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1.
J Chem Theory Comput ; 2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34592101

RESUMO

With the advent of make-on-demand commercial libraries, the number of purchasable compounds available for virtual screening and assay has grown explosively in recent years, with several libraries eclipsing one billion compounds. Today's screening libraries are larger and more diverse, enabling the discovery of more-potent hit compounds and unlocking new areas of chemical space, represented by new core scaffolds. Applying physics-based in silico screening methods in an exhaustive manner, where every molecule in the library must be enumerated and evaluated independently, is increasingly cost-prohibitive. Here, we introduce a protocol for machine learning-enhanced molecular docking based on active learning to dramatically increase throughput over traditional docking. We leverage a novel selection protocol that strikes a balance between two objectives: (1) identifying the best scoring compounds and (2) exploring a large region of chemical space, demonstrating superior performance compared to a purely greedy approach. Together with automated redocking of the top compounds, this method captures almost all the high scoring scaffolds in the library found by exhaustive docking. This protocol is applied to our recent virtual screening campaigns against the D4 and AMPC targets that produced dozens of highly potent, novel inhibitors, and a blind test against the MT1 target. Our protocol recovers more than 80% of the experimentally confirmed hits with a 14-fold reduction in compute cost, and more than 90% of the hit scaffolds in the top 5% of model predictions, preserving the diversity of the experimentally confirmed hit compounds.

2.
Proc Natl Acad Sci U S A ; 118(36)2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34475217

RESUMO

Protein flexibility remains a major challenge in library docking because of difficulties in sampling conformational ensembles with accurate probabilities. Here, we use the model cavity site of T4 lysozyme L99A to test flexible receptor docking with energy penalties from molecular dynamics (MD) simulations. Crystallography with larger and smaller ligands indicates that this cavity can adopt three major conformations: open, intermediate, and closed. Since smaller ligands typically bind better to the cavity site, we anticipate an energy penalty for the cavity opening. To estimate its magnitude, we calculate conformational preferences from MD simulations. We find that including a penalty term is essential for retrospective ligand enrichment; otherwise, high-energy states dominate the docking. We then prospectively docked a library of over 900,000 compounds for new molecules binding to each conformational state. Absent a penalty term, the open conformation dominated the docking results; inclusion of this term led to a balanced sampling of ligands against each state. High ranked molecules were experimentally tested by Tm upshift and X-ray crystallography. From 33 selected molecules, we identified 18 ligands and determined 13 crystal structures. Most interesting were those bound to the open cavity, where the buried site opens to bulk solvent. Here, highly unusual ligands for this cavity had been predicted, including large ligands with polar tails; these were confirmed both by binding and by crystallography. In docking, incorporating protein flexibility with thermodynamic weightings may thus access new ligand chemotypes. The MD approach to accessing and, crucially, weighting such alternative states may find general applicability.

3.
Nat Protoc ; 16(10): 4799-4832, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34561691

RESUMO

Structure-based docking screens of large compound libraries have become common in early drug and probe discovery. As computer efficiency has improved and compound libraries have grown, the ability to screen hundreds of millions, and even billions, of compounds has become feasible for modest-sized computer clusters. This allows the rapid and cost-effective exploration and categorization of vast chemical space into a subset enriched with potential hits for a given target. To accomplish this goal at speed, approximations are used that result in undersampling of possible configurations and inaccurate predictions of absolute binding energies. Accordingly, it is important to establish controls, as are common in other fields, to enhance the likelihood of success in spite of these challenges. Here we outline best practices and control docking calculations that help evaluate docking parameters for a given target prior to undertaking a large-scale prospective screen, with exemplification in one particular target, the melatonin receptor, where following this procedure led to direct docking hits with activities in the subnanomolar range. Additional controls are suggested to ensure specific activity for experimentally validated hit compounds. These guidelines should be useful regardless of the docking software used. Docking software described in the outlined protocol (DOCK3.7) is made freely available for academic research to explore new hits for a range of targets.

4.
J Chem Inf Model ; 61(9): 4331-4341, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34467754

RESUMO

While small molecule internal strain is crucial to molecular docking, using it in evaluating ligand scores has remained elusive. Here, we investigate a technique that calculates strain using relative torsional populations in the Cambridge Structural Database, enabling fast precalculation of these energies. In retrospective studies of large docking screens of the dopamine D4 receptor and of AmpC ß-lactamase, where close to 600 docking hits were tested experimentally, including such strain energies improved hit rates by preferentially reducing the ranks of strained high-scoring decoy molecules. In a 40-target subset of the DUD-E benchmark, we found two thresholds that usefully distinguished between ligands and decoys: one based on the total strain energy of the small molecules and another based on the maximum strain allowed for any given torsion within them. Using these criteria, about 75% of the benchmark targets had improved enrichment after strain filtering. Relying on precalculated population distributions, this approach is rapid, taking less than 0.04 s to evaluate a conformation on a standard core, making it pragmatic for precalculating strain in even ultralarge libraries. Since it is scoring function agnostic, it may be useful to multiple docking approaches; it is openly available at http://tldr.docking.org.

5.
bioRxiv ; 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34494023

RESUMO

To fight the SARS-CoV-2 pandemic, much effort has been directed toward drug repurposing, testing investigational and approved drugs against several viral or human proteins in vitro . Here we investigate the impact of colloidal aggregation, a common artifact in early drug discovery, in these repurposing screens. We selected 56 drugs reported to be active in biochemical assays and tested them for aggregation by both dynamic light scattering and by enzyme counter screening with and without detergent; seventeen of these drugs formed colloids at concentrations similar to their literature reported IC 50 s. To investigate the occurrence of colloidal aggregators more generally in repurposing libraries, we further selected 15 drugs that had physical properties resembling known aggregators from a common repurposing library, and found that 6 of these aggregated at micromolar concentrations. An attraction of repurposing is that drugs active on one target are considered de-risked on another. This study suggests not only that many of the drugs repurposed for SARS-CoV-2 in biochemical assays are artifacts, but that, more generally, when screened at relevant concentrations, drugs can act artifactually via colloidal aggregation. Understanding the role of aggregation, and detecting its effects rapidly, will allow the community to focus on those drugs and leads that genuinely have potential for treating COVID-19.

6.
Science ; 373(6554): 541-547, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34326236

RESUMO

Repurposing drugs as treatments for COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drawn much attention. Beginning with sigma receptor ligands and expanding to other drugs from screening in the field, we became concerned that phospholipidosis was a shared mechanism underlying the antiviral activity of many repurposed drugs. For all of the 23 cationic amphiphilic drugs we tested, including hydroxychloroquine, azithromycin, amiodarone, and four others already in clinical trials, phospholipidosis was monotonically correlated with antiviral efficacy. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the physicochemical properties of drugs and does not reflect specific target-based activities-rather, it may be considered a toxic confound in early drug discovery. Early detection of phospholipidosis could eliminate these artifacts, enabling a focus on molecules with therapeutic potential.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Reposicionamento de Medicamentos , Lipidoses/induzido quimicamente , Fosfolipídeos/metabolismo , SARS-CoV-2/efeitos dos fármacos , Células A549 , Animais , Antivirais/química , Antivirais/uso terapêutico , Antivirais/toxicidade , COVID-19/virologia , Cátions , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Testes de Sensibilidade Microbiana , SARS-CoV-2/fisiologia , Tensoativos/química , Tensoativos/farmacologia , Tensoativos/toxicidade , Células Vero , Replicação Viral/efeitos dos fármacos
7.
bioRxiv ; 2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33791693

RESUMO

Repurposing drugs as treatments for COVID-19 has drawn much attention. A common strategy has been to screen for established drugs, typically developed for other indications, that are antiviral in cells or organisms. Intriguingly, most of the drugs that have emerged from these campaigns, though diverse in structure, share a common physical property: cationic amphiphilicity. Provoked by the similarity of these repurposed drugs to those inducing phospholipidosis, a well-known drug side effect, we investigated phospholipidosis as a mechanism for antiviral activity. We tested 23 cationic amphiphilic drugs-including those from phenotypic screens and others that we ourselves had found-for induction of phospholipidosis in cell culture. We found that most of the repurposed drugs, which included hydroxychloroquine, azithromycin, amiodarone, and four others that have already progressed to clinical trials, induced phospholipidosis in the same concentration range as their antiviral activity; indeed, there was a strong monotonic correlation between antiviral efficacy and the magnitude of the phospholipidosis. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the gross physical properties of drugs, and does not reflect specific target-based activities, rather it may be considered a confound in early drug discovery. Understanding its role in infection, and detecting its effects rapidly, will allow the community to better distinguish between drugs and lead compounds that more directly impact COVID-19 from the large proportion of molecules that manifest this confounding effect, saving much time, effort and cost. One Sentence Summary: Drug-induced phospholipidosis is a single mechanism that may explain the in vitro efficacy of a wide-variety of therapeutics repurposed for COVID-19.

8.
J Med Chem ; 64(7): 4109-4116, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33761256

RESUMO

Small molecule colloidal aggregates adsorb and partially denature proteins, inhibiting them artifactually. Oddly, this inhibition is typically time-dependent. Two mechanisms might explain this: low concentrations of the colloid and enzyme might mean low encounter rates, or colloid-based protein denaturation might impose a kinetic barrier. These two mechanisms should have different concentration dependencies. Perplexingly, when enzyme concentration was increased, incubation times actually lengthened, inconsistent with both models and with classical chemical kinetics of solution species. We therefore considered molecular crowding, where colloids with lower protein surface density demand a shorter incubation time than more crowded colloids. To test this, we grew and shrank colloid surface area. As the surface area shrank, the incubation time lengthened, while as it increased, the converse was true. These observations support a crowding effect on protein binding to colloidal aggregates. Implications for drug delivery and for detecting aggregation-based inhibition will be discussed.


Assuntos
Proteínas de Bactérias/metabolismo , Coloides/metabolismo , Malato Desidrogenase/metabolismo , beta-Lactamases/metabolismo , Adsorção , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Coloides/química , Ensaios Enzimáticos , Fulvestranto/química , Cinética , Malato Desidrogenase/antagonistas & inibidores , Malato Desidrogenase/química , Ligação Proteica , Sorafenibe/química , beta-Lactamases/química
9.
J Chem Inf Model ; 61(2): 699-714, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33494610

RESUMO

Enrichment of ligands versus property-matched decoys is widely used to test and optimize docking library screens. However, the unconstrained optimization of enrichment alone can mislead, leading to false confidence in prospective performance. This can arise by over-optimizing for enrichment against property-matched decoys, without considering the full spectrum of molecules to be found in a true large library screen. Adding decoys representing charge extrema helps mitigate over-optimizing for electrostatic interactions. Adding decoys that represent the overall characteristics of the library to be docked allows one to sample molecules not represented by ligands and property-matched decoys but that one will encounter in a prospective screen. An optimized version of the DUD-E set (DUDE-Z), as well as Extrema and sets representing broad features of the library (Goldilocks), is developed here. We also explore the variability that one can encounter in enrichment calculations and how that can temper one's confidence in small enrichment differences. The new tools and new decoy sets are freely available at http://tldr.docking.org and http://dudez.docking.org.


Assuntos
Benchmarking , Ligantes , Modelos Moleculares , Estudos Prospectivos , Ligação Proteica
10.
bioRxiv ; 2020 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-33269349

RESUMO

The SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of diverse fragment libraries resulted in 214 unique macrodomain-binding fragments, out of 2,683 screened. An additional 60 molecules were selected from docking over 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several crystallographic and docking fragment hits were validated for solution binding using three biophysical techniques (DSF, HTRF, ITC). Overall, the 234 fragment structures presented explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

11.
Biochemistry ; 59(38): 3594-3614, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32865988

RESUMO

GPR68, an orphan G-protein coupled receptor, senses protons, couples to multiple G-proteins, and is also activated or inhibited by divalent metal ions. It has seven extracellular histidine residues, although it is not clear how these histidine residues play a role in both proton-sensing and metal ion modulation. Here we demonstrate that divalent metal ions are allosteric modulators that can activate or inhibit proton activity in a concentration- and pH-dependent manner. We then show that single histidine mutants have differential and varying degrees of effects on proton-sensing and metal ion modulation. Some histidine residues play dual roles in proton-sensing and metal ion modulation, while others are important in one or the other but not both. Two extracellular disulfide bonds are predicted to constrain histidine residues to be spatially close to each other. Combining histidine mutations leads to reduced proton activity and resistance to metal ion modulation, while breaking the less conserved disulfide bond results in a more severe reduction in proton-sensing over metal modulation. The small-molecule positive allosteric modulators (PAMs) ogerin and lorazepam are not affected by these mutations and remain active at mutants with severely reduced proton activity or are resistant to metal ion modulation. These results suggest GPR68 possesses two independent allosteric modulation systems, one through interaction with divalent metal ions at the extracellular surface and another through small-molecule PAMs in the transmembrane domains. A new GPR68 model is developed to accommodate the findings which could serve as a template for further studies and ligand discovery by virtual ligand docking.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Cálcio/farmacologia , Magnésio/farmacologia , Metais Pesados/farmacologia , Prótons , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Células HEK293 , Histidina/química , Humanos , Concentração de Íons de Hidrogênio , Mutação , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética
12.
Cell ; 182(6): 1574-1588.e19, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32946782

RESUMO

Hallucinogens like lysergic acid diethylamide (LSD), psilocybin, and substituted N-benzyl phenylalkylamines are widely used recreationally with psilocybin being considered as a therapeutic for many neuropsychiatric disorders including depression, anxiety, and substance abuse. How psychedelics mediate their actions-both therapeutic and hallucinogenic-are not understood, although activation of the 5-HT2A serotonin receptor (HTR2A) is key. To gain molecular insights into psychedelic actions, we determined the active-state structure of HTR2A bound to 25-CN-NBOH-a prototypical hallucinogen-in complex with an engineered Gαq heterotrimer by cryoelectron microscopy (cryo-EM). We also obtained the X-ray crystal structures of HTR2A complexed with the arrestin-biased ligand LSD or the inverse agonist methiothepin. Comparisons of these structures reveal determinants responsible for HTR2A-Gαq protein interactions as well as the conformational rearrangements involved in active-state transitions. Given the potential therapeutic actions of hallucinogens, these findings could accelerate the discovery of more selective drugs for the treatment of a variety of neuropsychiatric disorders.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Alucinógenos/química , Receptor 5-HT2A de Serotonina/química , Receptor 5-HT2A de Serotonina/metabolismo , Animais , Microscopia Crioeletrônica , Cristalografia por Raios X , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Células HEK293 , Alucinógenos/farmacologia , Alucinógenos/uso terapêutico , Humanos , Ligantes , Dietilamida do Ácido Lisérgico/química , Dietilamida do Ácido Lisérgico/farmacologia , Metiotepina/química , Metiotepina/metabolismo , Modelos Químicos , Mutação , Conformação Proteica em alfa-Hélice , Receptor 5-HT2A de Serotonina/genética , Proteínas Recombinantes , Serotonina/metabolismo , Spodoptera
13.
Science ; 369(6502): 403-413, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32703874

RESUMO

Excipients, considered "inactive ingredients," are a major component of formulated drugs and play key roles in their pharmacokinetics. Despite their pervasiveness, whether they are active on any targets has not been systematically explored. We computed the likelihood that approved excipients would bind to molecular targets. Testing in vitro revealed 25 excipient activities, ranging from low-nanomolar to high-micromolar concentration. Another 109 activities were identified by testing against clinical safety targets. In cellular models, five excipients had fingerprints predictive of system-level toxicity. Exposures of seven excipients were investigated, and in certain populations, two of these may reach levels of in vitro target potency, including brain and gut exposure of thimerosal and its major metabolite, which had dopamine D3 receptor dissociation constant K d values of 320 and 210 nM, respectively. Although most excipients deserve their status as inert, many approved excipients may directly modulate physiologically relevant targets.


Assuntos
Composição de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Excipientes/farmacologia , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/normas , Excipientes/efeitos adversos , Humanos , Terapia de Alvo Molecular
14.
Nat Chem Biol ; 16(7): 749-755, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483378

RESUMO

Most drugs acting on G-protein-coupled receptors target the orthosteric binding pocket where the native hormone or neurotransmitter binds. There is much interest in finding allosteric ligands for these targets because they modulate physiologic signaling and promise to be more selective than orthosteric ligands. Here we describe a newly developed allosteric modulator of the ß2-adrenergic receptor (ß2AR), AS408, that binds to the membrane-facing surface of transmembrane segments 3 and 5, as revealed by X-ray crystallography. AS408 disrupts a water-mediated polar network involving E1223.41 and the backbone carbonyls of V2065.45 and S2075.46. The AS408 binding site is adjacent to a previously identified molecular switch for ß2AR activation formed by I3.40, P5.50 and F6.44. The structure reveals how AS408 stabilizes the inactive conformation of this switch, thereby acting as a negative allosteric modulator for agonists and positive allosteric modulator for inverse agonists.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2/química , Antagonistas Adrenérgicos beta/química , Alprenolol/química , Norepinefrina/química , Receptores Adrenérgicos beta 2/química , Xinafoato de Salmeterol/química , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Regulação Alostérica , Sítio Alostérico , Alprenolol/farmacologia , Células HEK293 , Humanos , Cinética , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Norepinefrina/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Receptores Adrenérgicos beta 2/metabolismo , Xinafoato de Salmeterol/farmacologia , Termodinâmica , Água/química
15.
Proc Natl Acad Sci U S A ; 117(27): 16009-16018, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571913

RESUMO

Food and drug products contain diverse and abundant small-molecule additives (excipients) with unclear impacts on human physiology, drug safety, and response. Here, we evaluate their potential impact on intestinal drug absorption. By screening 136 unique compounds for inhibition of the key intestinal transporter OATP2B1 we identified and validated 24 potent OATP2B1 inhibitors, characterized by higher molecular weight and hydrophobicity compared to poor or noninhibitors. OATP2B1 inhibitors were also enriched for dyes, including 8 azo (R-N=N-R') dyes. Pharmacokinetic studies in mice confirmed that FD&C Red No. 40, a common azo dye excipient and a potent inhibitor of OATP2B1, decreased the plasma level of the OATP2B1 substrate fexofenadine, suggesting that FD&C Red No. 40 has the potential to block drug absorption through OATP2B1 inhibition in vivo. However, the gut microbiomes of multiple unrelated healthy individuals as well as diverse human gut bacterial isolates were capable of inactivating the identified azo dye excipients, producing metabolites that no longer inhibit OATP2B1 transport. These results support a beneficial role for the microbiome in limiting the unintended effects of food and drug additives in the intestine and provide a framework for the data-driven selection of excipients. Furthermore, the ubiquity and genetic diversity of gut bacterial azoreductases coupled to experiments in conventionally raised and gnotobiotic mice suggest that variations in gut microbial community structure may be less important to consider relative to the high concentrations of azo dyes in food products, which have the potential to saturate gut bacterial enzymatic activity.


Assuntos
Bactérias/metabolismo , Excipientes/metabolismo , Aditivos Alimentares/metabolismo , Alimentos , Microbioma Gastrointestinal/fisiologia , Absorção Intestinal/fisiologia , Transportadores de Ânions Orgânicos/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Antialérgicos/metabolismo , Antialérgicos/farmacocinética , Compostos Azo , Bactérias/isolamento & purificação , Excipientes/farmacocinética , Feminino , Aditivos Alimentares/farmacocinética , Antagonistas não Sedativos dos Receptores H1 da Histamina/metabolismo , Antagonistas não Sedativos dos Receptores H1 da Histamina/farmacocinética , Humanos , Absorção Intestinal/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Terfenadina/análogos & derivados
16.
J Am Chem Soc ; 142(11): 4960-4964, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32105459

RESUMO

Eukaryotic translation initiation factor 4E (eIF4E) binds the m7GTP cap structure at the 5'-end of mRNAs, stimulating the translation of proteins implicated in cancer cell growth and metastasis. eIF4E is a notoriously challenging target, and most of the reported inhibitors are negatively charged guanine analogues with negligible cell permeability. To overcome these challenges, we envisioned a covalent targeting strategy. As there are no cysteines near the eIF4E cap binding site, we developed a covalent docking approach focused on lysine. Taking advantage of a "make-on-demand" virtual library, we used covalent docking to identify arylsulfonyl fluorides that target a noncatalytic lysine (Lys162) in eIF4E. Guided by cocrystal structures, we elaborated arylsulfonyl fluoride 2 to 12, which to our knowledge is the first covalent eIF4E inhibitor with cellular activity. In addition to providing a new tool for acutely inactivating eIF4E in cells, our computational approach may offer a general strategy for developing selective lysine-targeted covalent ligands.


Assuntos
Fator de Iniciação 4E em Eucariotos/antagonistas & inibidores , Lisina/química , Sulfonamidas/farmacologia , Sítios de Ligação , Descoberta de Drogas , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/metabolismo , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Sulfonamidas/metabolismo
17.
Nature ; 579(7800): 609-614, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32040955

RESUMO

The neuromodulator melatonin synchronizes circadian rhythms and related physiological functions through the actions of two G-protein-coupled receptors: MT1 and MT2. Circadian release of melatonin at night from the pineal gland activates melatonin receptors in the suprachiasmatic nucleus of the hypothalamus, synchronizing the physiology and behaviour of animals to the light-dark cycle1-4. The two receptors are established drug targets for aligning circadian phase to this cycle in disorders of sleep5,6 and depression1-4,7-9. Despite their importance, few in vivo active MT1-selective ligands have been reported2,8,10-12, hampering both the understanding of circadian biology and the development of targeted therapeutics. Here we docked more than 150 million virtual molecules to an MT1 crystal structure, prioritizing structural fit and chemical novelty. Of these compounds, 38 high-ranking molecules were synthesized and tested, revealing ligands with potencies ranging from 470 picomolar to 6 micromolar. Structure-based optimization led to two selective MT1 inverse agonists-which were topologically unrelated to previously explored chemotypes-that acted as inverse agonists in a mouse model of circadian re-entrainment. Notably, we found that these MT1-selective inverse agonists advanced the phase of the mouse circadian clock by 1.3-1.5 h when given at subjective dusk, an agonist-like effect that was eliminated in MT1- but not in MT2-knockout mice. This study illustrates the opportunities for modulating melatonin receptor biology through MT1-selective ligands and for the discovery of previously undescribed, in vivo active chemotypes from structure-based screens of diverse, ultralarge libraries.


Assuntos
Ritmo Circadiano/fisiologia , Ligantes , Receptores de Melatonina/agonistas , Receptores de Melatonina/metabolismo , Animais , Ritmo Circadiano/efeitos dos fármacos , Escuridão , Avaliação Pré-Clínica de Medicamentos , Agonismo Inverso de Drogas , Feminino , Humanos , Luz , Masculino , Camundongos , Camundongos Knockout , Simulação de Acoplamento Molecular , Receptor MT1 de Melatonina/agonistas , Receptor MT1 de Melatonina/deficiência , Receptor MT1 de Melatonina/genética , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/agonistas , Receptor MT2 de Melatonina/deficiência , Receptor MT2 de Melatonina/genética , Receptor MT2 de Melatonina/metabolismo , Receptores de Melatonina/deficiência , Receptores de Melatonina/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato/genética
18.
Mol Pharm ; 17(3): 748-756, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31990564

RESUMO

Mechanistic-understanding-based selection of excipients may improve formulation development strategies for generic drug products and potentially accelerate their approval. Our study aimed at investigating the effects of molecular excipients present in orally administered FDA-approved drug products on the intestinal efflux transporter, BCRP (ABCG2), which plays a critical role in drug absorption with potential implications on drug safety and efficacy. We determined the interactions of 136 oral molecular excipients with BCRP in isolated membrane vesicles and identified 26 excipients as BCRP inhibitors with IC50 values less than 5 µM using 3H-cholecystokinin octapeptide (3H-CCK8). These BCRP inhibitors belonged to three functional categories of excipients: dyes, surfactants, and flavoring agents. Compared with noninhibitors, BCRP inhibitors had significantly higher molecular weights and SLogP values. The inhibitory effects of excipients identified in membrane vesicles were also evaluated in BCRP-overexpressing HEK293 cells at similar concentrations. Only 1 of the 26 inhibitors of BCRP identified in vesicles inhibited BCRP-mediated 3H-oxypurinol uptake by more than 50%, consistent with the notion that BCRP inhibition depends on transmembrane or intracellular availability of the inhibitors. Collectively, the results of this study provide new information on excipient selection during the development of drug products with active pharmaceutical ingredients that are BCRP substrates.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Corantes/metabolismo , Excipientes/metabolismo , Aromatizantes/metabolismo , Proteínas de Neoplasias/metabolismo , Tensoativos/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Administração Oral , Corantes/química , Corantes/farmacologia , Composição de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Excipientes/química , Excipientes/farmacologia , Feminino , Aromatizantes/química , Aromatizantes/farmacologia , Células HEK293 , Humanos , Concentração Inibidora 50 , Absorção Intestinal/efeitos dos fármacos , Peso Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Transdução de Sinais/genética , Tensoativos/química , Tensoativos/farmacologia , Transfecção
19.
J Biol Chem ; 294(50): 19246-19254, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31628191

RESUMO

Adhesion G protein-coupled receptors (aGPCRs) represent a distinct family of GPCRs that regulate several developmental and physiological processes. Most aGPCRs undergo GPCR autoproteolysis-inducing domain-mediated protein cleavage, which produces a cryptic tethered agonist (termed Stachel (stinger)), and cleavage-dependent and -independent aGPCR signaling mechanisms have been described. aGPCR G1 (ADGRG1 or G protein-coupled receptor 56 (GPR56)) has pleiotropic functions in the development of multiple organ systems, which has broad implications for human diseases. To date, two natural GPR56 ligands, collagen III and tissue transglutaminase (TG2), and one small-molecule agonist, 3-α-acetoxydihydrodeoxygedunin (3-α-DOG), have been identified, in addition to a synthetic peptide, P19, that contains seven amino acids of the native Stachel sequence. However, the mechanisms by which these natural and small-molecule agonists signal through GPR56 remain unknown. Here we engineered a noncleavable receptor variant that retains signaling competence via the P19 peptide. We demonstrate that both natural and small-molecule agonists can activate only cleaved GPR56. Interestingly, TG2 required both receptor cleavage and the presence of a matrix protein, laminin, to activate GPR56, whereas collagen III and 3-α-DOG signaled without any cofactors. On the other hand, both TG2/laminin and collagen III activate the receptor by dissociating the N-terminal fragment from its C-terminal fragment, enabling activation by the Stachel sequence, whereas P19 and 3-α-DOG initiate downstream signaling without disengaging the N-terminal fragment from its C-terminal fragment. These findings deepen our understanding of how GPR56 signals via natural ligands, and a small-molecule agonist may be broadly applicable to other aGPCR family members.


Assuntos
Limoninas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Humanos , Ligantes , Limoninas/química , Masculino , Camundongos , Camundongos Knockout , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
20.
J Med Chem ; 62(21): 9593-9599, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31589047

RESUMO

Small-molecule aggregates are a leading cause of artifacts in early drug discovery, but little is known about their interactions with proteins, nor why some proteins are more susceptible to inhibition than others. A possible reason for this apparent selectivity is that aggregation-based inhibition, as a stoichiometric process, is sensitive to protein concentration, which varies across assays. Alternatively, local protein unfolding by aggregates may lead to selectivity since stability varies among proteins. To deconvolute these effects, we used differentially stable point mutants of a single protein, TEM-1 ß-lactamase. Broadly, destabilized mutants had higher affinities for and were more potently inhibited by aggregates versus more stable variants. The addition of the irreversible inhibitor moxalactam destabilized several mutants, and these typically bound tighter to a colloidal particle, while the only mutant it stabilized bound weaker. These results suggest that less-stable enzymes are more easily sequestered and inhibited by colloidal aggregates.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , beta-Lactamases/metabolismo , Mutação , Estabilidade Proteica , Termodinâmica , beta-Lactamases/genética
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