RESUMEN
Molecular dynamics (MD) simulation is a powerful tool for characterizing ligand-protein conformational dynamics and offers significant advantages over docking and other rigid structure-based computational methods. However, setting up, running, and analyzing MD simulations continues to be a multi-step process making it cumbersome to assess a library of ligands in a protein binding pocket using MD. We present an automated workflow that streamlines setting up, running, and analyzing Desmond MD simulations for protein-ligand complexes using machine learning (ML) models. The workflow takes a library of pre-docked ligands and a prepared protein structure as input, sets up and runs MD with each protein-ligand complex, and generates simulation fingerprints for each ligand. Simulation fingerprints (SimFP) capture protein-ligand compatibility, including stability of different ligand-pocket interactions and other useful metrics that enable easy rank-ordering of the ligand library for pocket optimization. SimFPs from a ligand library are used to build & deploy ML models that predict binding assay outcomes and automatically infer important interactions. Unlike relative free-energy methods that are constrained to assess ligands with high chemical similarity, ML models based on SimFPs can accommodate diverse ligand sets. We present two case studies on how SimFP helps delineate structure-activity relationship (SAR) trends and explain potency differences across matched-molecular pairs of (1) cyclic peptides targeting PD-L1 and (2) small molecule inhibitors targeting CDK9.
Asunto(s)
Aprendizaje Automático , Simulación de Dinámica Molecular , Unión Proteica , Proteínas , Ligandos , Proteínas/química , Proteínas/metabolismo , Sitios de Unión , Simulación del Acoplamiento Molecular , Conformación Proteica , Flujo de Trabajo , Humanos , Diseño de Fármacos , Programas InformáticosRESUMEN
A novel method for exploring macrocycle conformational space, Prime macrocycle conformational sampling (Prime-MCS), is introduced and evaluated in the context of other available algorithms (Molecular Dynamics, LowModeMD in MOE, and MacroModel Baseline Search). The algorithms were benchmarked on a data set of 208 macrocycles which was curated for diversity from the Cambridge Structural Database, the Protein Data Bank, and the Biologically Interesting Molecule Reference Dictionary. The algorithms were evaluated in terms of accuracy (ability to reproduce the crystal structure), diversity (coverage of conformational space), and computational speed. Prime-MCS most reliably reproduced crystallographic structures for RMSD thresholds >1.0 Å, most often produced the most diverse conformational ensemble, and was most often the fastest algorithm. Detailed analysis and examination of both typical and outlier cases were performed to reveal characteristics, shortcomings, expected performance, and complementarity of the methods.
Asunto(s)
Compuestos Macrocíclicos/química , Simulación de Dinámica Molecular , Conformación Molecular , Termodinámica , Factores de TiempoRESUMEN
XIAP (X-chromosome-linked inhibitor of apoptosis protein) is a central apoptosis regulator that blocks cell death by inhibiting caspase-3, caspase-7, and caspase-9 via binding interactions with the XIAP BIR2 and BIR3 domains (where BIR is baculovirus IAP repeat). Smac protein, in its dimeric form, effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. Here we describe the development of highly sensitive homogeneous time-resolved fluorescence resonance energy transfer (HTRF) assays to measure binding affinities of potent bivalent peptidomimetic inhibitors of XIAP. Our results indicate that these assays can differentiate Smac-mimetic inhibitors with a wide range of binding affinities down to the picomolar range. Furthermore, we demonstrate the utility of these fluorescent tools for characterization of inhibitor off-rates, which as a crucial determinant of target engagement and cellular potency is another important parameter to guide optimization in a structure-based drug discovery effort. Our study also explores how increased inhibitor valency can lead to enhanced potency at multimeric proteins such as IAP.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Peptidomiméticos/farmacología , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Animales , Caspasa 3/metabolismo , Línea Celular , Humanos , Ratones Endogámicos BALB C , Peptidomiméticos/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína Inhibidora de la Apoptosis Ligada a X/químicaRESUMEN
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.
Asunto(s)
Cristalografía por Rayos X/métodos , Diseño de Fármacos , Proteasa del VIH/química , Proteasa del VIH/metabolismo , VIH-1/enzimología , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Proteínas Quinasas p38 Activadas por Mitógenos/química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Bivalent heterodimeric IAP antagonists that incorporate (R)-tetrahydroisoquinoline in the P3' subunit show high affinity for the BIR2 domain and demonstrated potent IAP inhibitory activities in biochemical and cellular assays. Potent in vivo efficacy was observed in a variety of human tumor xenograft models. The bivalent heterodimeric molecule 3 with a P3-P3' benzamide linker induced pharmacodynamic markers of apoptosis and was efficacious when administered intravenously at a dose of 1mg/kg to mice harboring A875 human melanoma tumors. Analog 5, with a polyamine group incorporated at the P2' thiovaline side chain exhibited antiproliferative activity against the P-gp expressing HCT116/VM46 cell line.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Tetrahidroisoquinolinas/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Sitios de Unión , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Moleculares , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PAR4 is a promising antithrombotic target with potential for separation of efficacy from bleeding risk relative to current antiplatelet therapies. In an effort to discover a novel PAR4 antagonist chemotype, a quinoxaline-based HTS hit 3 with low µM potency was identified. Optimization of the HTS hit through the use of positional SAR scanning and the design of conformationally constrained cores led to the discovery of a quinoxaline-benzothiazole series as potent and selective PAR4 antagonists. The lead compound 48, possessing a 2 nM IC50 against PAR4 activation by γ-thrombin in platelet-rich plasma (PRP) and greater than 2500-fold selectivity versus PAR1, demonstrated robust antithrombotic efficacy and minimal bleeding in the cynomolgus monkey models.
Asunto(s)
Fibrinolíticos , Trombosis , Animales , Fibrinolíticos/farmacología , Fibrinolíticos/uso terapéutico , Macaca fascicularis , Quinoxalinas/farmacología , Quinoxalinas/uso terapéutico , Receptores de Trombina , Trombina , Hemorragia , Trombosis/tratamiento farmacológico , Trombosis/prevención & control , Receptor PAR-1 , Plaquetas , Agregación PlaquetariaRESUMEN
We describe an extension to the matched molecular pairs approach that merges pairwise activity differences with three-dimensional contextual information derived from X-ray crystal structures and binding pose predictions. The incorporation of 3D binding poses allows the direct comparison of structural changes to diverse chemotypes in particular binding pockets, facilitating the transfer of SAR from one series to another. Integrating matched pair data with the receptor structure can also highlight activity patterns within the binding site--for example, "hot spot" regions can be visualized where changes in the ligand structure are more likely to impact activity. The method is illustrated using P38α structural and activity data to generate novel hybrid ligands, identify SAR transfer networks, and annotate the receptor binding site.
Asunto(s)
Diseño de Fármacos , Proteína Quinasa 14 Activada por Mitógenos/química , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Anotación de Secuencia Molecular , Sitios de Unión , Bases de Datos Farmacéuticas , Humanos , Ligandos , Modelos Moleculares , Conformación Molecular , Unión Proteica , Relación Estructura-ActividadRESUMEN
The toll-like receptors (TLRs) play key roles in activation of the innate immune system. Aberrant activation of TLR7 and TLR8 pathways can occur in the context of autoimmune disorders due to the elevated presence and recognition of self-RNA as activating ligands. Control of this unintended activation via inhibition of TLR7/8 signaling holds promise for the treatment of diseases such as psoriasis, arthritis, and lupus. Optimization of a 2-pyridinylindole series of compounds led to the identification of potent dual inhibitors of TLR7 and TLR8, which demonstrated good selectivity against TLR9 and other family members. The in vitro characterization and in vivo evaluation in rodent pharmacokinetic/pharmacodynamic and efficacy studies of BMS-905 is detailed, along with structural information obtained through X-ray cocrystallographic studies.
RESUMEN
Protease-activated receptor 4 (PAR4) is a G-protein coupled receptor that is expressed on human platelets and activated by the coagulation enzyme thrombin. PAR4 plays a key role in blood coagulation, and its importance in pathological thrombosis has been increasingly recognized in recent years. Herein, we describe the optimization of a series of imidazothiadiazole PAR4 antagonists to a first-in-class clinical candidate, BMS-986120 (43), and a backup clinical candidate, BMS-986141 (49). Both compounds demonstrated excellent antithrombotic efficacy and minimal bleeding time prolongation in monkey models relative to the clinically important antiplatelet agent clopidogrel and provide a potential opportunity to improve the standard of care in the treatment of arterial thrombosis.
Asunto(s)
Agregación Plaquetaria , Trombosis , Benzofuranos , Plaquetas , Humanos , Imidazoles , Morfolinas , Receptor PAR-1 , Receptores de Trombina , Tiazoles , Trombina , Trombosis/tratamiento farmacológicoRESUMEN
The identification of agonists of the stimulator of interferon genes (STING) pathway has been an area of intense research due to their potential to enhance innate immune response and tumor immunogenicity in the context of immuno-oncology therapy. Initial efforts to identify STING agonists focused on the modification of 2',3'-cGAMP (1) (an endogenous STING activator ligand) and other closely related cyclic dinucleotides (CDNs). While these efforts have successfully identified novel CDNs that have progressed into the clinic, their utility is currently limited to patients with solid tumors that STING agonists can be delivered to intratumorally. Herein, we report the discovery of a unique class of non-nucleotide small-molecule STING agonists that demonstrate antitumor activity when dosed intratumorally in a syngeneic mouse model.
Asunto(s)
Proteínas de la Membrana/agonistas , Animales , Cristalografía por Rayos X , AMP Cíclico/química , AMP Cíclico/farmacología , GMP Cíclico/química , GMP Cíclico/farmacología , Femenino , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunoterapia/métodos , Proteínas de la Membrana/química , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Neoplasias/inmunología , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas PequeñasRESUMEN
The toll-like receptor (TLR) family is an evolutionarily conserved component of the innate immune system, responsible for the early detection of foreign or endogenous threat signals. In the context of autoimmunity, the unintended recognition of self-motifs as foreign promotes initiation or propagation of disease. Overactivation of TLR7 and TLR9 have been implicated as factors contributing to autoimmune disorders such as psoriasis, arthritis, and lupus. In our search for small molecule antagonists of TLR7/9, 7f was identified as possessing excellent on-target potency for human TLR7/9 as well as for TLR8, with selectivity against other representative TLR family members. Good pharmacokinetic properties and a relatively balanced potency against TLR7 and TLR9 in mouse systems (systems which lack functional TLR8) made this an excellent in vivo tool compound, and efficacy from oral dosing in preclinical models of autoimmune disease was demonstrated.
RESUMEN
In an effort to identify novel antithrombotics, we have investigated protease-activated receptor 4 (PAR4) antagonism by developing and evaluating a tool compound, UDM-001651, in a monkey thrombosis model. Beginning with a high-throughput screening hit, we identified an imidazothiadiazole-based PAR4 antagonist chemotype. Detailed structure-activity relationship studies enabled optimization to a potent, selective, and orally bioavailable PAR4 antagonist, UDM-001651. UDM-001651 was evaluated in a monkey thrombosis model and shown to have robust antithrombotic efficacy and no prolongation of kidney bleeding time. This combination of excellent efficacy and safety margin strongly validates PAR4 antagonism as a promising antithrombotic mechanism.
Asunto(s)
Benzofuranos/farmacología , Fibrinolíticos/farmacología , Hemorragia/prevención & control , Receptores de Trombina/antagonistas & inhibidores , Trombosis/prevención & control , Animales , Benzofuranos/química , Benzofuranos/farmacocinética , Disponibilidad Biológica , Modelos Animales de Enfermedad , Fibrinolíticos/química , Fibrinolíticos/farmacocinética , Células HEK293 , Hemorragia/metabolismo , Humanos , Macaca fascicularis , Modelos Químicos , Estructura Molecular , Agregación Plaquetaria/efectos de los fármacos , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Relación Estructura-Actividad , Trombosis/metabolismoRESUMEN
Affinity selection screening of macrocycle libraries derived from DNA-programmed chemistry identified XIAP BIR2 and BIR3 domain inhibitors that displace bound pro-apoptotic caspases. X-ray cocrystal structures of key compounds with XIAP BIR2 suggested potency-enhancing structural modifications. Optimization of dimeric macrocycles with similar affinity for both domains were potent pro-apoptotic agents in cancer cell lines and efficacious in shrinking tumors in a mouse xenograft model.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/uso terapéutico , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Animales , Antineoplásicos/farmacocinética , Mama/efectos de los fármacos , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Caspasa 3/metabolismo , Línea Celular Tumoral , Cristalografía por Rayos X , Descubrimiento de Drogas , Femenino , Biblioteca de Genes , Humanos , Compuestos Macrocíclicos/farmacocinética , Ratones , Modelos Moleculares , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
A series of dimeric macrocyclic compounds were prepared and evaluated as antagonists for inhibitor of apoptosis proteins. The most potent analogue 11, which binds to XIAP and c-IAP proteins with high affinity and induces caspase-3 activation and ultimately cell apoptosis, inhibits growth of human melanoma and colorectal cell lines at low nanomolar concentrations. Furthermore, compound 11 demonstrated significant antitumor activity in the A875 human melanoma xenograft model at doses as low as 2 mg/kg on a q3d schedule.
RESUMEN
A kinome-wide selectivity screen of >20000 compounds with a rich representation of many structural classes has been completed. Analysis of the selectivity patterns for each class shows that a broad spectrum of structural scaffolds can achieve specificity for many kinase families. Kinase selectivity and potency are inversely correlated, a trend that is also found in a large set of kinase functional data. Although selective and nonselective compounds are mostly similar in their physicochemical characteristics, we identify specific features that are present more frequently in compounds that bind to many kinases. Our results support a scaffold-oriented approach for building compound collections to screen kinase targets.