RESUMEN

Modeling fluid cycloalkanes with molecular dynamics simulations has proven to be a very challenging task partly because of lacking a reliable force field based on quantum chemistry calculations. In this paper, we construct an ab initio force field for fluid cyclopropane using the second-order Møller-Plesset perturbation theory. We consider 15 conformers of the cyclopropane dimer for the orientation sampling. Single-point energies at important geometries are calibrated by the coupled cluster with single, double, and perturbative triple excitation method. Dunning's correlation consistent basis sets (up to aug-cc-pVTZ) are used in extrapolating the interaction energies at the complete basis set limit. The force field parameters in a 9-site Lennard-Jones model are regressed by the calculated interaction energies without using empirical data. With this ab initio force field, we perform molecular dynamics simulations of fluid cyclopropane and calculate both the structural and dynamical properties. We compare the simulation results with those using an empirical force field and obtain a quantitative agreement for the detailed atom-wise radial distribution functions. The experimentally observed gross radial distribution function (extracted from the neutron scattering measurements) is well reproduced in our simulation. Moreover, the calculated self-diffusion coefficients and shear viscosities are in good agreement with the experimental data over a wide range of thermodynamic conditions. To the best of our knowledge, this is the first ab initio force field which is capable of competing with empirical force fields for simulating fluid cyclopropane.

RESUMEN

NAMPT inhibitors may show potential as therapeutics for oncology. Throughout our NAMPT inhibitor program, we found that exposed pyridines or related heterocyclic systems in the left-hand portion of the inhibitors are necessary pharmacophores for potent cellular NAMPT inhibition. However, when combined with a benzyl group in the center of the inhibitors, such pyridine-like moieties also led to consistent and potent inhibition of CYP2C9. In an attempt to reduce CYP2C9 inhibition, a parallel synthesis approach was used to identify central benzyl group replacements with increased Fsp3. A spirocyclic central motif was thus discovered that was combined with left-hand pyridines (or pyridine-like systems) to provide cellularly potent NAMPT inhibitors with minimal CYP2C9 inhibition. Further optimization of potency and ADME properties led to the discovery of compound 68, a highly potent NAMPT inhibitor with outstanding efficacy in a mouse tumor xenograft model and lacking measurable CYP2C9 inhibition at the concentrations tested.

Asunto(s)

Citocromo P-450 CYP2C9/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Piridinas/química , Piridinas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Inhibidores del Citocromo P-450 CYP2C9/química , Inhibidores del Citocromo P-450 CYP2C9/farmacología , Descubrimiento de Drogas , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Nicotinamida Fosforribosiltransferasa/metabolismo , Piridinas/uso terapéutico

RESUMEN

Herein we report the optimization efforts to ameliorate the potent CYP3A4 time-dependent inhibition (TDI) and low aqueous solubility exhibited by a previously identified lead compound from our NAMPT inhibitor program (1, GNE-617). Metabolite identification studies pinpointed the imidazopyridine moiety present in 1 as the likely source of the TDI signal, and replacement with other bicyclic systems was found to reduce or eliminate the TDI finding. A strategy of reducing the number of aromatic rings and/or lowering cLogD7.4 was then employed to significantly improve aqueous solubility. These efforts culminated in the discovery of 42, a compound with no evidence of TDI, improved aqueous solubility, and robust efficacy in tumor xenograft studies.

Asunto(s)

Citocromo P-450 CYP3A/química , Inhibidores Enzimáticos/química , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Animales , Sitios de Unión , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Citocromo P-450 CYP3A/metabolismo , Inhibidores del Citocromo P-450 CYP3A/química , Inhibidores del Citocromo P-450 CYP3A/farmacocinética , Inhibidores del Citocromo P-450 CYP3A/toxicidad , Perros , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/uso terapéutico , Femenino , Semivida , Humanos , Cinética , Células de Riñón Canino Madin Darby , Ratones , Ratones Desnudos , Simulación de Dinámica Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Nicotinamida Fosforribosiltransferasa/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Pirimidinas/química , Pirimidinas/uso terapéutico , Pirimidinas/toxicidad , Solubilidad , Relación Estructura-Actividad , Termodinámica , Trasplante Heterólogo , Agua/química

RESUMEN

Inhibiting NAD biosynthesis by blocking the function of nicotinamide phosphoribosyl transferase (NAMPT) is an attractive therapeutic strategy for targeting tumor metabolism. However, the development of drug resistance commonly limits the efficacy of cancer therapeutics. This study identifies mutations in NAMPT that confer resistance to a novel NAMPT inhibitor, GNE-618, in cell culture and in vivo, thus demonstrating that the cytotoxicity of GNE-618 is on target. We determine the crystal structures of six NAMPT mutants in the apo form and in complex with various inhibitors and use cellular, biochemical and structural data to elucidate two resistance mechanisms. One is the surprising finding of allosteric modulation by mutation of residue Ser165, resulting in unwinding of an α-helix that binds the NAMPT substrate 5-phosphoribosyl-1-pyrophosphate (PRPP). The other mechanism is orthosteric blocking of inhibitor binding by mutations of Gly217. Furthermore, by evaluating a panel of diverse small molecule inhibitors, we unravel inhibitor structure activity relationships on the mutant enzymes. These results provide valuable insights into the design of next generation NAMPT inhibitors that offer improved therapeutic potential by evading certain mechanisms of resistance.

Asunto(s)

Antineoplásicos/farmacología , Citocinas/antagonistas & inhibidores , Citocinas/química , Resistencia a Antineoplásicos , Inhibidores Enzimáticos/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/química , Dominio Catalítico , Línea Celular Tumoral , Citocinas/genética , Humanos , Modelos Moleculares , Mutación , Nicotinamida Fosforribosiltransferasa/genética

RESUMEN

Prolonged inhibition of nicotinamide phosphoribosyltransferase (NAMPT) is a strategy for targeting cancer metabolism. Many NAMPT inhibitors undergo NAMPT-catalyzed phosphoribosylation (pRib), a property often correlated with their cellular potency. To understand this phenomenon and facilitate drug design, we analyzed a potent cellularly active NAMPT inhibitor (GNE-617). A crystal structure of pRib-GNE-617 in complex with NAMPT protein revealed a relaxed binding mode. Consistently, the adduct formation resulted in tight binding and strong product inhibition. In contrast, a biochemically equipotent isomer of GNE-617 (GNE-643) also formed pRib adducts but displayed significantly weaker cytotoxicity. Structural analysis revealed an altered ligand conformation of GNE-643, thus suggesting weak association of the adducts with NAMPT. Our data support a model for cellularly active NAMPT inhibitors that undergo NAMPT-catalyzed phosphoribosylation to produce pRib adducts that retain efficient binding to the enzyme.

Asunto(s)

Antineoplásicos/farmacología , Biocatálisis , Citocinas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Compuestos Heterocíclicos con 2 Anillos/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Sulfonas/farmacología , Animales , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Perros , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Compuestos Heterocíclicos con 2 Anillos/química , Humanos , Modelos Moleculares , Estructura Molecular , Nicotinamida Fosforribosiltransferasa/metabolismo , Permeabilidad/efectos de los fármacos , Fosforribosil Pirofosfato/metabolismo , Relación Estructura-Actividad , Sulfonas/química

RESUMEN

The fragment-based identification of two novel and potent biochemical inhibitors of the nicotinamide phosphoribosyltransferase (NAMPT) enzyme is described. These compounds (51 and 63) incorporate an amide moiety derived from 3-aminopyridine, and are thus structurally distinct from other known anti-NAMPT agents. Each exhibits potent inhibition of NAMPT biochemical activity (IC50=19 and 15 nM, respectively) as well as robust antiproliferative properties in A2780 cell culture experiments (IC50=121 and 99 nM, respectively). However, additional biological studies indicate that only inhibitor 51 exerts its A2780 cell culture effects via a NAMPT-mediated mechanism. The crystal structures of both 51 and 63 in complex with NAMPT are also independently described.

Asunto(s)

Amidas/síntesis química , Amidas/farmacología , Aminopiridinas/síntesis química , Citocinas/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Amidas/química , Aminopiridinas/química , Aminopiridinas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Relación Estructura-Actividad

RESUMEN

Potent, trans-2-(pyridin-3-yl)cyclopropanecarboxamide-containing inhibitors of the human nicotinamide phosphoribosyltransferase (NAMPT) enzyme were identified using fragment-based screening and structure-based design techniques. Multiple crystal structures were obtained of initial fragment leads, and this structural information was utilized to improve the biochemical and cell-based potency of the associated molecules. Many of the optimized compounds exhibited nanomolar antiproliferative activities against human tumor lines in in vitro cell culture experiments. In a key example, a fragment lead (13, KD = 51 µM) was elaborated into a potent NAMPT inhibitor (39, NAMPT IC50 = 0.0051 µM, A2780 cell culture IC50 = 0.000 49 µM) which demonstrated encouraging in vivo efficacy in an HT-1080 mouse xenograft tumor model.

Asunto(s)

Amidas/síntesis química , Antineoplásicos/síntesis química , Ciclopropanos/síntesis química , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Piridinas/síntesis química , Sulfonas/síntesis química , Amidas/química , Amidas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X , Ciclopropanos/química , Ciclopropanos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Trasplante de Neoplasias , Conformación Proteica , Piridinas/química , Piridinas/farmacología , Estereoisomerismo , Relación Estructura-Actividad , Sulfonas/química , Sulfonas/farmacología

RESUMEN

A co-crystal structure of amide-containing compound (4) in complex with the nicotinamide phosphoribosyltransferase (Nampt) protein and molecular modeling were utilized to design and discover a potent novel cyanoguanidine-containing inhibitor bearing a sulfone moiety (5, Nampt Biochemical IC50=2.5nM, A2780 cell proliferation IC50=9.7nM). Further SAR exploration identified several additional cyanoguanidine-containing compounds with high potency and good microsomal stability. Among these, compound 15 was selected for in vivo profiling and demonstrated good oral exposure in mice. It also exhibited excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model. The co-crystal structure of this compound in complex with the NAMPT protein was also determined.

Asunto(s)

Antineoplásicos/farmacología , Citocinas/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Neoplasias Ováricas/tratamiento farmacológico , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/química , Femenino , Guanidinas/administración & dosificación , Guanidinas/química , Humanos , Ratones , Modelos Moleculares , Estructura Molecular , Nicotinamida Fosforribosiltransferasa/metabolismo , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Potent, 1H-pyrazolo[3,4-b]pyridine-containing inhibitors of the human nicotinamide phosphoribosyltransferase (NAMPT) enzyme were identified using structure-based design techniques. Many of these compounds exhibited nanomolar antiproliferation activities against human tumor lines in in vitro cell culture experiments, and a representative example (compound 26) demonstrated encouraging in vivo efficacy in a mouse xenograft tumor model derived from the A2780 cell line. This molecule also exhibited reduced rat retinal exposures relative to a previously studied imidazo-pyridine-containing NAMPT inhibitor. Somewhat surprisingly, compound 26 was only weakly active in vitro against mouse and monkey tumor cell lines even though it was a potent inhibitor of NAMPT enzymes derived from these species. The compound also exhibited only minimal effects on in vivo NAD levels in mice, and these changes were considerably less profound than those produced by an imidazo-pyridine-containing NAMPT inhibitor. The crystal structures of compound 26 and the corresponding PRPP-derived ribose adduct in complex with NAMPT were also obtained.

Asunto(s)

Amidas/química , Ácidos Carboxílicos/química , Citocinas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Niacinamida/análogos & derivados , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Pirazoles/química , Piridinas/química , Sulfonas/química , Amidas/síntesis química , Amidas/farmacocinética , Animales , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Citocinas/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Femenino , Semivida , Haplorrinos , Humanos , Ratones , Ratones Desnudos , NAD/metabolismo , Niacinamida/sangre , Niacinamida/química , Niacinamida/farmacocinética , Nicotinamida Fosforribosiltransferasa/metabolismo , Estructura Terciaria de Proteína , Pirazoles/sangre , Pirazoles/farmacocinética , Ratas , Retina/efectos de los fármacos , Retina/metabolismo , Relación Estructura-Actividad , Sulfonas/sangre , Sulfonas/farmacocinética , Trasplante Heterólogo

RESUMEN

Potent nicotinamide phosphoribosyltransferase (NAMPT) inhibitors containing 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas were identified using structure-based design techniques. The new compounds displayed improved aqueous solubilities, determined using a high-throughput solubility assessment, relative to previously disclosed urea and amide-containing NAMPT inhibitors. An optimized 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived compound exhibited potent anti-NAMPT activity (18; BC NAMPT IC50 = 11 nM; PC-3 antiproliferative IC50 = 36 nM), satisfactory mouse PK properties, and was efficacious in a PC-3 mouse xenograft model. The crystal structure of another optimized compound (29; NAMPT IC50 = 10nM; A2780 antiproliferative IC50 = 7 nM) in complex with the NAMPT protein was also determined.

Asunto(s)

Antineoplásicos/química , Antineoplásicos/uso terapéutico , Citocinas/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Piridinas/química , Piridinas/uso terapéutico , Urea/química , Urea/uso terapéutico , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Línea Celular Tumoral , Citocinas/metabolismo , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Nicotinamida Fosforribosiltransferasa/metabolismo , Piridinas/farmacocinética , Piridinas/farmacología , Relación Estructura-Actividad , Urea/farmacocinética , Urea/farmacología

RESUMEN

Crystal structures of several urea- and thiourea-derived compounds in complex with the nicotinamide phosphoribosyltransferase (Nampt) protein were utilized to design a potent amide-containing inhibitor bearing an aza-indole moiety (7, Nampt BC IC50 = 9.0 nM, A2780 cell proliferation IC50 = 10 nM). The Nampt-7 cocrystal structure was subsequently obtained and enabled the design of additional amide-containing inhibitors which incorporated various other fused 6,5-heterocyclic moieties and biaryl sulfone or sulfonamide motifs. Additional modifications of these molecules afforded many potent biaryl sulfone-containing Nampt inhibitors which also exhibited favorable in vitro ADME properties (microsomal and hepatocyte stability, MDCK permeability, plasma protein binding). An optimized compound (58) was a potent inhibitor of multiple cancer cell lines (IC50 <10 nM vs U251, HT1080, PC3, MiaPaCa2, and HCT116 lines), displayed acceptable mouse PK properties (F = 41%, CL = 52.4 mL/min/kg), and exhibited robust efficacy in a U251 mouse xenograft model.

Asunto(s)

Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Animales , Inhibidores Enzimáticos/farmacocinética , Espectroscopía de Resonancia Magnética , Ratones , Ratones Desnudos , Modelos Moleculares , Estructura Molecular , Espectrometría de Masa por Ionización de Electrospray

RESUMEN

Potent, reversible inhibition of the cytochrome P450 CYP2C9 isoform was observed in a series of urea-containing nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. This unwanted property was successfully removed from the described inhibitors through a combination of structure-based design and medicinal chemistry activities. An optimized compound which did not inhibit CYP2C9 exhibited potent anti-NAMPT activity (17; BC NAMPT IC50=3 nM; A2780 antiproliferative IC50=70 nM), good mouse PK properties, and was efficacious in an A2780 mouse xenograft model. The crystal structure of this compound in complex with the NAMPT protein is also described.

Asunto(s)

Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Urea/análogos & derivados , Urea/farmacología , Animales , Hidrocarburo de Aril Hidroxilasas/química , Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2C9 , Humanos , Ratones , Ratones Endogámicos BALB C , Nicotinamida Fosforribosiltransferasa/química , Nicotinamida Fosforribosiltransferasa/metabolismo , Urea/síntesis química

RESUMEN

Nicotinamide phosphoribosyltransferase (Nampt) is a promising anticancer target. Virtual screening identified a thiourea analogue, compound 5, as a novel highly potent Nampt inhibitor. Guided by the cocrystal structure of 5, SAR exploration revealed that the corresponding urea compound 7 exhibited similar potency with an improved solubility profile. These studies also indicated that a 3-pyridyl group was the preferred substituent at one inhibitor terminus and also identified a urea moiety as the optimal linker to the remainder of the inhibitor structure. Further SAR optimization of the other inhibitor terminus ultimately yielded compound 50 as a urea-containing Nampt inhibitor which exhibited excellent biochemical and cellular potency (enzyme IC50 = 0.007 µM; A2780 IC50 = 0.032 µM). Compound 50 also showed excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model (TGI of 97% was observed on day 17).

Asunto(s)

Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Urea/química , Urea/farmacología , Humanos , Concentración 50 Inhibidora , Nicotinamida Fosforribosiltransferasa/química , Conformación Proteica , Relación Estructura-Actividad