Structure of a Hallucinogen-Activated Gq-Coupled 5-HT2A Serotonin Receptor.

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.

Structures of the σ2 receptor enable docking for bioactive ligand discovery.

The σ2 receptor has attracted intense interest in cancer imaging1, psychiatric disease2, neuropathic pain3-5 and other areas of biology6,7. Here we determined the crystal structure of this receptor in complex with the clinical candidate roluperidone2 and the tool compound PB288. These structures templated a large-scale docking screen of 490 million virtual molecules, of which 484 compounds were synthesized and tested. We identified 127 new chemotypes with affinities superior to 1 µM, 31 of which had affinities superior to 50 nM. The hit rate fell smoothly and monotonically with docking score. We optimized three hits for potency and selectivity, and achieved affinities that ranged from 3 to 48 nM, with up to 250-fold selectivity versus the σ1 receptor. Crystal structures of two ligands bound to the σ2 receptor confirmed the docked poses. To investigate the contribution of the σ2 receptor in pain, two potent σ2-selective ligands and one potent σ1/σ2 non-selective ligand were tested for efficacy in a mouse model of neuropathic pain. All three ligands showed time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model9, suggesting that the σ2 receptor has a role in nociception. This study illustrates the opportunities for rapid discovery of in vivo probes through structure-based screens of ultra large libraries, enabling study of underexplored areas of biology.

Modeling the expansion of virtual screening libraries.

Recently, 'tangible' virtual libraries have made billions of molecules readily available. Prioritizing these molecules for synthesis and testing demands computational approaches, such as docking. Their success may depend on library diversity, their similarity to bio-like molecules and how receptor fit and artifacts change with library size. We compared a library of 3 million 'in-stock' molecules with billion-plus tangible libraries. The bias toward bio-like molecules in the tangible library decreases 19,000-fold versus those 'in-stock'. Similarly, thousands of high-ranking molecules, including experimental actives, from five ultra-large-library docking campaigns are also dissimilar to bio-like molecules. Meanwhile, better-fitting molecules are found as the library grows, with the score improving log-linearly with library size. Finally, as library size increases, so too do rare molecules that rank artifactually well. Although the nature of these artifacts changes from target to target, the expectation of their occurrence does not, and simple strategies can minimize their impact.

Ultra-large library docking for discovering new chemotypes.

Despite intense interest in expanding chemical space, libraries containing hundreds-of-millions to billions of diverse molecules have remained inaccessible. Here we investigate structure-based docking of 170 million make-on-demand compounds from 130 well-characterized reactions. The resulting library is diverse, representing over 10.7 million scaffolds that are otherwise unavailable. For each compound in the library, docking against AmpC ß-lactamase (AmpC) and the D4 dopamine receptor were simulated. From the top-ranking molecules, 44 and 549 compounds were synthesized and tested for interactions with AmpC and the D4 dopamine receptor, respectively. We found a phenolate inhibitor of AmpC, which revealed a group of inhibitors without known precedent. This molecule was optimized to 77 nM, which places it among the most potent non-covalent AmpC inhibitors known. Crystal structures of this and other AmpC inhibitors confirmed the docking predictions. Against the D4 dopamine receptor, hit rates fell almost monotonically with docking score, and a hit-rate versus score curve predicted that the library contained 453,000 ligands for the D4 dopamine receptor. Of 81 new chemotypes discovered, 30 showed submicromolar activity, including a 180-pM subtype-selective agonist of the D4 dopamine receptor.

Property-Unmatched Decoys in Docking Benchmarks.

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.

Antiproliferative and apoptosis-inducing activities of novel naphthalimide-cyclam conjugates through dual topoisomerase (topo) I/II inhibition.

A novel series of naphthalimide-cyclam conjugates were designed and synthesized. Among them, compounds 4c, 4d, 8c and 8d which bearing long lipophilic alkyl chains, displayed comparable or more potent cytotoxic activities against human tumor cell lines than amonafide. Furthermore, the four compounds were proved to possess strong inhibition against both topoisomerase I and II. The representative compound 8c exhibited moderate DNA intercalation activity. Molecular modeling studies identified the possible interaction of compound 8c with the molecular target by forming topoisomerase/DNA/drug ternary complex. Finally, derivatives with long lipophilic alkyl chains could efficiently induce apoptosis.

Small vs. Large Library Docking for Positive Allosteric Modulators of the Calcium Sensing Receptor.

Drugs acting as positive allosteric modulators (PAMs) to enhance the activation of the calcium sensing receptor (CaSR) and to suppress parathyroid hormone (PTH) secretion can treat hyperparathyroidism but suffer from side effects including hypocalcemia and arrhythmias. Seeking new CaSR modulators, we docked libraries of 2.7 million and 1.2 billion molecules against transforming pockets in the active-state receptor dimer structure. Consistent with simulations suggesting that docking improves with library size, billion-molecule docking found new PAMs with a hit rate that was 2.7-fold higher than the million-molecule library and with hits up to 37-fold more potent. Structure-based optimization of ligands from both campaigns led to nanomolar leads, one of which was advanced to animal testing. This PAM displays 100-fold the potency of the standard of care, cinacalcet, in ex vivo organ assays, and reduces serum PTH levels in mice by up to 80% without the hypocalcemia typical of CaSR drugs. Cryo-EM structures with the new PAMs show that they induce residue rearrangements in the binding pockets and promote CaSR dimer conformations that are closer to the G-protein coupled state compared to established drugs. These findings highlight the promise of large library docking for therapeutic leads, especially when combined with experimental structure determination and mechanism.

AlphaFold2 structures template ligand discovery.

AlphaFold2 (AF2) and RosettaFold have greatly expanded the number of structures available for structure-based ligand discovery, even though retrospective studies have cast doubt on their direct usefulness for that goal. Here, we tested unrefined AF2 models prospectively, comparing experimental hit-rates and affinities from large library docking against AF2 models vs the same screens targeting experimental structures of the same receptors. In retrospective docking screens against the σ2 and the 5-HT2A receptors, the AF2 structures struggled to recapitulate ligands that we had previously found docking against the receptors' experimental structures, consistent with published results. Prospective large library docking against the AF2 models, however, yielded similar hit rates for both receptors versus docking against experimentally-derived structures; hundreds of molecules were prioritized and tested against each model and each structure of each receptor. The success of the AF2 models was achieved despite differences in orthosteric pocket residue conformations for both targets versus the experimental structures. Intriguingly, against the 5-HT2A receptor the most potent, subtype-selective agonists were discovered via docking against the AF2 model, not the experimental structure. To understand this from a molecular perspective, a cryoEM structure was determined for one of the more potent and selective ligands to emerge from docking against the AF2 model of the 5-HT2A receptor. Our findings suggest that AF2 models may sample conformations that are relevant for ligand discovery, much extending the domain of applicability of structure-based ligand discovery.

AlphaFold2 structures guide prospective ligand discovery.

AlphaFold2 (AF2) models have had wide impact but mixed success in retrospective ligand recognition. We prospectively docked large libraries against unrefined AF2 models of the σ2 and serotonin 2A (5-HT2A) receptors, testing hundreds of new molecules and comparing results with those obtained from docking against the experimental structures. Hit rates were high and similar for the experimental and AF2 structures, as were affinities. Success in docking against the AF2 models was achieved despite differences between orthosteric residue conformations in the AF2 models and the experimental structures. Determination of the cryo-electron microscopy structure for one of the more potent 5-HT2A ligands from the AF2 docking revealed residue accommodations that resembled the AF2 prediction. AF2 models may sample conformations that differ from experimental structures but remain low energy and relevant for ligand discovery, extending the domain of structure-based drug design.

Covalent Targeting of Splicing in T Cells.

Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide chemical probe EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and availability of functional mRNA pools that can be depleted due to extensive alternative splicing. We further introduce a comprehensive list of proteins involved in splicing and leverage both cysteine- and protein-directed activity-based protein profiling (ABPP) data with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in primary human T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry.

Large library docking for novel SARS-CoV-2 main protease non-covalent and covalent inhibitors.

Antiviral therapeutics to treat SARS-CoV-2 are needed to diminish the morbidity of the ongoing COVID-19 pandemic. A well-precedented drug target is the main viral protease (MPro ), which is targeted by an approved drug and by several investigational drugs. Emerging viral resistance has made new inhibitor chemotypes more pressing. Adopting a structure-based approach, we docked 1.2 billion non-covalent lead-like molecules and a new library of 6.5 million electrophiles against the enzyme structure. From these, 29 non-covalent and 11 covalent inhibitors were identified in 37 series, the most potent having an IC50 of 29 and 20 µM, respectively. Several series were optimized, resulting in low micromolar inhibitors. Subsequent crystallography confirmed the docking predicted binding modes and may template further optimization. While the new chemotypes may aid further optimization of MPro inhibitors for SARS-CoV-2, the modest success rate also reveals weaknesses in our approach for challenging targets like MPro versus other targets where it has been more successful, and versus other structure-based techniques against MPro itself.

A practical guide to large-scale docking.

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.

Phospholipidosis is a shared mechanism underlying the in vitro antiviral activity of many repurposed drugs against SARS-CoV-2.

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.

Drug-induced phospholipidosis confounds drug repurposing for SARS-CoV-2.

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.

Selectivity Challenges in Docking Screens for GPCR Targets and Antitargets.

To investigate large library docking's ability to find molecules with joint activity against on-targets and selectivity versus antitargets, the dopamine D2 and serotonin 5-HT2A receptors were targeted, seeking selectivity against the histamine H1 receptor. In a second campaign, κ-opioid receptor ligands were sought with selectivity versus the µ-opioid receptor. While hit rates ranged from 40% to 63% against the on-targets, they were just as good against the antitargets, even though the molecules were selected for their putative lack of binding to the off-targets. Affinities, too, were often as good or better for the off-targets. Even though it was occasionally possible to find selective molecules, such as a mid-nanomolar D2/5-HT2A ligand with 21-fold selectivity versus the H1 receptor, this was the exception. Whereas false-negatives are tolerable in docking screens against on-targets, they are intolerable against antitargets; addressing this problem may demand new strategies in the field.

Design, Synthesis, and Biological Evaluation of Pyrimido[4,5- d]pyrimidine-2,4(1 H,3 H)-diones as Potent and Selective Epidermal Growth Factor Receptor (EGFR) Inhibitors against L858R/T790M Resistance Mutation.

First-generation epidermal growth factor receptor (EGFR) inhibitors, gefitinib and erlotinib, have achieved initially marked clinical efficacy for nonsmall cell lung cancer (NSCLC) patients with EGFR activating mutations. However, their clinical benefit was limited by the emergence of acquired resistance mutations. In most cases (approximately 60%), the resistance was caused by the secondary EGFR T790M gatekeeper mutation. Thus, it is still desirable to develop novel third-generation EGFR inhibitors to overcome T790M mutation while sparing wild-type (WT) EGFR. Herein, a series of pyrimido[4,5- d]pyrimidine-2,4(1 H,3 H)-dione derivatives were designed and synthesized, among which the most potent compound 20g not only demonstrated significant inhibitory activity and selectivity for EGFRL858R/T790M and H1975 cells in vitro but also displayed outstanding antitumor efficiency in H1975 xenograft mouse model. The encouraging mutant-selective results at both in vitro and in vivo levels suggested that 20g might be used as a promising lead compound for further structural optimization as potent and selective EGFRL858R/T790M inhibitors.

Structure-Guided Design of C4-alkyl-1,4-dihydro-2H-pyrimido[4,5-d][1,3]oxazin-2-ones as Potent and Mutant-Selective Epidermal Growth Factor Receptor (EGFR) L858R/T790M Inhibitors.

Epidermal growth factor receptor (EGFR) T790M acquired drug-resistance mutation has become a major clinical challenge for the therapy of non-small cell lung cancer. Here, we applied a structure-guided approach on the basis of the previous reported EGFR inhibitor (compound 9), and designed a series of C4-alkyl-1,4-dihydro-2H-pyrimido[4,5-d][1,3]oxazin-2-one derivatives as novel mutant-selective EGFR inhibitors. Finally, the most representative compound 20a was identified, which showed high selectivity at both enzymatic and cellular levels against EGFRL858R/T790M (H1975 cell lines) over EGFRWT (A431 cell lines). The representative compound 20a also showed promising antitumor efficiency in the in vivo antitumor efficacy study of H1975 xenograft mouse model driven by EGFRL858R/T790M. These results provide a new scaffold for the treatment of dual-mutant-driven non-small cell lung cancer.

Discovery of peptide inhibitors targeting human programmed death 1 (PD-1) receptor.

Blocking the interaction of human programmed death 1 (hPD-1) and its ligand hPD-L1 has been a promising immunotherapy in cancer treatment. In this paper, using a computational de novo peptide design method, we designed several hPD-1 binding peptides. The most potent peptide Ar5Y_4 showed a KD value of 1.38 ± 0.39 µM, comparable to the binding affinity of the cognate hPD-L1. A Surface Plasmon Resonance (SPR) competitive binding assay result indicated that Ar5Y_4 could inhibit the interaction of hPD-1/hPD-L1. Moreover, Ar5Y_4 could restore the function of Jurkat T cells which had been suppressed by stimulated HCT116 cells. Peptides described in this paper provide promising biologic candidates for cancer immunotherapy or diagnostics.

Discovery and Rational Design of Pteridin-7(8H)-one-Based Inhibitors Targeting FMS-like Tyrosine Kinase 3 (FLT3) and Its Mutants.

FLT3 has been validated as a therapeutic target for the treatment of acute myeloid leukemia (AML). In this paper, we describe for the first time, pteridin-7(8H)-one as a scaffold for potent FLT3 inhibitors derived from structural optimizations on irreversible EGFR inhibitors. The representative inhibitor (31) demonstrates single-digit nanomolar inhibition against FLT3 and subnanomolar KD for drug-resistance FLT3 mutants. In profiling of the in vitro tumor cell lines, it shows good selectivity against AML cells harboring FLT3-ITD mutations over other leukemia and solid tumor cell lines. The mechanism of action study illustrates that pteridin-7(8H)-one derivatives suppress the phosphorylation of FLT3 and its downstream pathways, thereby inducing G0/G1 cell cycle arrest and apoptosis in AML cells. In in vivo studies, 31 significantly suppresses the tumor growth in MV4-11 xenograft model. Overall, we provide a structurally distinct chemical scaffold with which to develop FLT3 mutants-selective inhibitors for AML treatment.