RESUMEN
There has been much debate around the success rates of various screening strategies to identify starting points for drug discovery. Although high-throughput target-based and phenotypic screening has been the focus of this debate, techniques such as fragment screening, virtual screening, and DNA-encoded library screening are also increasingly reported as a source of new chemical equity. Here, we provide examples in which integration of more than one screening approach has improved the campaign outcome and discuss how strengths and weaknesses of various methods can be used to build a complementary toolbox of approaches, giving researchers the greatest probability of successfully identifying leads. Among others, we highlight case studies for receptor-interacting serine/threonine-protein kinase 1 and the bromo- and extra-terminal domain family of bromodomains. In each example, the unique insight or chemistries individual approaches provided are described, emphasizing the synergy of information obtained from the various tactics employed and the particular question each tactic was employed to answer. We conclude with a short prospective discussing how screening strategies are evolving, what this screening toolbox might look like in the future, how to maximize success through integration of multiple tactics, and scenarios that drive selection of one combination of tactics over another.
Asunto(s)
Diseño de Fármacos , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos , Animales , Descubrimiento de Drogas/normas , Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/normas , Ensayos Analíticos de Alto Rendimiento , Humanos , Bibliotecas de Moléculas Pequeñas , Relación Estructura-ActividadRESUMEN
Discovery of potent renin inhibitors which contain a simplified alkylamino Asp-binding group and exhibit improved selectivity for renin over Cyp3A4 is described. Structure-function results in this series are rationalized based on analysis of selected compounds bound to renin, and the contribution of each molecular feature leading to the reduced P450 inhibition is quantified.
Asunto(s)
Ácido Aspártico/metabolismo , Citocromo P-450 CYP3A/metabolismo , Inhibidores de Proteasas/química , Renina/antagonistas & inhibidores , Ácido Aspártico/química , Sitios de Unión , Cristalografía por Rayos X , Citocromo P-450 CYP3A/química , Inhibidores del Citocromo P-450 CYP3A/química , Inhibidores del Citocromo P-450 CYP3A/metabolismo , Evaluación Preclínica de Medicamentos , Humanos , Concentración 50 Inhibidora , Simulación de Dinámica Molecular , Inhibidores de Proteasas/metabolismo , Unión Proteica , Renina/metabolismo , Relación Estructura-ActividadRESUMEN
1-(1,3,5-Triazin-yl)piperidine-4-carboxamide inhibitors of soluble epoxide hydrolase were identified from high through-put screening using encoded library technology. The triazine heterocycle proved to be a critical functional group, essential for high potency and P450 selectivity. Phenyl group substitution was important for reducing clearance, and establishing good oral exposure. Based on this lead optimization work, 1-[4-methyl-6-(methylamino)-1,3,5-triazin-2-yl]-N-{[[4-bromo-2-(trifluoromethoxy)]-phenyl]methyl}-4-piperidinecarboxamide (27) was identified as a useful tool compound for in vivo investigation. Robust effects on a serum biomarker, 9, 10-epoxyoctadec-12(Z)-enoic acid (the epoxide derived from linoleic acid) were observed, which provided evidence of robust in vivo target engagement and the suitability of 27 as a tool compound for study in various disease models.
Asunto(s)
Amidas/química , Amidas/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Epóxido Hidrolasas/antagonistas & inhibidores , Piperidinas/química , Piperidinas/farmacología , Amidas/síntesis química , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Epóxido Hidrolasas/metabolismo , Humanos , Modelos Moleculares , Piperidinas/síntesis química , Relación Estructura-Actividad , Triazinas/síntesis química , Triazinas/química , Triazinas/farmacologíaRESUMEN
The metalloprotease ADAMTS-5 is considered a potential target for the treatment of osteoarthritis. To identify selective inhibitors of ADAMTS-5, we employed encoded library technology (ELT), which enables affinity selection of small molecule binders from complex mixtures by DNA tagging. Selection of ADAMTS-5 against a four-billion member ELT library led to a novel inhibitor scaffold not containing a classical zinc-binding functionality. One exemplar, (R)-N-((1-(4-(but-3-en-1-ylamino)-6-(((2-(thiophen-2-yl)thiazol-4-yl)methyl)amino)-1,3,5-triazin-2-yl)pyrrolidin-2-yl)methyl)-4-propylbenzenesulfonamide (8), inhibited ADAMTS-5 with IC(50) = 30 nM, showing >50-fold selectivity against ADAMTS-4 and >1000-fold selectivity against ADAMTS-1, ADAMTS-13, MMP-13, and TACE. Extensive SAR studies showed that potency and physicochemical properties of the scaffold could be further improved. Furthermore, in a human osteoarthritis cartilage explant study, compounds 8 and 15f inhibited aggrecanase-mediated (374)ARGS neoepitope release from aggrecan and glycosaminoglycan in response to IL-1ß/OSM stimulation. This study provides the first small molecule evidence for the critical role of ADAMTS-5 in human cartilage degradation.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Cartílago Articular/efectos de los fármacos , Bases de Datos de Compuestos Químicos , Osteoartritis/patología , Sulfonamidas/síntesis química , Triazinas/síntesis química , Proteína ADAMTS5 , Agrecanos/metabolismo , Animales , Cartílago Articular/metabolismo , Cartílago Articular/patología , Endopeptidasas/metabolismo , Epítopos , Glicosaminoglicanos/metabolismo , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Osteoartritis/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Bibliotecas de Moléculas Pequeñas , Estereoisomerismo , Relación Estructura-Actividad , Sulfonamidas/farmacocinética , Sulfonamidas/farmacología , Triazinas/farmacocinética , Triazinas/farmacologíaRESUMEN
A new class of selective MMP-12 inhibitors have been identified via high throughput screening. Crystallization with MMP-12 confirmed the mode of binding and allowed initial optimization to be carried out using classical structure based design.
Asunto(s)
Ácidos Carboxílicos/química , Inhibidores de la Metaloproteinasa de la Matriz , Inhibidores de Proteasas/química , Animales , Sitios de Unión , Ácidos Carboxílicos/síntesis química , Ácidos Carboxílicos/farmacología , Cristalografía por Rayos X , Diseño de Fármacos , Cobayas , Humanos , Metaloproteinasa 12 de la Matriz/metabolismo , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Relación Estructura-ActividadRESUMEN
Explorations of the S(1') subsite of ACE2 via modifications of the P(1') methylene biphenyl moiety of thiol-based metalloprotease inhibitors led to improvements in ACE2 selectivity versus ACE and NEP, while maintaining potent ACE2 inhibition.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/química , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Peptidil-Dipeptidasa A/metabolismo , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/farmacología , Enzima Convertidora de Angiotensina 2 , Sitios de Unión , Humanos , Modelos Moleculares , Estructura Molecular , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
Screening of a metalloprotease library led to the identification of a thiol-based dual ACE/NEP inhibitor as a potent ACE2 inhibitor. Modifications of the P(1) benzyl moiety led to improvements in ACE2 potency as well as to increased selectivity versus ACE and NEP.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/química , Peptidil-Dipeptidasa A/efectos de los fármacos , Compuestos de Sulfhidrilo/química , Enzima Convertidora de Angiotensina 2 , Inhibidores de la Enzima Convertidora de Angiotensina/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Peptidil-Dipeptidasa A/genética , Relación Estructura-Actividad , Especificidad por Sustrato , Compuestos de Sulfhidrilo/metabolismo , Compuestos de Sulfhidrilo/farmacologíaAsunto(s)
Enzimas/metabolismo , Proteínas de Unión al ARN , Proteínas de Saccharomyces cerevisiae , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Factores de Transcripción , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Glucosa/análogos & derivados , Glucosa/metabolismo , Hidroliasas/química , Hidroliasas/metabolismo , Estructura Molecular , Proteínas Nucleares , Oligonucleótidos/análisis , Oligosacáridos/análisis , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/metabolismo , Schizosaccharomyces/enzimología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentaciónRESUMEN
dTDP-D-glucose 4,6-dehydratase (RmlB) was first identified in the L-rhamnose biosynthetic pathway, where it catalyzes the conversion of dTDP-D-glucose into dTDP-4-keto-6-deoxy-D-glucose. The structures of RmlB from Salmonella enterica serovar Typhimurium in complex with substrate deoxythymidine 5'-diphospho-D-glucose (dTDP-D-glucose) and deoxythymidine 5'-diphosphate (dTDP), and RmlB from Streptococcus suis serotype 2 in complex with dTDP-D-glucose, dTDP, and deoxythymidine 5'-diphospho-D-pyrano-xylose (dTDP-xylose) have all been solved at resolutions between 1.8 A and 2.4 A. The structures show that the active sites are highly conserved. Importantly, the structures show that the active site tyrosine functions directly as the active site base, and an aspartic and glutamic acid pairing accomplishes the dehydration step of the enzyme mechanism. We conclude that the substrate is required to move within the active site to complete the catalytic cycle and that this movement is driven by the elimination of water. The results provide insight into members of the SDR superfamily.
Asunto(s)
Hidroliasas/química , Hidroliasas/metabolismo , Estructura Terciaria de Proteína , Salmonella typhimurium/enzimología , Streptococcus suis/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Glucosa/análogos & derivados , Glucosa/metabolismo , Hidroliasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Alineación de SecuenciaRESUMEN
The conversion of dTDP-glucose into dTDP-4-keto-6-deoxyglucose by Escherichia coli dTDP-glucose 4,6-dehydratase (4,6-dehydratase) takes place in the active site in three steps: dehydrogenation to dTDP-4-ketoglucose, dehydration to dTDP-4-ketoglucose-5,6-ene, and rereduction of C6 to the methyl group. The 4,6-dehydratase makes use of tightly bound NAD(+) as the coenzyme for transiently oxidizing the substrate, activating it for the dehydration step. Dehydration may occur by either of two mechanisms, enolization of the dTDP-4-ketoglucose intermediate, followed by elimination [as proposed for beta-eliminations by Gerlt, J. A., and Gassman, P. G. (1992) J. Am. Chem. Soc. 114, 5928-5934], or a concerted 5,6-elimination of water from the intermediate. To assign one of these two mechanisms, a simultaneous kinetic characterization of glucosyl C5((1)H/(2)H) solvent hydrogen and C6((16)OH/(18)OH) solvent oxygen exchange was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The reaction of the wild-type enzyme is shown to proceed through a concerted dehydration mechanism. Interestingly, mutation of Asp135, the acid catalyst, to Asn or Ala alters the mechanism, allowing enolization to occur to varying extents. While aspartic acid 135 is the acid catalyst for dehydration in the wild-type enzyme, the differential enolization capabilities of D135N and D135A dehydratases suggest an additional role for this residue. We postulate that the switch from a concerted to stepwise dehydration mechanism observed in the aspartic acid variants is due to the loss of control over the glucosyl C5-C6 bond rotation in the active site.