General Medicine ,0104 chemical sciences ,Medical Laboratory Technology ,Macaca fascicularis ,Liver ,Metabolite profiling ,Chromatography, Liquid - Abstract
Aim: Advancements in RNA interference therapeutics have triggered development of improved bioanalytical methods for oligonucleotide metabolite profiling and high-throughput quantification in biological matrices. Results & methodology: HPLC coupled with high-resolution mass spectrometry (LC-HRMS) methods were developed to investigate the metabolism of a REVERSIR™ molecule in vivo. Plasma and tissue samples were extracted using solid-phase extraction followed by LC-HRMS analysis for metabolite profiling and quantification. The method was qualified from 10 to 5000 ng/ml (plasma) and 100 to 50000 ng/g (liver and kidney). In rat liver, intra and interday accuracy ranged from 80.9 to 118.5% and 88.4 to 111.9%, respectively, with acceptable precision (
- Published
- 2019
26. Structural basis for the synergy of 4′- and 2′-modifications on siRNA nuclease resistance, thermal stability and RNAi activity
- Author
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Kallanthottathil G. Rajeev, Anna Bisbe, Gopal R Bommineni, Martin Egli, Lydia Perkins, Shigeo Matsuda, Martin Maier, Klaus Charisse, Dale C. Guenther, Muthiah Manoharan, Donald Foster, Joel M. Harp, Nate Taneja, and Ivan Zlatev
- Subjects
Models, Molecular ,0301 basic medicine ,Exonuclease ,Small interfering RNA ,Stereochemistry ,RNA Stability ,Ribose ,Oligonucleotides ,Biology ,Phosphates ,03 medical and health sciences ,chemistry.chemical_compound ,Ribonucleases ,RNA interference ,otorhinolaryngologic diseases ,Genetics ,Humans ,Nucleotide ,RNA, Small Interfering ,Uridine ,chemistry.chemical_classification ,Nuclease ,Oligonucleotide ,RNA ,030104 developmental biology ,chemistry ,biology.protein ,Nucleic Acid Conformation ,Thermodynamics ,RNA Interference - Abstract
Chemical modification is a prerequisite of oligonucleotide therapeutics for improved metabolic stability, uptake and activity, irrespective of their mode of action, i.e. antisense, RNAi or aptamer. Phosphate moiety and ribose C2'/O2' atoms are the most common sites for modification. Compared to 2'-O-substituents, ribose 4'-C-substituents lie in proximity of both the 3'- and 5'-adjacent phosphates. To investigate potentially beneficial effects on nuclease resistance we combined 2'-F and 2'-OMe with 4'-Cα- and 4'-Cβ-OMe, and 2'-F with 4'-Cα-methyl modification. The α- and β-epimers of 4'-C-OMe-uridine and the α-epimer of 4'-C-Me-uridine monomers were synthesized and incorporated into siRNAs. The 4'α-epimers affect thermal stability only minimally and show increased nuclease stability irrespective of the 2'-substituent (H, F, OMe). The 4'β-epimers are strongly destabilizing, but afford complete resistance against an exonuclease with the phosphate or phosphorothioate backbones. Crystal structures of RNA octamers containing 2'-F,4'-Cα-OMe-U, 2'-F,4'-Cβ-OMe-U, 2'-OMe,4'-Cα-OMe-U, 2'-OMe,4'-Cβ-OMe-U or 2'-F,4'-Cα-Me-U help rationalize these observations and point to steric and electrostatic origins of the unprecedented nuclease resistance seen with the chain-inverted 4'β-U epimer. We used structural models of human Argonaute 2 in complex with guide siRNA featuring 2'-F,4'-Cα-OMe-U or 2'-F,4'-Cβ-OMe-U at various sites in the seed region to interpret in vitro activities of siRNAs with the corresponding 2'-/4'-C-modifications.
- Published
- 2018
27. Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity
- Author
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Adam Castoreno, Carole E. Harbison, Kallanthottathil G. Rajeev, Huilei Xu, Svetlana Shulga-Morskaya, Vasant Jadhav, Mark K Schlegel, Vedat O. Yilmaz, Yongfeng Jiang, Martin Maier, Natalie D. Keirstead, Muthiah Manoharan, Ivan Zlatev, Maja M. Janas, and Rubina G. Parmar
- Subjects
Male ,0301 basic medicine ,Small interfering RNA ,Acetylgalactosamine ,Science ,General Physics and Astronomy ,Endogeny ,Conjugated system ,Article ,General Biochemistry, Genetics and Molecular Biology ,Rats, Sprague-Dawley ,03 medical and health sciences ,0302 clinical medicine ,RNA interference ,Animals ,RNA, Small Interfering ,lcsh:Science ,Multidisciplinary ,Chemistry ,Oligonucleotide ,fungi ,RNA ,food and beverages ,General Chemistry ,Rats ,Cell biology ,030104 developmental biology ,Liver ,030220 oncology & carcinogenesis ,Toxicity ,RNA Interference ,lcsh:Q ,Intracellular - Abstract
Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of indications. The typical development candidate selection process includes evaluation of the most active compounds for toxicity in rats at pharmacologically exaggerated doses. The subset of GalNAc-siRNAs that show rat hepatotoxicity is not advanced to clinical development. Potential mechanisms of hepatotoxicity can be associated with the intracellular accumulation of oligonucleotides and their metabolites, RNA interference (RNAi)-mediated hybridization-based off-target effects, and/or perturbation of endogenous RNAi pathways. Here we show that rodent hepatotoxicity observed at supratherapeutic exposures can be largely attributed to RNAi-mediated off-target effects, but not chemical modifications or the perturbation of RNAi pathways. Furthermore, these off-target effects can be mitigated by modulating seed-pairing using a thermally destabilizing chemical modification, which significantly improves the safety profile of a GalNAc-siRNA in rat and may minimize the occurrence of hepatotoxic siRNAs across species., A subset of chemically-modified siRNAs conjugated to trivalent GalNAc may fail during nonclinical development due to rat hepatotoxicity. Here, the authors show that hepatotoxicity may be accounted for by microRNA-like off-target effects of siRNA and can be mitigated by a thermally destabilizing modification in the siRNA seed region.
- Published
- 2018
28. Facile Synthesis, Geometry, and 2′-Substituent-Dependent in Vivo Activity of 5′-(E)- and 5′-(Z)-Vinylphosphonate-Modified siRNA Conjugates
- Author
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Klaus Charisse, Kallanthottathil G. Rajeev, Vasant Jadhav, Christopher R. Brown, Shigeo Matsuda, Martin Egli, Muthiah Manoharan, Jennifer L. S. Willoughby, Martin Maier, Donald Foster, Ivan Zlatev, Christopher S. Theile, and Rubina G. Parmar
- Subjects
Models, Molecular ,0301 basic medicine ,Small interfering RNA ,Stereochemistry ,Organophosphonates ,Stereoisomerism ,Chemistry Techniques, Synthetic ,Pivaloyloxymethyl ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Domains ,Drug Discovery ,Sense (molecular biology) ,Animals ,Moiety ,Nucleotide ,Gene Silencing ,RNA, Small Interfering ,chemistry.chemical_classification ,Base Sequence ,Chemistry ,Oligonucleotide ,Phosphonate ,030104 developmental biology ,Argonaute Proteins ,Molecular Medicine - Abstract
(E)-Vinylphosphonate ((E)-VP), a metabolically stable phosphate mimic at the 5'-end of the antisense strand, enhances the in vivo potency of siRNA. Here we describe a straightforward synthetic approach to incorporate a nucleotide carrying a vinylphosphonate (VP) moiety at the 5'-end of oligonucleotides under standard solid-phase synthesis and deprotection conditions by utilizing pivaloyloxymethyl (POM) protected VP-nucleoside phosphoramidites. The POM protection enhances scope and scalability of 5'-VP-modified oligonucleotides and, in a broader sense, the synthesis of oligonucleotides modified with phosphonate moieties. Trivalent N-acetylgalactosamine-conjugated small interfering RNA (GalNAc-siRNA) comprising (E)-geometrical isomer of VP showed improved RISC loading with robust RNAi-mediated gene silencing in mice compared to the corresponding (Z)-isomer despite similar tissue accumulation. We also obtained structural insights into why bulkier 2'-ribosugar substitutions such as 2'-O-[2-(methylamino)-2-oxoethyl] are well tolerated only when combined with 5'-(E)-VP.
- Published
- 2018
29. 4′-C-Methoxy-2′-deoxy-2′-fluoro Modified Ribonucleotides Improve Metabolic Stability and Elicit Efficient RNAi-Mediated Gene Silencing
- Author
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Shigeo Matsuda, Martin Egli, Anna Bisbe, Kallanthottathil G. Rajeev, Johans Fakhoury, Masad J. Damha, Rajar M. Manoharan, Mihai Burai Patrascu, Martin Maier, Donald Foster, Lydia Perkins, Ivan Zlatev, Klaus Charisse, Dale C. Guenther, Nate Taneja, Elise Malek-Adamian, Joel M. Harp, Nicolas Moitessier, Muthiah Manoharan, and Saúl Martínez-Montero
- Subjects
0301 basic medicine ,Small interfering RNA ,RNA Stability ,Stereochemistry ,Substituent ,Nucleic Acid Denaturation ,Hyperconjugation ,Biochemistry ,Catalysis ,03 medical and health sciences ,chemistry.chemical_compound ,Organophosphorus Compounds ,Colloid and Surface Chemistry ,Nucleotide ,Gene Silencing ,RNA, Small Interfering ,Uridine ,chemistry.chemical_classification ,Nuclease ,biology ,Chemistry ,RNA ,General Chemistry ,Ribonucleotides ,RNAi Therapeutics ,030104 developmental biology ,biology.protein ,Thermodynamics ,RNA Interference ,Epimer - Abstract
We designed novel 4′-modified 2′-deoxy-2′-fluorouridine (2′-F U) analogues with the aim to improve nuclease resistance and potency of therapeutic siRNAs by introducing 4′-C-methoxy (4′-OMe) as the alpha (C4′α) or beta (C4′β) epimers. The C4′α epimer was synthesized by a stereoselective route in six steps; however, both α and β epimers could be obtained by a nonstereoselective approach starting from 2′-F U. 1H NMR analysis and computational investigation of the α-epimer revealed that the 4′-OMe imparts a conformational bias toward the North-East sugar pucker, due to intramolecular hydrogen bonding and hyperconjugation effects. The α-epimer generally conceded similar thermal stability as unmodified nucleotides, whereas the β-epimer led to significant destabilization. Both 4′-OMe epimers conferred increased nuclease resistance, which can be explained by the close proximity between 4′-OMe substituent and the vicinal 5′- and 3′-phosphate group, as seen in the X-ray crystal structure of modified RNA. siRNAs con...
- Published
- 2017
30. Abstract P2031: Control Of Antihypertensive Effect Of Small Interfering RNA Targeting Angiotensinogen
- Author
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A.H. Jan Danser, Estrellita Uijl, Ewout J. Hoorn, Ivan Zlatev, Stephen Huang, Don Foster, Katrina M Mirabito Colafella, Lauren Melton, and Jae B. Kim
- Subjects
Small interfering RNA ,business.industry ,Internal Medicine ,Medicine ,Vasoconstrictor Agents ,Pharmacology ,business ,Hypertension experimental - Abstract
A single dose of small interfering ribonucleic acids (siRNA) targeting angiotensinogen (AGT) provides a strong and sustained antihypertensive effect, lasting weeks to months in preclinical models. It is not known whether conventional vasopressors are effective to reverse RNAi-mediated blood pressure lowering. Here, we investigated whether conventional vasopressors can elevate arterial pressure under conditions of AGT depletion. Spontaneously hypertensive rats on a low-salt diet (
- Published
- 2019
31. Abstract P2030: DOCA-Salt Diminishes Brain RAS Activity In Parallel With Plasma And Renal RAS Activity - No Evidence For Selective Brain RAS Activation
- Author
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Estrellita Uijl, Katrina M Mirabito Colafella, Ivan Zlatev, Oliver Domenig, Stephen Huang, A.H. Jan Danser, Lauren Melton, Don Foster, Marko Poglitsch, Jae B. Kim, and Liwei Ren
- Subjects
medicine.medical_specialty ,Increase blood pressure ,Chemistry ,Doca salt ,Angiotensin II ,Endocrinology ,Downregulation and upregulation ,Internal medicine ,parasitic diseases ,Internal Medicine ,medicine ,Deoxycorticosterone acetate ,Hypertension experimental ,hormones, hormone substitutes, and hormone antagonists - Abstract
Deoxycorticosterone acetate (DOCA)-salt is suggested to increase blood pressure via selective upregulation of the brain renin-angiotensin system (RAS), while suppressing the circulating RAS. Yet, we have observed parallel downregulation of plasma and brain renin in mice. Here, we quantified brainstem angiotensinogen (AGT) and angiotensin (Ang) levels in rats exposed to DOCA (200mg; 60-day release) and 0.9% NaCl as drinking water for 7 weeks. To determine the contribution of AGT (liver vs. kidney/brain), Ang II type 1 receptors (AT1R) and blood pressure to tissue Ang II content, rats received vehicle, liver-targeted AGT siRNA (10 mg/kg fortnightly; s.c.), valsartan (31 mg/kg/day; s.c.) or spironolactone (80 mg/kg/day; s.c.) during the final 3 weeks, the latter fully normalizing blood pressure (n=3-9 per group). Plasma renin and AGT were determined by enzyme-kinetic assay, tissue AGT by western blotting, and Ang I and II by LC-MS/MS. Plasma renin, AGT, Ang I and II in rats not exposed to DOCA-salt were 24±8ng Ang I/mL per h, 731±51nM, 123±38pM and 109±43pM. AGT was present in liver, kidney and brainstem. Kidney Ang I and II were 540±135and 510±97fmol/g, while brainstem Ang I and II were undetectable in all and 50% of the rats, respectively (
- Published
- 2019
32. Strong and Sustained Antihypertensive Effect of Small Interfering RNA Targeting Liver Angiotensinogen
- Author
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René de Vries, Katrina M Mirabito Colafella, Ewout J. Hoorn, Estrellita Uijl, Ivan Zlatev, Ingrid M. Garrelds, Yuan Sun, Don Foster, A.H. Jan Danser, Marko Poglitsch, Jae B. Kim, Liwei Ren, Richard van Veghel, and Internal Medicine
- Subjects
0301 basic medicine ,Male ,Small interfering RNA ,Injections, Subcutaneous ,Angiotensinogen ,Blood Pressure ,030204 cardiovascular system & hematology ,Pharmacology ,RNAi Therapeutics ,03 medical and health sciences ,0302 clinical medicine ,Rats, Inbred SHR ,Renin–angiotensin system ,Internal Medicine ,Medicine ,Animals ,RNA, Small Interfering ,business.industry ,Acute kidney injury ,medicine.disease ,Rats ,Disease Models, Animal ,030104 developmental biology ,Gene Expression Regulation ,Liver ,Hypertension ,RNA ,business - Abstract
Small interfering RNAs (siRNAs) targeting hepatic angiotensinogen ( Agt ) may provide long-lasting antihypertensive effects, but the optimal approach remains unclear. Here, we assessed the efficacy of a novel AGT siRNA in spontaneously hypertensive rats. Rats were treated with vehicle, siRNA (10 mg/kg fortnightly; subcutaneous), valsartan (31 mg/kg per day; oral), captopril (100 mg/kg per day; oral), valsartan+siRNA, or captopril+valsartan for 4 weeks (all groups, n=8). Mean arterial pressure (recorded via radiotelemetry) was lowered the most by valsartan+siRNA (−68±4 mm Hg), followed by captopril+valsartan (−54±4 mm Hg), captopril (−23±2 mm Hg), siRNA (−14±2 mm Hg), and valsartan (−10±2 mm Hg). siRNA and captopril monotherapies improved cardiac hypertrophy equally, but less than the dual therapies, which also lowered NT-proBNP (N-terminal pro-B-type natriuretic peptide). Glomerular filtration rate, urinary NGAL (neutrophil gelatinase-associated lipocalin), and albuminuria were unaffected by treatment. siRNA lowered circulating AGT by 97.9±1.0%, and by 99.8±0.1% in combination with valsartan. Although siRNA greatly reduced renal Ang (angiotensin) I, only valsartan+siRNA suppressed circulating and renal Ang II. This coincided with decreased renal sodium hydrogen exchanger type 3 and phosphorylated sodium chloride cotransporter abundances. Renin and plasma K + increased with every treatment, but especially during valsartan+siRNA; no effects on aldosterone were observed. Collectively, these data indicate that Ang II elimination requires >99% suppression of circulating AGT. Maximal blockade of the renin-angiotensin system, achieved by valsartan+siRNA, yielded the greatest reduction in blood pressure and cardiac hypertrophy, whereas AGT lowering alone was as effective as conventional renin-angiotensin system inhibitors. Given its stable and sustained efficacy, lasting weeks, RNA interference may offer a unique approach to improving therapy adherence and treating hypertension.
- Published
- 2019
33. DOCA-SALT DIMINISHES BRAIN RAS ACTIVITY IN PARALLEL WITH PLASMA AND RENAL RAS ACTIVITY – NO EVIDENCE FOR SELECTIVE BRAIN RAS ACTIVATION
- Author
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Stephen Huang, Ivan Zlatev, Don Foster, Estrellita Uijl, Jae B. Kim, Oliver Domenig, Lauren Melton, Liwei Ren, A.H. Jan Danser, Marko Poglitsch, and Katrina M Mirabito Colafella
- Subjects
medicine.medical_specialty ,Endocrinology ,Physiology ,business.industry ,Internal medicine ,Internal Medicine ,medicine ,Cardiology and Cardiovascular Medicine ,business ,Doca salt - Published
- 2021
34. CONTROL OF ANTIHYPERTENSIVE EFFECT MEDIATED BY SMALL INTERFERING RNA TARGETING ANGIOTENSINOGEN
- Author
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Don Foster, Ewout J. Hoorn, Katrina M Mirabito Colafella, Ingrid M. Garrelds, Ivan Zlatev, A.H. Jan Danser, Estrellita Uijl, Liwei Ren, Richard van Veghel, Lauren Melton, Jae B. Kim, and Stephen Huang
- Subjects
Small interfering RNA ,Physiology ,business.industry ,Internal Medicine ,Medicine ,Pharmacology ,Cardiology and Cardiovascular Medicine ,business - Published
- 2021
35. 5′-C-Malonyl RNA: Small Interfering RNAs Modified with 5′-Monophosphate Bioisostere Demonstrate Gene Silencing Activity
- Author
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Kallanthottathil G. Rajeev, Muthiah Manoharan, Martin Egli, Jingxuan Liu, Donald Foster, Klaus Charisse, Martin Maier, Ivan Zlatev, Benjamin Brigham, Rubina G. Parmar, and Vasant Jadhav
- Subjects
0301 basic medicine ,Small interfering RNA ,RNA-induced transcriptional silencing ,RNA-induced silencing complex ,Trans-acting siRNA ,RNA ,General Medicine ,Argonaute ,Biology ,Biochemistry ,Malonates ,Mice ,03 medical and health sciences ,RNA silencing ,030104 developmental biology ,Animals ,Molecular Medicine ,Gene silencing ,Gene Silencing ,Phosphorylation ,RNA, Small Interfering ,Cells, Cultured - Abstract
5'-Phosphorylation is a critical step in the cascade of events that leads to loading of small interfering RNAs (siRNAs) into the RNA-induced silencing complex (RISC) to elicit gene silencing. 5'-Phosphorylation of exogenous siRNAs is generally accomplished by a cytosolic Clp1 kinase, and in most cases, the presence of a 5'-monophosphate on synthetic siRNAs is not a prerequisite for activity. Chemically introduced, metabolically stable 5'-phosphate mimics can lead to higher metabolic stability, increased RISC loading, and higher gene silencing activities of chemically modified siRNAs. In this study, we report the synthesis of 5'-C-malonyl RNA, a 5'-monophosphate bioisostere. A 5'-C-malonyl-modified nucleotide was incorporated at the 5'-terminus of chemically modified RNA oligonucleotides using solid-phase synthesis. In vitro silencing activity, in vitro metabolic stability, and in vitro RISC loading of 5'-C-malonyl siRNA was compared to corresponding 5'-phosphorylated and 5'-nonphosphorylated siRNAs. The 5'-C-malonyl siRNAs showed sustained or improved in vitro gene silencing and high levels of Ago2 loading and conferred dramatically improved metabolic stability to the antisense strand of the siRNA duplexes. In silico modeling studies indicate a favorable fit of the 5'-C-malonyl group within the 5'-phosphate binding pocket of human Ago2MID domain.
- Published
- 2016
36. Reversal of siRNA-mediated gene silencing in vivo
- Author
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Svetlana Shulga-Morskaya, Shigeo Matsuda, Muthiah Manoharan, Akin Akinc, Huilei Xu, Martin Maier, Kallanthottathil G. Rajeev, Scott Waldron, Ivan Zlatev, Christopher R. Brown, June Qin, Adam Castoreno, Mark K Schlegel, Rohan Degaonkar, Swati Gupta, and Vasant Jadhav
- Subjects
0301 basic medicine ,Small interfering RNA ,Acetylgalactosamine ,Biomedical Engineering ,Oligonucleotides ,Bioengineering ,Ligands ,Applied Microbiology and Biotechnology ,03 medical and health sciences ,Mice ,RNA interference ,In vivo ,Gene silencing ,Animals ,Humans ,Gene Silencing ,RNA, Small Interfering ,Cells, Cultured ,Base Sequence ,Oligonucleotide ,Chemistry ,RNA ,Cell biology ,Mice, Inbred C57BL ,030104 developmental biology ,Nucleic acid ,Hepatocytes ,Molecular Medicine ,Female ,RNA Interference ,Conjugate ,Biotechnology - Abstract
We report rapid, potent reversal of GalNAc-siRNA-mediated RNA interference (RNAi) activity in vivo with short, synthetic, high-affinity oligonucleotides complementary to the siRNA guide strand. We found that 9-mers with five locked nucleic acids (LNAs) have the highest potency across several targets. Our modular, sequence-specific approach, named REVERSIR, may enhance the therapeutic profile of any long-acting GalNAc-siRNA (short interfering RNA) conjugate by enabling control of RNAi pharmacology.
- Published
- 2017
37. Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides
- Author
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Luciano H. Apponi, Ivan Zlatev, Dinah W.Y. Sah, Stephany Michelle Standley, Nenad Svrzikapa, Gregory L. Verdine, Chandra Vargeese, David Butler, Meena, Genliang Lu, Jason Jingxin Zhang, Naoki Iwamoto, Susovan Mohapatra, and Maria Frank-Kamenetsky
- Subjects
0301 basic medicine ,Male ,RNase P ,Stereochemistry ,Mipomersen ,Ribonuclease H ,Biomedical Engineering ,Oligonucleotides ,Phosphorothioate Oligonucleotides ,Bioengineering ,Stereoisomerism ,Mice, Transgenic ,010402 general chemistry ,01 natural sciences ,Applied Microbiology and Biotechnology ,Rats, Sprague-Dawley ,03 medical and health sciences ,Mice ,Drug Stability ,Animals ,Humans ,Oligonucleotide ,Chemistry ,Genetic Therapy ,Oligonucleotides, Antisense ,Small molecule ,In vitro ,0104 chemical sciences ,Rats ,Mice, Inbred C57BL ,030104 developmental biology ,Nucleic acid ,Molecular Medicine ,Female ,RNA Cleavage ,Hydrophobic and Hydrophilic Interactions ,Biotechnology - Abstract
Whereas stereochemical purity in drugs has become the standard for small molecules, stereoisomeric mixtures containing as many as a half million components persist in antisense oligonucleotide (ASO) therapeutics because it has been feasible neither to separate the individual stereoisomers, nor to synthesize stereochemically pure ASOs. Here we report the development of a scalable synthetic process that yields therapeutic ASOs having high stereochemical and chemical purity. Using this method, we synthesized rationally designed stereopure components of mipomersen, a drug comprising 524,288 stereoisomers. We demonstrate that phosphorothioate (PS) stereochemistry substantially affects the pharmacologic properties of ASOs. We report that Sp-configured PS linkages are stabilized relative to Rp, providing stereochemical protection from pharmacologic inactivation of the drug. Further, we elucidated a triplet stereochemical code in the stereopure ASOs, 3'-SpSpRp, that promotes target RNA cleavage by RNase H1 in vitro and provides a more durable response in mice than stereorandom ASOs.
- Published
- 2016
38. A Glu-urea-Lys Ligand-conjugated Lipid Nanoparticle/siRNA System Inhibits Androgen Receptor Expression In Vivo
- Author
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Kallanthottathil G. Rajeev, Paul S. Rennie, Ivan Zlatev, Kaixin Zhang, Ying K. Tam, Yuen Yi C. Tam, Paulo Jc Lin, Justin B. Lee, Yan Liu, Sam Chen, Pieter R. Cullis, Muthiah Manoharan, Jayaprakash K. Nair, and Joslyn Quick
- Subjects
0301 basic medicine ,liposomes ,Small interfering RNA ,Biology ,lipid nanoparticles ,03 medical and health sciences ,Prostate cancer ,0302 clinical medicine ,In vivo ,androgen receptor ,Drug Discovery ,medicine ,Liposome ,Messenger RNA ,lcsh:RM1-950 ,prostate specific membrane antigen ,Ligand (biochemistry) ,medicine.disease ,prostate cancer ,3. Good health ,Androgen receptor ,lcsh:Therapeutics. Pharmacology ,030104 developmental biology ,Biochemistry ,030220 oncology & carcinogenesis ,siRNA ,Cancer research ,Systemic administration ,Molecular Medicine ,Original Article - Abstract
The androgen receptor plays a critical role in the progression of prostate cancer. Here, we describe targeting the prostate-specific membrane antigen using a lipid nanoparticle formulation containing small interfering RNA designed to silence expression of the messenger RNA encoding the androgen receptor. Specifically, a Glu-urea-Lys PSMA-targeting ligand was incorporated into the lipid nanoparticle system formulated with a long alkyl chain polyethylene glycol-lipid to enhance accumulation at tumor sites and facilitate intracellular uptake into tumor cells following systemic administration. Through these features, and by using a structurally refined cationic lipid and an optimized small interfering RNA payload, a lipid nanoparticle system with improved potency and significant therapeutic potential against prostate cancer and potentially other solid tumors was developed. Decreases in serum prostate-specific antigen, tumor cellular proliferation, and androgen receptor levels were observed in a mouse xenograft model following intravenous injection. These results support the potential clinical utility of a prostate-specific membrane antigen–targeted lipid nanoparticle system to silence the androgen receptor in advanced prostate cancer.
- Published
- 2016
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39. 3′-Deoxy Phosphoramidate Dinucleosides as Improved Inhibitors of Hepatitis C Virus Subgenomic Replicon and NS5B Polymerase Activity
- Author
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Karine Alvarez, Hélène Dutartre, Johan Neyts, Ivan Zlatev, Jean-Jacques Vasseur, François Morvan, Stéphane Priet, Katrien Geerts, Pieter Leyssen, Ivan Barvík, Bruno Canard, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Department of Microbiology and Immunology, Rega Institute for Medical Research, and Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven)
- Subjects
Hepatitis C virus ,Genome, Viral ,Hepacivirus ,Molecular Dynamics Simulation ,Viral Nonstructural Proteins ,Virus Replication ,medicine.disease_cause ,Antiviral Agents ,01 natural sciences ,Structure-Activity Relationship ,03 medical and health sciences ,Organophosphorus Compounds ,Cell Line, Tumor ,Drug Discovery ,medicine ,Side chain ,[CHIM]Chemical Sciences ,Humans ,Potency ,IC50 ,ComputingMilieux_MISCELLANEOUS ,Polymerase ,030304 developmental biology ,EC50 ,0303 health sciences ,biology ,010405 organic chemistry ,Chemistry ,virus diseases ,Nucleosides ,Stereoisomerism ,Phosphoramidate ,DNA-Directed RNA Polymerases ,Molecular biology ,digestive system diseases ,In vitro ,3. Good health ,0104 chemical sciences ,Biochemistry ,biology.protein ,RNA, Viral ,Molecular Medicine ,Replicon - Abstract
Phosphoramidate dinucleosides named "GC 3'-OH" series, carrying various phosphoramidate linkages have been previously reported as hepatitis C virus (HCV) inhibitors. To enhance the efficacy of these dinucleotides, we synthesized a novel "GC 3'-H" series as potential chain terminators. We showed that their inhibition potency is strongly increased by the introduction of novel neutral and bis-negatively charged phosphoramidate side chains. Their inhibitory effect on HCV NS5B polymerase was evaluated in vitro and in HCV subgenomic replicon containing Huh-6 cells. As expected, 3'-H compounds are more potent than their 3'-OH counterparts to inhibit HCV polymerase activity. The most potent inhibitor, a 5'-phosphorylated dinucleotide bearing a bis-negatively charged amino side chain (7), exhibits an IC(50) value of 8 μM in vitro and EC(50) value of 2.6 μM in the HCV subgenomic replicon system. A molecular structure model is presented to propose an interpretation of the gain afforded by the of 3'-H-cytidine modification.
- Published
- 2010
40. Influenza A virus-generated small RNAs regulate the switch from transcription to replication
- Author
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Ivan Zlatev, Adolfo García-Sastre, Andrew Varble, Benjamin R. tenOever, Muthiah Manoharan, Ravi Sachidanandam, and Jasmine T. Perez
- Subjects
Multidisciplinary ,biology ,viruses ,DNA replication ,RNA ,RNA-dependent RNA polymerase ,medicine.disease_cause ,Virology ,Virus ,chemistry.chemical_compound ,chemistry ,Transcription (biology) ,RNA polymerase ,biology.protein ,Influenza A virus ,medicine ,Polymerase - Abstract
The discovery of regulatory small RNAs continues to reshape paradigms in both molecular biology and virology. Here we describe examples of influenza A virus-derived small viral RNAs (svRNAs). svRNAs are 22–27 nt in length and correspond to the 5′ end of each of the viral genomic RNA (vRNA) segments. Expression of svRNA correlates with the accumulation of vRNA and a bias in RNA-dependent RNA polymerase (RdRp) activity from transcription toward genome replication. Synthesis of svRNA requires the RdRp, nucleoprotein and the nuclear export protein NS2. In addition, svRNA is detectable during replication of various influenza A virus subtypes across multiple host species and associates physically with the RdRp. We demonstrate that depletion of svRNA has a minimal impact on mRNA and complementary vRNA (cRNA) but results in a dramatic loss of vRNA in a segment-specific manner. We propose that svRNA triggers the viral switch from transcription to replication through interactions with the viral polymerase machinery. Taken together, the discovery of svRNA redefines the mechanistic switch of influenza virus transcription/replication and provides a potential target for broad-range, anti-influenza virus-based therapeutics.
- Published
- 2010
41. A Small-RNA Enhancer of Viral Polymerase Activity
- Author
-
Benjamin R. tenOever, Sai Lakshmi Subramanian, Muthiah Manoharan, Shilpa Aggarwal, Ivan Zlatev, Ravi Sachidanandam, Baek Kim, and Jasmine T. Perez
- Subjects
Small RNA ,viruses ,Immunology ,Blotting, Western ,RNA-dependent RNA polymerase ,Genome, Viral ,Microbiology ,Cell Line ,chemistry.chemical_compound ,Viral entry ,Transcription (biology) ,Virology ,RNA polymerase ,RNA polymerase I ,Humans ,Immunoprecipitation ,Polymerase ,biology ,RNA-Dependent RNA Polymerase ,Genome Replication and Regulation of Viral Gene Expression ,chemistry ,Influenza A virus ,Insect Science ,biology.protein ,Small nuclear RNA - Abstract
Influenza A virus (IAV) is an unremitting virus that results in significant morbidity and mortality worldwide. Key to the viral life cycle is the RNA-dependent RNA polymerase (RdRp), a heterotrimeric complex responsible for both transcription and replication of the segmented genome. Here, we demonstrate that the viral polymerase utilizes a small RNA enhancer to regulate enzymatic activity and maintain stoichiometric balance of the viral genome. We demonstrate that IAV synthesizes small viral RNAs (svRNAs) that interact with the viral RdRp in order to promote genome replication in a segment-specific manner. svRNAs localize to the nucleus, the site of IAV replication, are synthesized from the positive-sense genomic intermediate, and interact within a novel RNA binding channel of the polymerase PA subunit. Synthetic svRNAs promote polymerase activity in vitro , while loss of svRNA inhibits viral RNA synthesis in a segment-specific manner. Taking these observations together, we mechanistically define svRNA as a small regulatory enhancer RNA, which functions to promote genome replication and maintain segment balance through allosteric modulation of polymerase activity.
- Published
- 2012
42. Automated parallel synthesis of 5'-triphosphate oligonucleotides and preparation of chemically modified 5'-triphosphate small interfering RNA
- Author
-
Richard G Duncan, Ligang Zhang, Ivan Zlatev, Kallanthottathil G. Rajeev, Muthiah Manoharan, Jeremy G. Lackey, Kathy McRae, Sarfraz Shaikh, and Amy Dell
- Subjects
Small interfering RNA ,Clinical Biochemistry ,Oligonucleotides ,Pharmaceutical Science ,Oligonucleotide synthesis ,Biochemistry ,chemistry.chemical_compound ,Automation ,Mice ,RNA interference ,Polyphosphates ,Drug Discovery ,Gene silencing ,Animals ,Humans ,RNA, Small Interfering ,Molecular Biology ,Cells, Cultured ,Molecular Structure ,Oligonucleotide ,Chemistry ,Organic Chemistry ,RNA ,Combinatorial chemistry ,In vitro ,Molecular Medicine ,DNA ,HeLa Cells - Abstract
A fully automated chemical method for the parallel and high-throughput solid-phase synthesis of 5'-triphosphate and 5'-diphosphate oligonucleotides is described. The desired full-length oligonucleotides were first constructed using standard automated DNA/RNA solid-phase synthesis procedures. Then, on the same column and instrument, efficient implementation of an uninterrupted sequential cycle afforded the corresponding unmodified or chemically modified 5'-triphosphates and 5'-diphosphates. The method was readily translated into a scalable and high-throughput synthesis protocol compatible with the current DNA/RNA synthesizers yielding a large variety of unique 5'-polyphosphorylated oligonucleotides. Using this approach, we accomplished the synthesis of chemically modified 5'-triphosphate oligonucleotides that were annealed to form small-interfering RNAs (ppp-siRNAs), a potentially interesting class of novel RNAi therapeutic tools. The attachment of the 5'-triphosphate group to the passenger strand of a siRNA construct did not induce a significant improvement in the in vitro RNAi-mediated gene silencing activity nor a strong specific in vitro RIG-I activation. The reported method will enable the screening of many chemically modified ppp-siRNAs, resulting in a novel bi-functional RNAi therapeutic platform.
- Published
- 2012
43. Efficient solid-phase chemical synthesis of 5'-triphosphates of DNA, RNA and their analogues
- Author
-
Thomas Lavergne, Françoise Debart, Jean-Jacques Vasseur, Ivan Zlatev, Muthiah Manoharan, François Morvan, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), and Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)
- Subjects
Organophosphonates ,01 natural sciences ,Biochemistry ,Chemical synthesis ,High-performance liquid chromatography ,03 medical and health sciences ,chemistry.chemical_compound ,Phase (matter) ,[CHIM]Chemical Sciences ,Nucleotide ,Solid phase extraction ,Physical and Theoretical Chemistry ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,010405 organic chemistry ,Oligonucleotide ,Solid Phase Extraction ,Organic Chemistry ,RNA ,DNA ,Combinatorial chemistry ,0104 chemical sciences ,chemistry ,Nucleic Acid Conformation - Abstract
A robust, reproducible, and scalable method for the solid-phase synthesis of 5'-triphosphates of DNA, RNA, and their chemically modified analogues using 5'-H-phosphonate intermediates is described. 5'-Triphosphates of oligonucleotides with varying lengths and sequences containing different 5'-terminal nucleotides, with and without internal sugar-backbone modifications, were efficiently prepared as crude products or further purified by HPLC.
- Published
- 2010
44. Delta-Di-carboxybutyl phosphoramidate of 2'-deoxycytidine-5'-monophosphate as substrate for DNA-polymerization by HIV-1 reverse transcriptase
- Author
-
Piet Herdewijn, Jean-Jacques Vasseur, François Morvan, Ivan Zlatev, Anne Giraut, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), and Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)
- Subjects
DNA Replication ,Stereochemistry ,Clinical Biochemistry ,Pharmaceutical Science ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Pyrophosphate ,Substrate Specificity ,chemistry.chemical_compound ,Drug Discovery ,Phosphoric Acids ,Nucleotide ,Molecular Biology ,Polymerase ,ComputingMilieux_MISCELLANEOUS ,chemistry.chemical_classification ,biology ,010405 organic chemistry ,[CHIM.ORGA]Chemical Sciences/Organic chemistry ,RNA-Directed DNA Polymerase ,Organic Chemistry ,Phosphoramidate ,Deoxycytidine Monophosphate ,Amides ,HIV Reverse Transcriptase ,Reverse transcriptase ,Amino Acids, Dicarboxylic ,0104 chemical sciences ,Enzyme ,chemistry ,biology.protein ,Molecular Medicine ,DNA - Abstract
The replacement of the pyrophosphate moiety of 2′-deoxynucleoside triphosphates by non natural δ-dicarboxylic butyl amino acid allows incorporation of natural 2′-deoxycytidine into DNA using HIV-1 reverse transcriptase (RT) as enzyme. In contrast, the 3′-deoxycytidine analogue was not a substrate of the HIV-1 RT.
- Published
- 2009
45. Phosphoramidate dinucleosides as hepatitis C virus polymerase inhibitors
- Author
-
Jean-Jacques Vasseur, François Morvan, Karine Alvarez, Bruno Canard, Hélène Dutartre, Johan Neyts, Ivan Barvík, Ivan Zlatev, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), and Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)
- Subjects
Magnetic Resonance Spectroscopy ,Stereochemistry ,Hepatitis C virus ,Lysine ,Genome, Viral ,Hepacivirus ,Viral Nonstructural Proteins ,medicine.disease_cause ,Virus Replication ,01 natural sciences ,03 medical and health sciences ,Organophosphorus Compounds ,Cell Line, Tumor ,Drug Discovery ,medicine ,Humans ,Enzyme Inhibitors ,Magnesium ion ,ComputingMilieux_MISCELLANEOUS ,Polymerase ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,[CHIM.ORGA]Chemical Sciences/Organic chemistry ,010405 organic chemistry ,Chemistry ,Active site ,Phosphoramidate ,Nucleosides ,3. Good health ,0104 chemical sciences ,Enzyme ,Biochemistry ,Enzyme inhibitor ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,biology.protein ,Molecular Medicine ,Oxidation-Reduction - Abstract
GC dinucleosides exhibiting a phosphoramidate internucleosidic linkage with neutral, amphiphile, positively or negatively charged side chains were synthesized. Their potential inhibitory effect on the hepatitis C virus (HCV) NS5B polymerase was evaluated in vitro and in HCV replicon containing cells. Whereas the amphiphile and the positively charged analogues were found to be inactive, the neutral (1) and the negatively charged (4) ones inhibited enzyme activity when tested as a diastereoisomeric mixture. The most potent inhibitor proved to be the Sp isomer of the 5'-thiophosphorylated dinucleotide bearing the carboxylic side chain (8) (IC 50 of 25 microM in vitro and an EC 50 of 9 microM in HCV subgenomic replicon). Molecular modeling suggests that the phosphoramidate dinucleoside (8) is stabilized in the active site by interactions with magnesium ions and lysine and arginine residues of the polymerase.
- Published
- 2008
46. Deoxygenation of 5
- Author
-
Jean-Jacques Vasseur, François Morvan, and Ivan Zlatev
- Subjects
Hydrogen ,3′-Deoxynucleosides ,Barton–McCombie deoxygenation ,chemistry.chemical_element ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Medicinal chemistry ,Article ,Thiocarbonylimidazolyl ,Drug Discovery ,Organic chemistry ,Glycosyl donor ,Deoxygenation ,ComputingMethodologies_COMPUTERGRAPHICS ,chemistry.chemical_classification ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,Glycoside ,3. Good health ,0104 chemical sciences ,Reagent ,Dimethyl phosphite - Abstract
Graphical abstract, An efficient radical deoxygenation reaction of thiocarbonylimidazolyl activated glycoside analogue using dimethyl phosphite as hydrogen source and radical chain carrier was performed as a key step in a multi step synthesis towards a common 3-deoxy glycosyl donor for 3′-deoxynucleosides. This method has safety and cost advantages compared to the generally used radical reduction reagents.
- Published
- 2008
47. Convenient synthesis of N2-isobutyryl-2'-O-methyl guanosine by efficient alkylation of O6-trimethylsilylethyl-3'-5'-di-tert-butylsilanediyl guanosine
- Author
-
Jean-Jacques Vasseur, Ivan Zlatev, François Morvan, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), and Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)
- Subjects
0303 health sciences ,Chemistry ,Organic Chemistry ,Guanosine ,Single step ,Methylation ,Alkylation ,010402 general chemistry ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,03 medical and health sciences ,chemistry.chemical_compound ,Reagent ,Yield (chemistry) ,Drug Discovery ,Organic chemistry ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,030304 developmental biology - Abstract
We present a novel route for the synthesis of N 2 -isobutyryl-2′- O -methyl guanosine, introducing 3′,5′-di- tert -butylsilyl and O 6 -trimethylsilylethyl groups as efficient protections during the 2′- O -methylation step with NaH/CH 3 I. These protections were then removed simultaneously in a single step with TBAF. The eight-step synthesis is easy to perform, employing convenient commercially available reagents; crude mixtures are of satisfying purity, so only three chromatography purifications were required. Title compound was obtained in 25% overall yield from guanosine.
- Published
- 2007
48. Use of a solid-supported coupling reagent for a selective phosphitylation of the primary alcohol of N2-isobutyryl-2'-deoxy or 2'-O-methyl guanosine
- Author
-
Yukiko Kato, Ivan Zlatev, Albert Meyer, Jean-Jacques Vasseur, François Morvan, Chimie organique biomoléculaire de synthèse (COBS), and Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
chemistry.chemical_classification ,Phosphoramidite ,010405 organic chemistry ,Organic Chemistry ,Guanosine ,Coupling reagent ,Primary alcohol ,010402 general chemistry ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,chemistry.chemical_compound ,chemistry ,Drug Discovery ,Organic chemistry ,Nucleotide ,Selectivity ,Nucleoside ,Phosphine - Abstract
We have developed a 5′-regioselective phosphitylation of 3′,5′-OH-guanosine derivatives thanks to a solid-supported coupling reagent with either a standard or a bulky phosphine. A 5′-phosphitylation up to a 95% selectivity was obtained with a quantitative conversion of starting nucleoside. After oxidation into thionophosphotriester or phosphotriester by means of solid-supported oxidizers, the 5′-phosphorylated N 2 - i -Bu-2′-OMe guanosines were isolated in good yields (70–80%).
- Published
- 2006
49. Correction to 3′-Deoxy Phosphoramidate Dinucleosides as Improved Inhibitors of Hepatitis C Virus Subgenomic Replicon and NS5B Polymerase Activity
- Author
-
Karine Alvarez, Ivan Barvík, Pieter Leyssen, Katrien Geerts, Stéphane Priet, Bruno Canard, Ivan Zlatev, François Morvan, Jean-Jacques Vasseur, Hélène Dutartre, and Johan Neyts
- Subjects
Chemistry ,Hepatitis C virus ,Drug Discovery ,medicine ,Molecular Medicine ,Phosphoramidate ,medicine.disease_cause ,Virology ,Ns5b polymerase ,Subgenomic replicon - Published
- 2011
50. Phosphoramidate Dinucleosides as Inhibitors of Hepatitis C Virus Subgenomic Replicon and NS5B Polymerase Activity
- Author
-
Ivan Barvík, Bruno Canard, Stéphane Priet, Hélène Dutartre, Johan Neyts, Karine Alvarez, Ivan Zlatev, and François Morvan
- Subjects
Pharmacology ,Chemistry ,Hepatitis B virus DNA polymerase ,Virology ,Hepatitis C virus ,medicine ,Phosphoramidate ,medicine.disease_cause ,Ns5b polymerase ,Subgenomic replicon - Published
- 2011
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