16 results on '"Verlinde CLMJ"'
Search Results
2. CRYSTALLOGRAPHIC AND MOLECULAR MODELING STUDIES ON TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE - A CRITICAL-ASSESSMENT OF THE PREDICTED AND OBSERVED STRUCTURES OF THE COMPLEX WITH 2-PHOSPHOGLYCERATE
- Author
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NOBLE, MEM, VERLINDE, CLMJ, GROENDIJK, H, KALK, KH, WIERENGA, RK, and HOL, WGJ
- Subjects
TRIOSE-PHOSPHATE ISOMERASE ,RESOLUTION ,REFINEMENT ,BRUCEI-BRUCEI ,CATALYSIS ,PROGRAM ,DRUG DESIGN ,COMPUTER-GRAPHICS ,ENZYMES - Abstract
In the continuation of a project aimed at the rational design of drugs against diseases caused by trypanosomes, the crystal structure of trypanosomal triosephosphate isomerase in complex with the active site inhibitor 2-phosphoglycerate has been determined. Two alternative modeling protocols have been attempted to predict the mode of binding of this ligand. In the first protocol, certain key interactions were restrained in the modeling procedure. In the second protocol, a full search of ligand conformational space was performed. In both cases the protein scaffold was kept static. Both protocols produced models which were reasonably close to the observed structure (rms difference
- Published
- 1991
3. ANION BINDING AT THE ACTIVE-SITE OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE - MONOHYDROGEN PHOSPHATE DOES NOT MIMIC SULFATE
- Author
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VERLINDE, CLMJ, NOBLE, MEM, KALK, KH, GROENDIJK, H, WIERENGA, RK, HOL, WGJ, and Groningen Biomolecular Sciences and Biotechnology
- Subjects
BRUCEI-BRUCEI ,CRYSTALLOGRAPHY ,ENZYMES ,PARAMETERS - Abstract
The three-dimensional structure of triosephosphate isomerase complexed with the competitive inhibitor HPO4(2-) was determined by X-ray crystallography to a resolution of 0.24 nm. A comparison with the native crystal structure, where SO4(2-) is bound, revealed five changes: (a) a 0.10-nm shift of the anion-binding site; (b) a further closing of the flexible loop of the enzyme; (c) a 'swinging in' of the side chain of the catalytic Glu, that is chi-1 changes from (+) to (-) synclinal; (d) an altered water structure; (e) a disappearance of the conformational heterogeneity at the C-terminus of strand beta-7. Some of these changes may be related to the different hydrogen-bond pattern about the two different anions. However, the distance of 0.10 nm between the sulphur and phosphorus positions is unexpected and remains intriguing.
- Published
- 1991
4. Synthesis and Structure-activity-relationships of Analogs of 2'-deoxy-2'-(3-methoxybenzamido)adenosine, a Selective Inhibitor of Trypanosomal Glycosomal Glyceraldehyde-3-phosphate Dehydrogenase
- Author
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UCL, Vancalenbergh, S., Verlinde, CLMJ., Soenens, J., Debruyn, A., Callens, M., Blaton, NM., Peeters, OM., Rozenski, J., Hol, WGJ., Herdewijn, P., UCL, Vancalenbergh, S., Verlinde, CLMJ., Soenens, J., Debruyn, A., Callens, M., Blaton, NM., Peeters, OM., Rozenski, J., Hol, WGJ., and Herdewijn, P.
- Abstract
In continuation of a project aimed at the structure-based design of drugs against sleeping sickness, analogs of 2'-deoxy-2'-(3-methoxybenzamido)adenosine (1) were synthesized and tested to establish structure-activity relationships for inhibiting glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Compound 1 was recently designed using the NAD: GAPDH complexes of the human enzyme and that of Trypanosoma brucei, the causative agent of sleeping sickness. In an effort to exploit an extra hydrophobic domain due to Val 207 of the parasite enzyme, several new 2'-amido-2'-deoxyadenosines were synthesized. Some of them displayed an interesting improvement in inhibitory activity compared to 1. Carbocyclic or acyclic analogs showed marked loss of activity, illustrating the importance of the typical (C-2'-endo) puckering of the ribose moiety. We also describe the synthesis of a pair of compounds that combine the beneficial effects of a 2- and 8-substituted adenine moiety on potency with the beneficial effect of a 2'-amido moiety on selectivity. Unfortunately, in both cases, IC50 values demonstrate the incompatibility of these combined modifications. Finally, introduction bf a hydrophobic 5'-amido group on 5'-deoxyadenosine enhances the inhibition of the protozoan enzyme significantly, although the gain in selectivity is mediocre.
- Published
- 1995
5. Spontaneous Selection of Cryptosporidium Drug Resistance in a Calf Model of Infection.
- Author
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Hasan MM, Stebbins EE, Choy RKM, Gillespie JR, de Hostos EL, Miller P, Mushtaq A, Ranade RM, Teixeira JE, Verlinde CLMJ, Sateriale A, Zhang Z, Osbourn DM, Griggs DW, Fan E, Buckner FS, and Huston CD
- Subjects
- Animals, Cattle, Child, Child, Preschool, Drug Resistance genetics, Feces, Humans, Cattle Diseases drug therapy, Cryptosporidiosis drug therapy, Cryptosporidium genetics, Cryptosporidium parvum genetics
- Abstract
The intestinal protozoan Cryptosporidium is a leading cause of diarrheal disease and mortality in young children. There is currently no fully effective treatment for cryptosporidiosis, which has stimulated interest in anticryptosporidial development over the last ∼10 years, with numerous lead compounds identified, including several tRNA synthetase inhibitors. Here, we report the results of a dairy calf efficacy trial of the methionyl-tRNA ( Cryptosporidium parvum MetRS [ Cp MetRS]) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with Cryptosporidium parvum initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day 5. Parasites shed by each recrudescent calf had different amino acid-altering mutations in the gene encoding Cp MetRS ( CpMetRS ), yielding either an aspartate 243-to-glutamate (D243E) or a threonine 246-to-isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246I Cp MetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant-expressing parasites, respectively, had 2093 half-maximal effective concentrations (EC
50 s) that were 613- and 128-fold that of transgenic parasites with wild-type Cp MetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC50 >170-fold. Structural modeling of Cp MetRS based on an inhibitor-bound Trypanosoma brucei MetRS crystal structure suggested that the resistance mutations reposition nearby hydrophobic residues, interfering with compound binding while minimally impacting substrate binding. This is the first report of naturally emerging Cryptosporidium drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence., (Copyright © 2021 American Society for Microbiology.)- Published
- 2021
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6. Repurposing Infectious Disease Hits as Anti- Cryptosporidium Leads.
- Author
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Hulverson MA, Choi R, McCloskey MC, Whitman GR, Ojo KK, Michaels SA, Somepalli M, Love MS, McNamara CW, Rabago LM, Barrett LK, Verlinde CLMJ, Arnold SLM, Striepen B, Jimenez-Alfaro D, Ballell L, Fernández E, Greenwood MN, Las Heras L, Calderón F, and Van Voorhis WC
- Subjects
- Drug Repositioning, Humans, Infant, Communicable Diseases, Cryptosporidiosis drug therapy, Cryptosporidium, Cryptosporidium parvum
- Abstract
New drugs are critically needed to treat Cryptosporidium infections, particularly for malnourished children under 2 years old in the developing world and persons with immunodeficiencies. Bioactive compounds from the Tres-Cantos GSK library that have activity against other pathogens were screened for possible repurposing against Cryptosporidium parvum growth. Nineteen compounds grouped into nine structural clusters were identified using an iterative process to remove excessively toxic compounds and screen related compounds from the Tres-Cantos GSK library. Representatives of four different clusters were advanced to a mouse model of C. parvum infection, but only one compound, an imidazole-pyrimidine, led to significant clearance of infection. This imidazole-pyrimidine compound had a number of favorable safety and pharmacokinetic properties and was maximally active in the mouse model down to 30 mg/kg given daily. Though the mechanism of action against C. parvum was not definitively established, this imidazole-pyrimidine compound inhibits the known C. parvum drug target, calcium-dependent protein kinase 1, with a 50% inhibitory concentration of 2 nM. This compound, and related imidazole-pyrimidine molecules, should be further examined as potential leads for Cryptosporidium therapeutics.
- Published
- 2021
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7. Structure-guided discovery of selective methionyl-tRNA synthetase inhibitors with potent activity against Trypanosoma brucei .
- Author
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Zhang Z, Barros-Álvarez X, Gillespie JR, Ranade RM, Huang W, Shibata S, Molasky NMR, Faghih O, Mushtaq A, Choy RKM, de Hostos E, Hol WGJ, Verlinde CLMJ, Buckner FS, and Fan E
- Abstract
Based on crystal structures of Trypanosoma brucei methionyl-tRNA synthetase ( Tb MetRS) bound to inhibitors, we designed, synthesized, and evaluated two series of novel Tb MetRS inhibitors targeting this parasite enzyme. One series has a 1,3-dihydro-imidazol-2-one containing linker, the other has a rigid fused aromatic ring in the linker. For both series of compounds, potent inhibition of parasite growth was achieved with EC
50 < 10 nM and most compounds exhibited low general toxicity to mammalian cells with CC50 s > 20 000 nM. Selectivity over human mitochondrial methionyl tRNA synthetase was also evaluated, using a cell-based mitochondrial protein synthesis assay, and selectivity in a range of 20-200-fold was achieved. The inhibitors exhibited poor permeability across the blood brain barrier, necessitating future efforts to optimize the compounds for use in late stage human African trypanosomiasis., (This journal is © The Royal Society of Chemistry 2020.)- Published
- 2020
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8. Optimization of Methionyl tRNA-Synthetase Inhibitors for Treatment of Cryptosporidium Infection.
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Buckner FS, Ranade RM, Gillespie JR, Shibata S, Hulverson MA, Zhang Z, Huang W, Choi R, Verlinde CLMJ, Hol WGJ, Ochida A, Akao Y, Choy RKM, Van Voorhis WC, Arnold SLM, Jumani RS, Huston CD, and Fan E
- Subjects
- Animals, Cryptosporidium parvum genetics, Cyclooxygenase 1 drug effects, Disease Models, Animal, Drug Discovery methods, Female, Hep G2 Cells, Humans, Imidazoles chemistry, Mice, Mice, Inbred C57BL, Mice, Knockout, Pyridines chemistry, Antiprotozoal Agents pharmacology, Cryptosporidiosis drug therapy, Cryptosporidium parvum drug effects, Imidazoles pharmacology, Methionine-tRNA Ligase antagonists & inhibitors, Pyridines pharmacology
- Abstract
Cryptosporidiosis is one of the leading causes of moderate to severe diarrhea in children in low-resource settings. The therapeutic options for cryptosporidiosis are limited to one drug, nitazoxanide, which unfortunately has poor activity in the most needy populations of malnourished children and HIV-infected persons. We describe here the discovery and early optimization of a class of imidazopyridine-containing compounds with potential for treating Cryptosporidium infections. The compounds target the Cryptosporidium methionyl-tRNA synthetase (MetRS), an enzyme that is essential for protein synthesis. The most potent compounds inhibited the enzyme with K
i values in the low picomolar range. Cryptosporidium cells in culture were potently inhibited with 50% effective concentrations as low as 7 nM and >1,000-fold selectivity over mammalian cells. A parasite persistence assay indicates that the compounds act by a parasiticidal mechanism. Several compounds were demonstrated to control infection in two murine models of cryptosporidiosis without evidence of toxicity. Pharmacological and physicochemical characteristics of compounds were investigated to determine properties that were associated with higher efficacy. The results indicate that MetRS inhibitors are excellent candidates for development for anticryptosporidiosis therapy., (Copyright © 2019 American Society for Microbiology.)- Published
- 2019
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9. The crystal structure of the drug target Mycobacterium tuberculosis methionyl-tRNA synthetase in complex with a catalytic intermediate.
- Author
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Barros-Álvarez X, Turley S, Ranade RM, Gillespie JR, Duster NA, Verlinde CLMJ, Fan E, Buckner FS, and Hol WGJ
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- Catalysis, Catalytic Domain, Crystallization, Crystallography, X-Ray, Models, Molecular, Protein Binding, Protein Conformation, Drug Design, Methionine-tRNA Ligase chemistry, Methionine-tRNA Ligase metabolism, Mycobacterium tuberculosis enzymology
- Abstract
Mycobacterium tuberculosis is a pathogenic bacterial infectious agent that is responsible for approximately 1.5 million human deaths annually. Current treatment requires the long-term administration of multiple medicines with substantial side effects. Lack of compliance, together with other factors, has resulted in a worrisome increase in resistance. New treatment options are therefore urgently needed. Here, the crystal structure of methionyl-tRNA synthetase (MetRS), an enzyme critical for protein biosynthesis and therefore a drug target, in complex with its catalytic intermediate methionyl adenylate is reported. Phenylalanine 292 of the M. tuberculosis enzyme is in an `out' conformation and barely contacts the adenine ring, in contrast to other MetRS structures where ring stacking occurs between the adenine and a protein side-chain ring in the `in' conformation. A comparison with human cytosolic MetRS reveals substantial differences in the active site as well as regarding the position of the connective peptide subdomain 1 (CP1) near the active site, which bodes well for arriving at selective inhibitors. Comparison with the human mitochondrial enzyme at the amino-acid sequence level suggests that arriving at inhibitors with higher affinity for the mycobacterial enzyme than for the mitochondrial enzyme might be achievable.
- Published
- 2018
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10. The calcium-dependent protein kinase 1 from Toxoplasma gondii as target for structure-based drug design.
- Author
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Cardew EM, Verlinde CLMJ, and Pohl E
- Subjects
- Animals, Humans, Life Cycle Stages drug effects, Malaria drug therapy, Models, Molecular, Plasmodium drug effects, Protein Kinases chemistry, Protozoan Proteins metabolism, Toxoplasmosis parasitology, Drug Design, Protein Kinases drug effects, Toxoplasma drug effects, Toxoplasma enzymology
- Abstract
The apicomplexan protozoan parasites include the causative agents of animal and human diseases ranging from malaria (Plasmodium spp.) to toxoplasmosis (Toxoplasma gondii). The complex life cycle of T. gondii is regulated by a unique family of calcium-dependent protein kinases (CDPKs) that have become the target of intensive efforts to develop new therapeutics. In this review, we will summarize structure-based strategies, recent successes and future directions in the pursuit of specific and selective inhibitors of T. gondii CDPK1.
- Published
- 2018
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11. Development of Methionyl-tRNA Synthetase Inhibitors as Antibiotics for Gram-Positive Bacterial Infections.
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Faghih O, Zhang Z, Ranade RM, Gillespie JR, Creason SA, Huang W, Shibata S, Barros-Álvarez X, Verlinde CLMJ, Hol WGJ, Fan E, and Buckner FS
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- Animals, Anti-Bacterial Agents metabolism, Anti-Bacterial Agents pharmacokinetics, Blood Proteins metabolism, Drug Resistance, Bacterial drug effects, Enzyme Inhibitors chemistry, Enzyme Inhibitors metabolism, Enzyme Inhibitors pharmacokinetics, Escherichia coli drug effects, Female, Gram-Positive Bacterial Infections drug therapy, Gram-Positive Bacterial Infections microbiology, Humans, Inactivation, Metabolic, Mice, Microbial Sensitivity Tests, Microsomes, Liver, Staphylococcus aureus drug effects, Anti-Bacterial Agents chemistry, Anti-Bacterial Agents pharmacology, Enzyme Inhibitors pharmacology, Gram-Positive Bacteria drug effects, Methionine-tRNA Ligase antagonists & inhibitors
- Abstract
Antibiotic-resistant bacteria are widespread and pose a growing threat to human health. New antibiotics acting by novel mechanisms of action are needed to address this challenge. The bacterial methionyl-tRNA synthetase (MetRS) enzyme is essential for protein synthesis, and the type found in Gram-positive bacteria is substantially different from its counterpart found in the mammalian cytoplasm. Both previously published and new selective inhibitors were shown to be highly active against Gram-positive bacteria with MICs of ≤1.3 μg/ml against Staphylococcus , Enterococcus , and Streptococcus strains. Incorporation of radioactive precursors demonstrated that the mechanism of activity was due to the inhibition of protein synthesis. Little activity against Gram-negative bacteria was observed, consistent with the fact that Gram-negative bacterial species contain a different type of MetRS enzyme. The ratio of the MIC to the minimum bactericidal concentration (MBC) was consistent with a bacteriostatic mechanism. The level of protein binding of the compounds was high (>95%), and this translated to a substantial increase in MICs when the compounds were tested in the presence of serum. Despite this, the compounds were very active when they were tested in a Staphylococcus aureus murine thigh infection model. Compounds 1717 and 2144, given by oral gavage, resulted in 3- to 4-log decreases in the bacterial load compared to that in vehicle-treated mice, which was comparable to the results observed with the comparator drugs, vancomycin and linezolid. In summary, the research describes MetRS inhibitors with oral bioavailability that represent a class of compounds acting by a novel mechanism with excellent potential for clinical development., (Copyright © 2017 American Society for Microbiology.)
- Published
- 2017
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12. Leishmania donovani tyrosyl-tRNA synthetase structure in complex with a tyrosyl adenylate analog and comparisons with human and protozoan counterparts.
- Author
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Barros-Álvarez X, Kerchner KM, Koh CY, Turley S, Pardon E, Steyaert J, Ranade RM, Gillespie JR, Zhang Z, Verlinde CLMJ, Fan E, Buckner FS, and Hol WGJ
- Subjects
- Adenosine Monophosphate metabolism, Amino Acid Sequence, Binding Sites, Humans, Protein Structure, Tertiary, Sequence Alignment, Single-Chain Antibodies, Tyrosine metabolism, Tyrosine-tRNA Ligase chemistry, Adenosine Monophosphate analogs & derivatives, Catalytic Domain, Leishmania donovani enzymology, Models, Molecular, Tyrosine analogs & derivatives, Tyrosine-tRNA Ligase metabolism
- Abstract
The crystal structure of Leishmania donovani tyrosyl-tRNA synthetase (LdTyrRS) in complex with a nanobody and the tyrosyl adenylate analog TyrSA was determined at 2.75 Å resolution. Nanobodies are the variable domains of camelid heavy chain-only antibodies. The nanobody makes numerous crystal contacts and in addition reduces the flexibility of a loop of LdTyrRS. TyrSA is engaged in many interactions with active site residues occupying the tyrosine and adenine binding pockets. The LdTyrRS polypeptide chain consists of two pseudo-monomers, each consisting of two domains. Comparing the two independent chains in the asymmetric unit reveals that the two pseudo-monomers of LdTyrRS can bend with respect to each other essentially as rigid bodies. This flexibility might be useful in the positioning of tRNA for catalysis since both pseudo-monomers in the LdTyrRS chain are needed for charging tRNA
Tyr . An "extra pocket" (EP) appears to be present near the adenine binding region of LdTyrRS. Since this pocket is absent in the two human homologous enzymes, the EP provides interesting opportunities for obtaining selective drugs for treating infections caused by L. donovani, a unicellular parasite causing visceral leishmaniasis, or kala azar, which claims 20,000 to 30,000 deaths per year. Sequence and structural comparisons indicate that the EP is a characteristic which also occurs in the active site of several other important pathogenic protozoa. Therefore, the structure of LdTyrRS could inspire the design of compounds useful for treating several different parasitic diseases., (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)- Published
- 2017
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13. Optimization of a binding fragment targeting the "enlarged methionine pocket" leads to potent Trypanosoma brucei methionyl-tRNA synthetase inhibitors.
- Author
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Huang W, Zhang Z, Ranade RM, Gillespie JR, Barros-Álvarez X, Creason SA, Shibata S, Verlinde CLMJ, Hol WGJ, Buckner FS, and Fan E
- Subjects
- Animals, Binding Sites drug effects, Brain metabolism, Cell Line, Cell Survival drug effects, Dose-Response Relationship, Drug, Enzyme Inhibitors administration & dosage, Enzyme Inhibitors chemistry, Hep G2 Cells, Humans, Methionine administration & dosage, Methionine chemistry, Methionine-tRNA Ligase metabolism, Mice, Molecular Structure, Structure-Activity Relationship, Enzyme Inhibitors pharmacology, Methionine pharmacology, Methionine-tRNA Ligase antagonists & inhibitors, Trypanosoma brucei brucei enzymology
- Abstract
Potent inhibitors of Trypanosoma brucei methionyl-tRNA synthetase were previously designed using a structure-guided approach. Compounds 1 and 2 were the most active compounds in the cyclic and linear linker series, respectively. To further improve cellular potency, SAR investigation of a binding fragment targeting the "enlarged methionine pocket" (EMP) was performed. The optimization led to the identification of a 6,8-dichloro-tetrahydroquinoline ring as a favorable fragment to bind the EMP. Replacement of 3,5-dichloro-benzyl group (the EMP binding fragment) of inhibitor 2 using this tetrahydroquinoline fragment resulted in compound 13, that exhibited an EC
50 of 4nM., (Copyright © 2017 Elsevier Ltd. All rights reserved.)- Published
- 2017
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14. Structure-guided design of novel Trypanosoma brucei Methionyl-tRNA synthetase inhibitors.
- Author
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Huang W, Zhang Z, Barros-Álvarez X, Koh CY, Ranade RM, Gillespie JR, Creason SA, Shibata S, Verlinde CLMJ, Hol WGJ, Buckner FS, and Fan E
- Subjects
- Animals, Brain metabolism, Chemistry Techniques, Synthetic, Enzyme Inhibitors metabolism, Enzyme Inhibitors toxicity, Hep G2 Cells, Humans, Methionine-tRNA Ligase chemistry, Methionine-tRNA Ligase metabolism, Mice, Permeability, Protein Conformation, Structure-Activity Relationship, Trypanocidal Agents metabolism, Trypanocidal Agents toxicity, Trypanosoma brucei brucei drug effects, Drug Design, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors pharmacology, Methionine-tRNA Ligase antagonists & inhibitors, Trypanocidal Agents chemical synthesis, Trypanocidal Agents pharmacology, Trypanosoma brucei brucei enzymology
- Abstract
A screening hit 1 against Trypanosoma brucei methionyl-tRNA synthetase was optimized using a structure-guided approach. The optimization led to the identification of two novel series of potent inhibitors, the cyclic linker and linear linker series. Compounds of both series were potent in a T. brucei growth inhibition assay while showing low toxicity to mammalian cells. The best compound of each series, 16 and 31, exhibited EC
50 s of 39 and 22 nM, respectively. Compounds 16 and 31 also exhibited promising PK properties after oral dosing in mice. Moreover, compound 31 had moderately good brain permeability, with a brain/plasma ratio of 0.27 at 60 min after IP injection. This study provides new lead compounds for arriving at new treatments of human African trypanosomiasis (HAT)., Competing Interests: The authors declare no conflict of interest., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)- Published
- 2016
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15. Multivalent drug design and inhibition of cholera toxin by specific and transient protein-ligand interactions.
- Author
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Liu J, Begley D, Mitchell DD, Verlinde CLMJ, Varani G, and Fan E
- Subjects
- Cholera drug therapy, Cholera Toxin chemistry, Cross-Linking Reagents chemistry, Ligands, Magnetic Resonance Spectroscopy methods, Structure-Activity Relationship, Cholera Toxin antagonists & inhibitors, Drug Design, Protein Binding
- Abstract
Multivalent inhibitors of the cholera toxin B pentamer are potential therapeutic drugs for treating cholera and serve as models for demonstrating multivalent ligand effects through a structure-based approach. A crucial yet often overlooked aspect of multivalent drug design is the length, rigidity and chemical composition of the linker used to connect multiple binding moieties. To specifically study the role of chemical linkers in multivalent ligand design, we have synthesized a series of compounds with one and two binding motifs connected by several different linkers. These compounds have affinity for and potency against the cholera toxin B pentamer despite the fact that none can simultaneously bind two toxin receptor sites. Results from saturation transfer difference NMR reveal transient, non-specific interactions between the cholera toxin and linker groups contribute significantly to overall binding affinity of monovalent compounds. However, the same random protein-ligand interactions do not appear to affect binding of bivalent molecules. Moreover, the binding affinities and potencies of these 'non-spanning' bivalent ligands appear to be wholly independent of linker length. Our detailed analysis identifies multiple effects that account for the improved inhibitory potencies of bivalent ligands and suggest approaches to further improve the activity of this class of compounds.
- Published
- 2008
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16. Rod and cone visual cycle consequences of a null mutation in the 11-cis-retinol dehydrogenase gene in man.
- Author
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Cideciyan AV, Haeseleer F, Fariss RN, Aleman TS, Jang GF, Verlinde CLMJ, Marmor MF, Jacobson SG, and Palczewski K
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- Adaptation, Ocular genetics, Alcohol Oxidoreductases chemistry, Amino Acid Sequence genetics, Antibody Specificity, DNA Mutational Analysis, Humans, Kinetics, Male, Middle Aged, Molecular Sequence Data, NADP metabolism, Oxidation-Reduction, Protein Folding, Protein Structure, Tertiary genetics, Retinal Cone Photoreceptor Cells pathology, Retinal Cone Photoreceptor Cells physiopathology, Retinal Rod Photoreceptor Cells pathology, Retinal Rod Photoreceptor Cells physiopathology, Retinitis Pigmentosa metabolism, Retinitis Pigmentosa physiopathology, Vision, Ocular genetics, Alcohol Oxidoreductases deficiency, Alcohol Oxidoreductases genetics, Mutation physiology, Retinal Cone Photoreceptor Cells enzymology, Retinal Rod Photoreceptor Cells enzymology, Retinitis Pigmentosa genetics
- Abstract
Vertebrate vision starts with photoisomerization of the 11-cis-retinal chromophore to all-trans-retinal. Biosynthesis of 11-cis-retinal is required to maintain vision. A key enzyme catalyzing the oxidation of 11-cis-retinol is 11-cis-retinol dehydrogenase (11-cis-RDH), which is encoded by the RDH5 gene. 11-cis-RDH is expressed in the RPE and not in the neural retina. The consequences of a lack of 11-cis-RDH were studied in a family with fundus albipunctatus. We identified the causative novel RDH5 mutation, Arg157Trp, that replaces an amino acid residue conserved among short-chain alcohol dehydrogenases. Three-dimensional structure modeling and in vitro experiments suggested that this mutation destabilizes proper folding and inactivates the enzyme. Studies using RPE membranes indicated the existence of an alternative oxidizing system for the production of 11-cis-retinal. In vivo visual consequences of this null mutation showed complex kinetics of dark adaptation. Rod and cone resensitization was extremely delayed following full bleaches; unexpectedly, the rate of cone recovery was slower than rods. Cones showed a biphasic recovery with an initial rapid component and an elevated final threshold. Other unanticipated results included normal rod recovery following 0.5% bleach and abnormal recovery following bleaches in the 2-12% range. These intermediate bleaches showed rapid partial recovery of rods with transitory plateaux. Pathways in addition to 11-cis-RDH likely provide 11-cis-retinal for rods and cones and can maintain normal kinetics of visual recovery but only under certain constraints and less efficiently for cone than rod function.
- Published
- 2000
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