RESUMO
The design and synthesis of potent, tripeptidic acylsulfonamide inhibitors of HCV NS3 protease that contain a difluoromethyl cyclopropyl amino acid at P1 are described. A cocrystal structure of 18 with a NS3/4A protease complex suggests the presence of a H-bond between the polarized C-H of the CHF2 moiety and the backbone carbonyl of Leu135 of the enzyme. Structure-activity relationship studies indicate that this H-bond enhances enzyme inhibitory potency by 13- and 17-fold compared to the CH3 and CF3 analogues, respectively, providing insight into the deployment of this unique amino acid.
RESUMO
Naphthalene-linked P2-P4 macrocycles within a tri-peptide-based acyl sulfonamide chemotype have been synthesized and found to inhibit HCV NS3 proteases representing genotypes 1a and 1b with single digit nanomolar potency. The pharmacokinetic profile of compounds in this series was optimized through structural modifications along the macrocycle tether as well as the P1 subsite. Ultimately a compound with oral bioavailability of 100% in rat, and a long half-life in plasma was obtained. However, compounds in this macrocyclic series exhibited cardiac effects in an isolated rabbit heart model and for this reason further optimization efforts were discontinued.
Assuntos
Antivirais/química , Compostos Macrocíclicos/química , Naftalenos/química , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Administração Oral , Animais , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Coração/efeitos dos fármacos , Coração/fisiologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Compostos Macrocíclicos/farmacocinética , Compostos Macrocíclicos/farmacologia , Microssomos Hepáticos/metabolismo , Conformação Molecular , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/farmacologia , Coelhos , Ratos , Proteínas não Estruturais Virais/metabolismoRESUMO
Often similar structures need to be compared to reveal local differences throughout the entire model or between related copies within the model. Therefore, a program to compare multiple structures and enable correction any differences not supported by the density map was written within the Phenix framework (Adams et al., Acta Cryst 2010; D66:213-221). This program, called Structure Comparison, can also be used for structures with multiple copies of the same protein chain in the asymmetric unit, that is, as a result of non-crystallographic symmetry (NCS). Structure Comparison was designed to interface with Coot(Emsley et al., Acta Cryst 2010; D66:486-501) and PyMOL(DeLano, PyMOL 0.99; 2002) to facilitate comparison of large numbers of related structures. Structure Comparison analyzes collections of protein structures using several metrics, such as the rotamer conformation of equivalent residues, displays the results in tabular form and allows superimposed protein chains and density maps to be quickly inspected and edited (via the tools in Coot) for consistency, completeness and correctness.
Assuntos
Modelos Moleculares , Proteínas/química , Interface Usuário-Computador , Proteínas/genéticaRESUMO
The design and synthesis of a series of tripeptide acylsulfonamides as potent inhibitors of the HCV NS3/4A serine protease is described. These analogues house a C4 aryl, C4 hydroxy-proline at the S2 position of the tripeptide scaffold. Information relating to structure-activity relationships as well as the pharmacokinetic and cardiovascular profiles of these analogues is provided.
Assuntos
Antivirais/química , Hepacivirus/enzimologia , Oligopeptídeos/química , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Meia-Vida , Coração/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Humanos , Técnicas In Vitro , Oligopeptídeos/farmacocinética , Oligopeptídeos/farmacologia , Prolina/química , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/farmacologia , Coelhos , Ratos , Relação Estrutura-Atividade , Sulfonamidas/química , Proteínas não Estruturais Virais/metabolismoRESUMO
The discovery of a back-up to the hepatitis C virus NS3 protease inhibitor asunaprevir (2) is described. The objective of this work was the identification of a drug with antiviral properties and toxicology parameters similar to 2, but with a preclinical pharmacokinetic (PK) profile that was predictive of once-daily dosing. Critical to this discovery process was the employment of an ex vivo cardiovascular (CV) model which served to identify compounds that, like 2, were free of the CV liabilities that resulted in the discontinuation of BMS-605339 (1) from clinical trials. Structure-activity relationships (SARs) at each of the structural subsites in 2 were explored with substantial improvement in PK through modifications at the P1 site, while potency gains were found with small, but rationally designed structural changes to P4. Additional modifications at P3 were required to optimize the CV profile, and these combined SARs led to the discovery of BMS-890068 (29).
Assuntos
Antivirais/química , Hepacivirus/efeitos dos fármacos , Isoquinolinas/uso terapêutico , Oligopeptídeos/química , Sulfonamidas/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/administração & dosagem , Antivirais/farmacocinética , Antivirais/farmacologia , Cães , Esquema de Medicação , Farmacorresistência Viral , Hepacivirus/genética , Macaca fascicularis , Masculino , Modelos Moleculares , Oligopeptídeos/administração & dosagem , Oligopeptídeos/farmacocinética , Oligopeptídeos/farmacologia , Coelhos , Ratos Sprague-Dawley , Replicon , Estereoisomerismo , Relação Estrutura-Atividade , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêuticoRESUMO
A scheduler has been developed for an integrated laboratory robot system that operates in an always-on mode. The integrated system is designed for imaging plates containing protein crystallization experiments, and it allows crystallographers to enter plates at any time and request that they be imaged at multiple time points in the future. The scheduler must rearrange tasks within the time it takes to image one plate, trading off the quality of the schedule for the speed of the computation. For this reason, the scheduler was based on a simulated annealing algorithm with an objective function that makes use of a linear programming solver. To optimize the scheduler, extensive computational simulations were performed involving a difficult but representative scheduling problem. The simulations explore multiple configurations of the simulated annealing algorithm, including both geometric and adaptive annealing schedules, 3 neighborhood functions, and 20 neighborhood diameters. An optimal configuration was found that produced the best results in less than 60 seconds, well within the window necessary to dynamically reschedule imaging tasks as new plates are entered into the system.
Assuntos
Automação Laboratorial/métodos , Pesquisa Biomédica/organização & administração , Robótica/métodos , Algoritmos , Simulação por Computador , Cristalização/métodos , Imagem Óptica/métodosRESUMO
The discovery of BMS-605339 (35), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropylacylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1' site of the protease. The identification of 35 was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound 35 which demonstrated antiviral activity in HCV-infected patients.
Assuntos
Antivirais/uso terapêutico , Descoberta de Drogas , Hepatite C/tratamento farmacológico , Isoquinolinas/uso terapêutico , Inibidores de Proteases/uso terapêutico , Sulfonamidas/uso terapêutico , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Cristalografia por Raios X , Cães , Avaliação Pré-Clínica de Medicamentos , Humanos , Isoquinolinas/química , Modelos Moleculares , Inibidores de Proteases/química , Sulfonamidas/químicaRESUMO
The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhibitor of the NS3/4A enzyme is currently in phase III clinical trials for the treatment of hepatitis C virus infection. The discovery of 24 was enabled by employing an isolated rabbit heart model to screen for the cardiovascular (CV) liabilities (changes to HR and SNRT) that were responsible for the discontinuation of an earlier lead from this chemical series, BMS-605339 (1), from clinical trials. The structure-activity relationships (SARs) developed with respect to CV effects established that small structural changes to the P2* subsite of the molecule had a significant impact on the CV profile of a given compound. The antiviral activity, preclincial PK profile, and toxicology studies in rat and dog supported clinical development of BMS-650032 (24).
Assuntos
Antivirais/uso terapêutico , Hepatite C/tratamento farmacológico , Isoquinolinas/uso terapêutico , Inibidores de Proteases/uso terapêutico , Sulfonamidas/uso terapêutico , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/sangue , Antivirais/química , Cães , Humanos , Isoquinolinas/sangue , Isoquinolinas/química , Modelos Moleculares , Inibidores de Proteases/sangue , Inibidores de Proteases/química , Coelhos , Ratos , Sulfonamidas/sangue , Sulfonamidas/químicaRESUMO
The process of iterative structure-based drug design involves the X-ray crystal structure determination of upwards of 100 ligands with the same general scaffold (i.e. chemotype) complexed with very similar, if not identical, protein targets. In conjunction with insights from computational models and assays, this collection of crystal structures is analyzed to improve potency, to achieve better selectivity and to reduce liabilities such as absorption, distribution, metabolism, excretion and toxicology. Current methods for modeling ligands into electron-density maps typically do not utilize information on how similar ligands bound in related structures. Even if the electron density is of sufficient quality and resolution to allow de novo placement, the process can take considerable time as the size, complexity and torsional degrees of freedom of the ligands increase. A new module, Guided Ligand Replacement (GLR), was developed in Phenix to increase the ease and success rate of ligand placement when prior protein-ligand complexes are available. At the heart of GLR is an algorithm based on graph theory that associates atoms in the target ligand with analogous atoms in the reference ligand. Based on this correspondence, a set of coordinates is generated for the target ligand. GLR is especially useful in two situations: (i) modeling a series of large, flexible, complicated or macrocyclic ligands in successive structures and (ii) modeling ligands as part of a refinement pipeline that can automatically select a reference structure. Even in those cases for which no reference structure is available, if there are multiple copies of the bound ligand per asymmetric unit GLR offers an efficient way to complete the model after the first ligand has been placed. In all of these applications, GLR leverages prior knowledge from earlier structures to facilitate ligand placement in the current structure.
Assuntos
Cristalografia por Raios X/métodos , Desenho de Fármacos , Protease de HIV/química , Protease de HIV/metabolismo , HIV-1/enzimologia , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
High-throughput drug-discovery and mechanistic studies often require the determination of multiple related crystal structures that only differ in the bound ligands, point mutations in the protein sequence and minor conformational changes. If performed manually, solution and refinement requires extensive repetition of the same tasks for each structure. To accelerate this process and minimize manual effort, a pipeline encompassing all stages of ligand building and refinement, starting from integrated and scaled diffraction intensities, has been implemented in Phenix. The resulting system is able to successfully solve and refine large collections of structures in parallel without extensive user intervention prior to the final stages of model completion and validation.
Assuntos
Cristalografia por Raios X/métodos , Proteínas/química , Animais , Desenho de Fármacos , Fator Xa/química , Fator Xa/metabolismo , Protease de HIV/química , Protease de HIV/metabolismo , HIV-1/enzimologia , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Proteínas/metabolismo , Trombina/química , Trombina/metabolismoRESUMO
Human ß-glucuronidase (GUS) cleaves ß-D-glucuronic acid residues from the non-reducing termini of glycosaminoglycan and its deficiency leads to mucopolysaccharidosis type VII (MPSVII). Here we report a high resolution crystal structure of human GUS at 1.7 Å resolution and present an extensive analysis of the structural features, unifying recent findings in the field of lysosome targeting and glycosyl hydrolases. The structure revealed several new details including a new glycan chain at Asn272, in addition to that previously observed at Asn173, and coordination of the glycan chain at Asn173 with Lys197 of the lysosomal targeting motif which is essential for phosphotransferase recognition. Analysis of the high resolution structure not only provided new insights into the structural basis for lysosomal targeting but showed significant differences between human GUS, which is medically important in its own right, and E. coli GUS, which can be selectively inhibited in the human gut to prevent prodrug activation and is also widely used as a reporter gene by plant biologists. Despite these differences, both human and E. coli GUS share a high structure homology in all three domains with most of the glycosyl hydrolases, suggesting that they all evolved from a common ancestral gene.
Assuntos
Glucuronidase/química , Glucuronidase/metabolismo , Lisossomos/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Glicosaminoglicanos/metabolismo , Humanos , Dados de Sequência MolecularRESUMO
Optimization of a 5-oxopyrrolopyridine series based upon structure-activity relationships (SARs) developed from our previous efforts on a number of related bicyclic series yielded compound 2s (BMS-767778) with an overall activity, selectivity, efficacy, PK, and developability profile suitable for progression into the clinic. SAR in the series and characterization of 2s are described.
Assuntos
Acetamidas/química , Acetamidas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/química , Inibidores da Dipeptidil Peptidase IV/farmacologia , Desenho de Fármacos , Pirróis/química , Pirróis/farmacologia , Acetamidas/síntese química , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Domínio Catalítico , Dipeptidil Peptidase 4/química , Inibidores da Dipeptidil Peptidase IV/síntese química , Teste de Tolerância a Glucose , Humanos , Masculino , Camundongos , Modelos Moleculares , Pirróis/síntese química , Especificidade por SubstratoRESUMO
The design, synthesis and characterization of a phosphonate inhibitor of N-acetylneuraminate-9-phosphate phosphatase (HDHD4) is described. Compound 3, where the substrate C-9 oxygen was replaced with a nonlabile CH2 group, inhibits HDHD4 with a binding affinity (IC50 11µM) in the range of the native substrate Neu5Ac-9-P (compound 1, Km 47µM). Combined SAR, modeling and NMR studies are consistent with the phosphonate group in inhibitor 3 forming a stable complex with native Mg(2+). In addition to this key interaction, the C-1 carboxylate of the sugar interacts with a cluster of basic residues, K141, R104 and R72. Comparative NMR studies of compounds 3 and 1 with Ca(2+) and Mg(2+) are indicative of a highly dynamic process in the active site for the HDHD4/Mg(2+)/3 complex. Possible explanations for this observation are discussed.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Ácidos Siálicos/síntese química , Fosfatos Açúcares/síntese química , Animais , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Monoéster Fosfórico Hidrolases/metabolismo , Estrutura Terciária de Proteína , Ratos , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Fosfatos Açúcares/química , Fosfatos Açúcares/metabolismoRESUMO
The design, synthesis and SAR of a novel class of valerolactam-based arylsulfonamides as potent and selective FXa inhibitors is reported. The arylsulfonamide-valerolactam scaffold was derived based on the proposed bioisosterism to the arylcyanoguanidine-caprolactam core in known FXa inhibitors. The SAR study led to compound 46 as the most potent FXa inhibitor in this series, with an IC(50) of 7 nM and EC(2×PT) of 1.7 µM. The X-ray structure of compound 40 bound to FXa shows that the sulfonamide-valerolactam scaffold anchors the aryl group in the S1 and the novel acylcytisine pharmacophore in the S4 pockets.
Assuntos
Anticoagulantes/química , Inibidores do Fator Xa , Piperidonas/química , Inibidores de Serina Proteinase/química , Anticoagulantes/síntese química , Anticoagulantes/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Fator Xa/metabolismo , Humanos , Lactamas/química , Conformação Molecular , Piperidonas/síntese química , Piperidonas/farmacologia , Estrutura Terciária de Proteína , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-AtividadeRESUMO
Design, synthesis, and SAR of 7-oxopyrrolopyridine-derived DPP4 inhibitors are described. The preferred stereochemistry of these atropisomeric biaryl analogs has been identified as Sa. Compound (+)-3t, with a K(i) against DPP4, DPP8, and DPP9 of 0.37 nM, 2.2, and 5.7 µM, respectively, showed a significant improvement in insulin response after single doses of 3 and 10 µmol/kg in ob/ob mice.
Assuntos
Citocromo P-450 CYP3A/metabolismo , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/química , Inibidores da Dipeptidil Peptidase IV/farmacologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Piridinas/química , Piridinas/farmacologia , Animais , Domínio Catalítico , Diabetes Mellitus/tratamento farmacológico , Inibidores da Dipeptidil Peptidase IV/farmacocinética , Humanos , Insulina/sangue , Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Piridinas/farmacocinética , Pirróis/química , Pirróis/farmacocinética , Pirróis/farmacologia , Ratos , EstereoisomerismoRESUMO
CARM1 (co-activator-associated arginine methyltransferase 1) is a PRMT (protein arginine N-methyltransferase) family member that catalyses the transfer of methyl groups from SAM (S-adenosylmethionine) to the side chain of specific arginine residues of substrate proteins. This post-translational modification of proteins regulates a variety of transcriptional events and other cellular processes. Moreover, CARM1 is a potential oncological target due to its multiple roles in transcription activation by nuclear hormone receptors and other transcription factors such as p53. Here, we present crystal structures of the CARM1 catalytic domain in complex with cofactors [SAH (S-adenosyl-L-homocysteine) or SNF (sinefungin)] and indole or pyazole inhibitors. Analysis of the structures reveals that the inhibitors bind in the arginine-binding cavity and the surrounding pocket that exists at the interface between the N- and C-terminal domains. In addition, we show using ITC (isothermal titration calorimetry) that the inhibitors bind to the CARM1 catalytic domain only in the presence of the cofactor SAH. Furthermore, sequence differences for select residues that interact with the inhibitors may be responsible for the CARM1 selectivity against PRMT1 and PRMT3. Together, the structural and biophysical information should aid in the design of both potent and specific inhibitors of CARM1.
Assuntos
Indóis/antagonistas & inibidores , Indóis/química , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/química , Pirazóis/antagonistas & inibidores , Pirazóis/química , Sequência de Aminoácidos , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/metabolismo , Dados de Sequência Molecular , Ligação Proteica/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/metabolismo , Pirazóis/metabolismoRESUMO
Continued structure-activity relationship (SAR) exploration within our previously disclosed azolopyrimidine containing dipeptidyl peptidase-4 (DPP4) inhibitors led us to focus on an imidazolopyrimidine series in particular. Further study revealed that by replacing the aryl substitution on the imidazole ring with a more polar carboxylic ester or amide, these compounds displayed not only increased DPP4 binding activity but also significantly reduced human ether-a-go-go related gene (hERG) and sodium channel inhibitory activities. Additional incremental adjustment of polarity led to permeable molecules which exhibited favorable pharmacokinetic (PK) profiles in preclinical animal species. The active site binding mode of these compounds was determined by X-ray crystallography as exemplified by amide 24c. A subsequent lead molecule from this series, (+)-6-(aminomethyl)-5-(2,4-dichlorophenyl)-N-(1-ethyl-1H-pyrazol-5-yl)-7-methylimidazo[1,2-a]pyrimidine-2-carboxamide (24s), emerged as a potent, selective DPP4 inhibitor that displayed excellent PK profiles and in vivo efficacy in ob/ob mice.
Assuntos
Inibidores da Dipeptidil Peptidase IV , Hipoglicemiantes/síntese química , Imidazóis/síntese química , Pirimidinas/síntese química , Animais , Domínio Catalítico , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/tratamento farmacológico , Dipeptidil Peptidase 4/química , Cães , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Humanos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/farmacologia , Imidazóis/farmacocinética , Imidazóis/farmacologia , Masculino , Camundongos , Camundongos Obesos , Modelos Moleculares , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ratos , Bloqueadores dos Canais de Sódio/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
We report the design and synthesis of a novel class of N,N'-disubstituted aroylguanidine-based lactam derivatives as potent and orally active FXa inhibitors. The structure-activity relationships (SAR) investigation led to the discovery of the nicotinoyl guanidine 22 as a potent FXa inhibitor (FXa IC(50)=4 nM, EC(2xPT)=7 microM). However, the potent CYP3A4 inhibition activity (IC(50)=0.3 microM) of 22 precluded its further development. Detailed analysis of the X-ray crystal structure of compound 22 bound to FXa indicated that the substituent at the 6-position of the nicotinoyl group of 22 would be solvent-exposed, suggesting that efforts to attenuate the unwanted CYP activity could focus at this position without affecting FXa potency significantly. Further SAR studies on the 6-substituted nicotinoyl guanidines resulted in the discovery of 6-(dimethylcarbamoyl) nicotinoyl guanidine 36 (BMS-344577, IC(50)=9 nM, EC(2xPT)=2.5 microM), which was found to be a selective, orally efficacious FXa inhibitor with an excellent in vitro liability profile, favorable pharmacokinetics and pharmacodynamics in animal models.
Assuntos
Anticoagulantes/química , Inibidores do Fator Xa , Guanidinas/química , Inibidores de Serina Proteinase/química , Anticoagulantes/farmacologia , Citocromo P-450 CYP3A , Inibidores do Citocromo P-450 CYP3A , Descoberta de Drogas , Guanidinas/farmacologia , Humanos , Concentração Inibidora 50 , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-AtividadeRESUMO
The N,N'-disubstituted cyanoguanidine is an excellent bioisostere of the thiourea and ketene aminal functional groups. We report the design and synthesis of a novel class of cyanoguanidine-based lactam derivatives as potent and orally active FXa inhibitors. The SAR studies led to the discovery of compound 4 (BMS-269223, K(i)=6.5nM, EC(2xPT)=32muM) as a selective, orally bioavailable FXa inhibitor with an excellent in vitro liability profile, favorable pharmacokinetics and pharmacodynamics in animal models. The X-ray crystal structure of 4 bound in FXa is presented and key ligand-protein interactions are discussed.