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Na v 1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 ( 1 ) and GDC-0310 ( 2 ), selective Na v 1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3 . This resulted in the discovery of GDC-0276 ( 1 ), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Na v 1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 ( 2 ), which possesses improved Na v selectivity and pharmacokinetic profile over 1 .

Abstract: The voltage-gated sodium channel NaV1.7 plays an important role in pain processing according to genetic data. Those data made NaV1.7 a popular drug target, especially since its relatively selective expression in nociceptors promised pain relief without the adverse effects associated with broader sodium channel blockade. Despite encouraging preclinical data in rodents, NaV1.7-selective inhibitors have not yet proven effective in clinical trials. Discrepancies between preclinical and clinical results should raise alarms. We reviewed preclinical and clinical reports on the analgesic efficacy of NaV1.7-selective inhibitors and found critical differences in several factors. Putting aside species differences, most preclinical studies tested young male rodents with limited genetic variability, inconsistent with the clinical population. Inflammatory pain was the most common preclinical chronic pain model whereas nearly all clinical trials focused on neuropathic pain despite some evidence suggesting NaV1.7 channels are not essential for neuropathic pain. Preclinical studies almost exclusively measured evoked pain whereas most clinical trials assessed average pain intensity without distinguishing between evoked and spontaneous pain. Nearly all preclinical studies gave a single dose of drug unlike the repeat dosing used clinically, thus precluding preclinical data from demonstrating whether tolerance or other slow processes occur. In summary, preclinical testing of NaV1.7-selective inhibitors aligned poorly with clinical testing. Beyond issues that have already garnered widespread attention in the pain literature, our results highlight the treatment regimen and choice of pain model as areas for improvement., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Association for the Study of Pain.)

Herein, we report the discovery and optimization of a series of orally bioavailable acyl sulfonamide Na V 1.7 inhibitors that are selective for Na V 1.7 over Na V 1.5 and highly efficacious in in vivo models of pain and hNa V 1.7 target engagement. An analysis of the physicochemical properties of literature Na V 1.7 inhibitors suggested that acyl sulfonamides with high f sp3 could overcome some of the pharmacokinetic (PK) and efficacy challenges seen with existing series. Parallel library syntheses lead to the identification of analogue 7, which exhibited moderate potency against Na V 1.7 and an acceptable PK profile in rodents, but relatively poor stability in human liver microsomes. Further, design strategy then focused on the optimization of potency against hNa V 1.7 and improvement of human metabolic stability, utilizing induced fit docking in our previously disclosed X-ray cocrystal of the Na V 1.7 voltage sensing domain. These investigations culminated in the discovery of tool compound 33, one of the most potent and efficacious Na V 1.7 inhibitors reported to date.

The sodium channel Na V 1.7 has emerged as a promising target for the treatment of pain based on strong genetic validation of its role in nociception. In recent years, a number of aryl and acyl sulfonamides have been reported as potent inhibitors of Na V 1.7, with high selectivity over the cardiac isoform Na V 1.5. Herein, we report on the discovery of a novel series of N-([1,2,4]triazolo[4,3- a]pyridin-3-yl)methanesulfonamides as selective Na V 1.7 inhibitors. Starting with the crystal structure of an acyl sulfonamide, we rationalized that cyclization to form a fused heterocycle would improve physicochemical properties, in particular lipophilicity. Our design strategy focused on optimization of potency for block of Na V 1.7 and human metabolic stability. Lead compounds 10, 13 (GNE-131), and 25 showed excellent potency, good in vitro metabolic stability, and low in vivo clearance in mouse, rat, and dog. Compound 13 also displayed excellent efficacy in a transgenic mouse model of induced pain.

We report on a novel series of aryl sulfonamides that act as nanomolar potent, isoform-selective inhibitors of the human sodium channel hNaV1.7. The optimization of these inhibitors is described. We aimed to improve potency against hNaV1.7 while minimizing off-target safety concerns and generated compound 3. This agent displayed significant analgesic effects in rodent models of acute and inflammatory pain and demonstrated that binding to the voltage sensor domain 4 site of NaV1.7 leads to an analgesic effect in vivo. Our findings corroborate the importance of hNaV1.7 as a drug target for the treatment of pain.

In light of the small ring strain of five/six-membered cyclic ethers, constructing complex molecules via ring-opening reactions has consistently been a highly challenging topic in organic synthesis. Induced by Lewis acids, the charge redistribution in cyclic ethers forms oxonium ylide intermediates, thereby activating the C–O bond and subsequently facilitating nucleophilic attack for ring opening. In recent years, a variety of novel Lewis acids, encompassing those with new metal centers and frustrated Lewis pairs (FLPs), have been effectively utilized to induce the formation of oxonium ylides, offering a diverse array of methods for the ring opening of five/six-membered cyclic ethers. This review conveys the extensive application advancements of diverse Lewis acid types for cyclic ether ring-opening reactions over the past two decades, originating from the perspective of the classification of Lewis acids. Furthermore, the substrate applicability and chemical transformation efficiency of these Lewis acids in the ring-opening reactions of cyclic ethers have also been discussed herein. [ABSTRACT FROM AUTHOR]

Various substituted D-hexopypyranosides units with nitrogen-containing functionalities are present in many important natural compounds and pharmaceutical substances. Since their complex structural diversity contributes to a broad spectrum of biological functions and activities, these derivatives are frequently studied. This review covers syntheses of D-hexopyranosides with vicinal nitrogen-containing functionalities since the 1960s, when the first articles emerged. The syntheses are arranged according to the positions of substitutions, to form a relative configuration of vicinal functionalities, and synthetic methodologies. [ABSTRACT FROM AUTHOR]

DNA (cytosine-5)-methyltransferases (DNMTs) catalyze the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the 5-position of cytosine residues and thereby silence transcription of regulated genes. DNMTs are important epigenetic targets. However, isolated DNMTs are weak catalysts and are difficult to assay. We report an ultrasensitive luciferase-linked continuous assay that converts the S-adenosyl-L-homocysteine product of DNA methylation to a quantifiable luminescent signal. Results with this assay are compared with the commonly used DNA labeling from [methyl-(3)H]AdoMet. A 5'-methylthioadenosine-adenosylhomocysteine nucleosidase is used to hydrolyze AdoHcy to adenine. Adenine phosphoribosyl transferase converts adenine to AMP and pyruvate orthophosphate dikinase converts AMP to ATP. Firefly luciferase gives a stable luminescent signal that results from continuous AMP recycling to ATP. This assay exhibits a broad dynamic range (0.1-1000 pmol of AdoHcy). The rapid response time permits continuous assays of DNA methylation detected by light output. The assay is suitable for high-throughput screening of chemical libraries for DNMT inhibition activity. The kinetic properties of human and bacterial CpG methyltransferases are characterized using this assay. Human catalytic domain DNMT3b activation by DNMT3L is shown to involve two distinct kinetic states that alter k(cat) but not K(m) for AdoMet. The assay is shown to be robust in the presence of high concentrations of the pyrimidine analogues 5-azacytidine and 5-azacytosine.

The S-adenosylmethionine (AdoMet) salvage enzyme 5'-methylthioadenosine phosphorylase (MTAP) has been implicated as both a cancer target and a tumor suppressor. We tested these hypotheses in mouse xenografts of human lung cancers. AdoMet recycling from 5'-methylthioadenosine (MTA) was blocked by inhibition of MTAP with methylthio-DADMe-Immucillin-A (MTDIA), an orally available, nontoxic, picomolar transition state analogue. Blood, urine, and tumor levels of MTA increased in response to MTDIA treatment. MTDIA treatment inhibited A549 (human non-small cell lung carcinoma) and H358 (human bronchioloalveolar non-small cell lung carcinoma cells) xenograft tumor growth in immunodeficient Rag2(-/-)γC(-/-) and NCr-nu mice. Systemic MTA accumulation is implicated as the tumor-suppressive metabolite because MTDIA is effective for in vivo treatment of A549 MTAP(-/-) and H358 MTAP(+/+) tumors. Tumors from treated mice showed increased MTA and decreased polyamines but little alteration in AdoMet, methionine, or adenine levels. Gene expression profiles of A549 tumors from treated and untreated mice revealed only modest alterations with 62 up-regulated and 63 down-regulated mRNAs (≥ 3-fold). MTDIA antitumor activity in xenografts supports MTAP as a target for lung cancer therapy.

The synthesis of 4-deoxy-4-nitrosialic acid (3,4,5-trideoxy-4-nitro-D-glycero-beta-D-galacto-non-2-ulopyranosonic acid, 5), was completed in seven steps starting from D-arabinose. Coupling of the 6-carbon fragment, 2-acetamido-1,2-dideoxy-1-nitro-D-mannitol (6) with ethyl alpha-(bromomethyl)acrylate afforded a 2 : 1 mixture of ethyl 5-acetamido-2,3,4,5-tetradeoxy-2-methylene-4-nitro-D-glycero-D-galacto-nononate (9a-S) and ethyl 5-acetamido-2,3,4,5-tetradeoxy-2-methylene-4-nitro-D-glycero-D-talo-nononate (9a-R). This mixture of enones was subjected to ozonolysis, and following reduction of the ozonide, the resultant products cyclised to the pyranosides. The target compound, ethyl 4-deoxy-4-nitrosialate (11a) was isolated by fractional crystallisation. Hydrolysis of the ethyl ester proved problematic; thus, the synthesis was modified by using tert-butyl alpha-(bromomethyl)acrylate. Following ozonolysis of the corresponding tert-butyl enoate esters and diastereomer separation, the tert-butyl ester of 4-nitrosialic acid (11b) could be deprotected under acidic conditions to afford . The target compound is a useful intermediate for synthesis of a variety of C-4 substituted sialic acid derivatives, and it is synthesised by a modular route.

Silylstannanes can be regioselectively added across terminal alkynes in a quantitative fashion in the presence of a palladium(0) catalyst immobilised in the [bmim]PF6 ionic liquid which can be recycled without loss of activity.

Background and Purpose: The voltage-gated sodium channel isoform Na V 1.7 is a high-interest target for the development of non-opioid analgesics due to its preferential expression in pain-sensing neurons. Na V 1.7 is also expressed in autonomic neurons, yet its contribution to involuntary visceral reflexes has received limited attention. The small molecule inhibitor ST-2560 was advanced into pain behaviour and cardiovascular models to understand the pharmacodynamic effects of selective inhibition of Na V 1.7., Experimental Approach: Potency of ST-2560 at Na V 1.7 and off-target ion channels was evaluated by whole-cell patch-clamp electrophysiology. Effects on nocifensive reflexes were assessed in non-human primate (NHP) behavioural models, employing the chemical capsaicin and mechanical stimuli. Cardiovascular parameters were monitored continuously in freely-moving, telemetered NHPs following administration of vehicle and ST-2560., Key Results: ST-2560 is a potent inhibitor (IC 50  = 39 nM) of Na V 1.7 in primates with ≥1000-fold selectivity over other isoforms of the human Na V 1.x family. Following systemic administration, ST-2560 (0.1-0.3 mg·kg -1 , s.c.) suppressed noxious mechanical- and chemical-evoked reflexes at free plasma concentrations threefold to fivefold above Na V 1.7 IC 50 . ST-2560 (0.1-1.0 mg·kg -1 , s.c.) also produced changes in haemodynamic parameters, most notably a 10- to 20-mmHg reduction in systolic and diastolic arterial blood pressure, at similar exposures., Conclusions and Implications: Acute pharmacological inhibition of Na V 1.7 is antinociceptive, but also has the potential to impact the cardiovascular system. Further work is merited to understand the role of Na V 1.7 in autonomic ganglia involved in the control of heart rate and blood pressure, and the effect of selective Na V 1.7 inhibition on cardiovascular function., (© 2024 British Pharmacological Society.)

Simple Summary: Many types of cancer cells utilize the common amino acid l-glutamine to maintain their metabolic demands for energy and the nitrogen required for DNA synthesis. Versatility to control energy metabolism from l-glutamine (anaplerosis) is promoted by the use of two distinct pathways. The first (pathway 1; the canonical pathway) is as follows: [l-glutamine → l-glutamate ⇆ α-ketoglutarate → tricarboxylic acid (TCA) cycle]. This pathway contrasts with the much less studied GTωA (glutamine transaminase—ω-amidase or glutaminase II) pathway (pathway 2): [l-glutamine ⇆ α-ketoglutaramate (KGM) → α-ketoglutarate → TCA cycle]. Our prior publications have emphasized the importance of regulation of both pathways, which enables cancer cells to maintain selective metabolic advantages and to conserve their reliance on glucose. This review summarizes the metabolic importance of the GTωA pathway in both cancerous and normal tissues and proposes that anti-cancer strategies, based on inhibition of l-glutamine metabolism, require consideration of both the canonical and GTωA pathways. Many cancers utilize l-glutamine as a major energy source. Often cited in the literature as "l-glutamine addiction", this well-characterized pathway involves hydrolysis of l-glutamine by a glutaminase to l-glutamate, followed by oxidative deamination, or transamination, to α-ketoglutarate, which enters the tricarboxylic acid cycle. However, mammalian tissues/cancers possess a rarely mentioned, alternative pathway (the glutaminase II pathway): l-glutamine is transaminated to α-ketoglutaramate (KGM), followed by ω-amidase (ωA)-catalyzed hydrolysis of KGM to α-ketoglutarate. The name glutaminase II may be confused with the glutaminase 2 (GLS2) isozyme. Thus, we recently renamed the glutaminase II pathway the "glutamine transaminase—ω-amidase (GTωA)" pathway. Herein, we summarize the metabolic importance of the GTωA pathway, including its role in closing the methionine salvage pathway, and as a source of anaplerotic α-ketoglutarate. An advantage of the GTωA pathway is that there is no net change in redox status, permitting α-ketoglutarate production during hypoxia, diminishing cellular energy demands. We suggest that the ability to coordinate control of both pathways bestows a metabolic advantage to cancer cells. Finally, we discuss possible benefits of GTωA pathway inhibitors, not only as aids to studying the normal biological roles of the pathway but also as possible useful anticancer agents. [ABSTRACT FROM AUTHOR]

Morpholine is a heterocyclic moiety that is widely used in medicinal chemistry as a building block. It has unique physicochemical properties, as it can improve both pharmacokinetic and pharmacodynamic properties of active pharmaceutical ingredients. However, the efficient synthesis of enantiomerically pure morpholine building blocks remains challenging. Herein, we report the synthesis of optically pure 3-hydroxymethylmorpholine building blocks, as well as their sulfamidates, exploiting a stereospecific strategy from chiral pool material. [ABSTRACT FROM AUTHOR]

A mouse model of human Familial Adenomatous Polyposis responds favorably to pharmacological inhibition of 5′-methylthioadenosine phosphorylase (MTAP). Methylthio-DADMe-Immucillin-A (MTDIA) is an orally available, transition state analogue inhibitor of MTAP. 5′-Methylthioadenosine (MTA), the substrate for MTAP, is formed in polyamine synthesis and is recycled by MTAP to S-adenosyl-l-methionine (SAM) via salvage pathways. MTDIA treatment causes accumulation of MTA, which inhibits growth of human head and neck (FaDu) and lung (H359, A549) cancers in immunocompromised mouse models. We investigated the efficacy of oral MTDIA as an anti-cancer therapeutic for intestinal adenomas in immunocompetent APCMin/+ mice, a murine model of human Familial Adenomatous Polyposis. Tumors in APCMin/+ mice were decreased in size by MTDIA treatment, resulting in markedly improved anemia and doubling of mouse lifespan. Metabolomic analysis of treated mice showed no changes in polyamine, methionine, SAM or ATP levels when compared with control mice but indicated an increase in MTA, the MTAP substrate. Generation of an MTDIA-resistant cell line in culture showed a four-fold amplification of the methionine adenosyl transferase (MAT2A) locus and expression of this enzyme. MAT2A is downstream of MTAP action and catalyzes synthesis of the SAM necessary for methylation reactions. Immunohistochemical analysis of treated mouse intestinal tissue demonstrated a decrease in symmetric dimethylarginine, a PRMT5-catalyzed modification. The anti-cancer effects of MTDIA indicate that increased cellular MTA inhibits PRMT5-mediated methylations resulting in attenuated tumor growth. Oral dosing of MTDIA as monotherapy has potential for delaying the onset and progression of colorectal cancers in Familial Adenomatous Polyposis (FAP) as well as residual duodenal tumors in FAP patients following colectomy. MTDIA causes a physiologic inactivation of MTAP and may also have efficacy in combination with inhibitors of MAT2A or PRMT5, known synthetic-lethal interactions in MTAP−/− cancer cell lines. [ABSTRACT FROM AUTHOR]

Phosphorus oxychloride, POCl3, an important phosphorus compound, has diverse applications in synthetic chemistry. It plays an important role in chlorination, Vilsmeier Haack reaction, dehydration and cyclization which have been highlighted here. Moreover, different reaction conditions implied and reported in the literature along with the general mechanism are provided briefly in this review. This review provides an insight into the recent applications of phosphorus oxychloride as a reagent in organic syntheses in the last two year i.e. 2018–19. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to identify critical genes involved in the tumor biology of lung cancer via datamining of The Cancer Genome Atlas (TCGA) with special focus on gene copy number variation. Methods: Genomic deletion and amplification were analyzed with cBioportal online tools. Relative expression of Cyclin Dependent Kinase Inhibitor 2A (CDKN2A) was analyzed by both real‐time polymerase chain reaction (PCR) and Western blot. The abundance of methylthioadenosine phosphorylase (MTAP) and epithelial‐mesenchymal transition markers were analyzed by real‐time PCR. Cell proliferation was determined by cell counting kit‐8 method and cell viability was measured with 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. The cell migration and invasion were measured with transwell chamber assay, and migrative capacity was further evaluated by wound healing assay. Results: We found the frequent loss of CDKN2A was associated with its downregulation in lung cancer, and siRNA‐mediated CDNKN2A knockdown significantly stimulated cell proliferation, invasion, and migration. Mechanistically, we unraveled that MTAP, which was positively correlated with CDKN2A, predominantly mediated the antitumoral function of CDKN2A in lung cancer. Conclusion: Our study consolidated the involvement of CDKN2A‐MTAP signaling in the context of lung cancer. [ABSTRACT FROM AUTHOR]

Functional group interconversions are essential chemical processes enabling synthesis. In this report, we describe a strategy to convert alkylidenemalononitriles into primary alcohols in one step. The reaction relies on a choreographed redox process involving alkylidene reduction, malononitrile oxidation, and acylcyanide reduction where molecular oxygen and NaBH4 work cooperatively. The method was applied to a variety of carbon skeletons and was utilized to synthesize complex terpenoid architectures. [ABSTRACT FROM AUTHOR]

Recapitulating human disease pathophysiology using genetic animal models is a powerful approach to enable mechanistic understanding of genotype--phenotype relationships for drug development. NaV1.7 is a sodium channel expressed in the peripheral nervous system with strong human genetic validation as a pain target. Efforts to identify novel analgesics that are nonaddictive resulted in industry exploration of a class of sulfonamide compounds that bind to the fourth voltage-sensor domain of NaV1.7. Due to sequence differences in this region, sulfonamide blockers generally are potent on human but not rat NaV1.7 channels. To test sulfonamide-based chemical matter in rat models of pain, we generated a humanized NaV1.7 rat expressing a chimeric NaV1.7 protein containing the sulfonamide-binding site of the human gene sequence as a replacement for the equivalent rat sequence. Unexpectedly, upon transcription, the human insert was spliced out, resulting in a premature stop codon. Using a validated antibody, NaV1.7 protein was confirmed to be lost in the brainstem, dorsal root ganglia, sciatic nerve, and gastrointestinal tissue but not in nasal turbinates or olfactory bulb in rats homozygous for the knock-in allele (HOM-KI). HOM-KI rats exhibited normal intraepidermal nerve fiber density with reduced tetrodotoxin-sensitive current density and action potential firing in small diameter dorsal root ganglia neurons. HOM-KI rats did not exhibit nociceptive pain responses in hot plate or capsaicin-induced flinching assays and did not exhibit neuropathic pain responses following spinal nerve ligation. Consistent with expression of chimeric NaV1.7 in olfactory tissue, HOM-KI rats retained olfactory function. This new genetic model highlights the necessity of NaV1.7 for pain behavior in rats and indicates that sufficient inhibition of NaV1.7 in humans may reduce pain in neuropathic conditions. Due to preserved olfactory function, this rat model represents an alternative to global NaV1.7 knockout mice that require time-intensive hand feeding during early postnatal development. [ABSTRACT FROM AUTHOR]

Lysine methylation, catalyzed by protein lysine methyltransferases (PKMTs), is a central post-translational modification regulating many signaling pathways. It has direct and indirect effects on chromatin structure and transcription. Accumulating evidence suggests that dysregulation of PKMT activity has a fundamental impact on the development of many pathologies. While most of these works involve in-depth analysis of methylation events in the context of histones, in recent years, it has become evident that methylation of non-histone proteins also plays a pivotal role in cell processes. This review highlights the importance of non-histone methylation, with focus on methylation events taking place in the nucleus. Known experimental platforms which were developed to identify new methylation events, as well as examples of specific lysine methylation signaling events which regulate key transcription factors, are presented. In addition, the role of these methylation events in normal and disease states is emphasized. [ABSTRACT FROM AUTHOR]

Sialic acids cap the glycans of cell surface glycoproteins and glycolipids. They are involved in a multitude of biological processes and aberrant sialic acid expression is associated with several pathologies. Sialic acids modulate the characteristics and functions of glycoproteins and regulate cell–cell as well as cell–extracellular matrix interactions. Pathogens such as influenza virus use sialic acids to infect host cells and cancer cells exploit sialic acids to escape from the host's immune system. The introduction of unnatural sialic acids with different functionalities into surface glycans enables the study of the broad biological functions of these sugars and presents a therapeutic option to intervene with pathological processes involving sialic acids. Multiple chemically modified sialic acid analogs can be directly utilized by cells for sialoglycan synthesis. Alternatively, analogs of the natural sialic acid precursor sugar N-Acetylmannosamine (ManNAc) can be introduced into the sialic acid biosynthesis pathway resulting in the intracellular conversion into the corresponding sialic acid analog. Both, ManNAc and sialic acid analogs, have been employed successfully for a large variety of glycoengineering applications such as glycan imaging, targeting toxins to tumor cells, inhibiting pathogen binding, or altering immune cell activity. However, there are significant differences between ManNAc and sialic acid analogs with respect to their chemical modification potential and cellular metabolism that should be considered in sialic acid glycoengineering experiments. [ABSTRACT FROM AUTHOR]

An efficient and viable synthesis of α-ketoesters from alkyl halides and α-carbonyl aldehydes has been reported under metal-free conditions. The present method involves oxidative esterification of α-carbonyl aldehydes with alkyl halide using TBAI as a promoter and TBHP as an oxidant to form α-ketoesters in good to excellent yields with versatile structural diversity. Use of commercially accessible and inexpensive substrates, broad substrate scope and good functional group tolerance are the key features of this protocol. [ABSTRACT FROM AUTHOR]

A metal-free oxidative coupling reaction of trifluoromethyl β-diketones with alcohols for the synthesis of α-keto esters in good to excellent yields has been developed. Preliminary mechanistic studies suggest that an I2/TBHP promoted sequential iodination, C-C bond cleavage, C-O bond formation and oxidation pathway is involved in this reaction. [ABSTRACT FROM AUTHOR]

Resveratrol is a natural polyphenol with plethora of biological activities. Resveratrol has previously shown to decrease DNA-methyltransferase (DNMT) enzymes expression and to reactivate silenced tumor suppressor genes. Currently, it seems that no resveratrol analogs have been developed as DNMT inhibitors. Recently, we reported the synthesis of resveratrol-salicylate derivatives and by examining the chemical structure of these analogs, we proposed that these compounds could exhibit DNMT inhibition especially that they resembled NSC 14778, a compound we previously identified as a DNMT inhibitor by virtual screening. Indeed, usingin vitroDNMT inhibition assay, some of the resveratrol-salicylate analogs we screened in this work that showed selective inhibition against DNMT3 enzymes which were greater than resveratrol. A molecular docking study revealed key binding interactions with DNMT3A and DNMT3B enzymes. In addition, the most active analog,10showed considerable cytotoxicity against three human cancer cells; HT-29, HepG2 and SK-BR-3, which was greater than resveratrol. Further studies are needed to understand the anticancer mechanisms of these derivatives. [ABSTRACT FROM AUTHOR]

Human DNA methyltransferase 1 (DNMT1) maintains the epigenetic state of DNA by replicating CpG methylation signatures from parent to daughter strands, producing heritable methylation patterns through cell divisions. The proposed catalytic mechanism of DNMT1 involves nucleophilic attack of Cys1226 to cytosine (Cyt) C6, methyl transfer from S-adenosyl-L-methionine (SAM) to Cyt C5, and proton abstraction from C5 to form methylated CpG in DNA. Here, we report the subangstrom geometric and electrostatic structure of the major transition state (TS) of the reaction catalyzed by human DNMT1. Experimental kinetic isotope effects were used to guide quantum mechanical calculations to solve the TS structure. Methyl transfer occurs after Cys1226 attack to Cyt C6, and the methyl transfer step is chemically rate-limiting for DNMT1. Electrostatic potential maps were compared for the TS and ground states, providing the electronic basis for interactions between the protein and reactants at the TS. Understanding the TS of DNMT1 demonstrates the possibility of using similar analysis to gain subangstrom geometric insight into the complex reactions of epigenetic modifications. [ABSTRACT FROM AUTHOR]

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Epigenetic regulation has been implicated in diverse diseases including cancer, diabetes, and inflammation, and high-throughput screening for histone methyltransferase (HMT) inhibitors is an area of intense drug discovery effort. HMTs catalyze the transfer of methyl group from S-adenosylmethionine (SAM) to lysine or arginine on histone tails forming the methylated products and S-adenosylhomocysteine (SAH). HMTs are challenging to incorporate into biochemical assays for a number of reasons. They have slow turnovers and low Km values for SAM, which leads to low levels of product formation, and thus requires very sensitive detection methods and/or high levels of enzyme. They also have diverse acceptor substrate requirements, ranging from peptides to intact nucleosomes. Additionally, some HMTs function as complexes of three or more proteins. Developing assays for individual HMTs, including sourcing and acquiring high quality enzymes and acceptor substrates, therefore can be laborious and expensive. We recently developed the Transcreener® EPIGEN Methyltransferase assay, a sensitive SAH detection method with a fluorescence polarization readout, to enable universal HMT detection independent of acceptor substrate. To facilitate screening and profiling of HMTs, we describe the development of turnkey assay systems for thirteen HMTs including identification of optimal acceptor substrates and their concentrations, optimization of detection reagents, determination of initial velocity enzyme concentrations, and measurement of inhibitor potencies. [ABSTRACT FROM AUTHOR]

Copyright of Canadian Journal of Chemistry is the property of Canadian Science Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)