Computationally driven discovery of SARS-CoV-2 Mpro inhibitors: from design to experimental validation

We report a fast-track computationally driven discovery of new SARS-CoV-2 main protease (M^pro) inhibitors whose potency ranges from mM for the initial non-covalent ligands to sub-μM for the final covalent compound (IC₅₀ = 830 ± 50 nM). The project extensively relied on high-resolution all-atom molecular dynamics simulations and absolute binding free energy calculations performed using the polarizable AMOEBA force field. The study is complemented by extensive adaptive sampling simulations that are used to rationalize the different ligand binding poses through the explicit reconstruction of the ligand–protein conformation space. Machine learning predictions are also performed to predict selected compound properties. While simulations extensively use high performance computing to strongly reduce the time-to-solution, they were systematically coupled to nuclear magnetic resonance experiments to drive synthesis and for in vitro characterization of compounds. Such a study highlights the power of in silico strategies that rely on structure-based approaches for drug design and allows the protein conformational multiplicity problem to be addressed. The proposed fluorinated tetrahydroquinolines open routes for further optimization of M^pro inhibitors towards low nM affinities.

The dominant binding mode of the QUB-00006-Int-07 main protease inhibitor during absolute binding free energy simulations.     

Allosteric Binding of MDMA to the Human Serotonin Transporter (hSERT) via Ensemble Binding Space Analysis with ΔG Calculations,Induced Fit Docking and Monte Carlo Simulations

Despite the recent promising results of MDMA (3,4-methylenedioxy-methamphetamine) as a psychotherapeutic agent and its history of misuse, little is known about its molecular mode of action. MDMA enhances monoaminergic neurotransmission in the brain and its valuable psychoactive effects are associated to a dual action on the 5-HT transporter (SERT). This drug inhibits the reuptake of 5-HT (serotonin) and reverses its flow, acting as a substrate for the SERT, which possesses a central binding site (S1) for antidepressants as well as an allosteric (S2) one. Previously, we characterized the spatial binding requirements for MDMA at S1. Here, we propose a structure-based mechanistic model of MDMA occupation and translocation across both binding sites, applying ensemble binding space analyses, electrostatic complementarity, and Monte Carlo energy perturbation theory. Computed results were correlated with experimental data (r = 0.93 and 0.86 for S1 and S2, respectively). Simulations on all hSERT available structures with Gibbs free energy estimations (ΔG) revealed a favourable and pervasive dual binding mode for MDMA at S2, i.e., adopting either a 5-HT or an escitalopram-like orientation. Intermediate ligand conformations were identified within the allosteric site and between the two sites, outlining an internalization pathway for MDMA. Among the strongest and more frequent interactions were salt bridges with Glu494 and Asp328, a H-bond with Thr497, a π-π with Phe556, and a cation-π with Arg104. Similitudes and differences with the allosteric binding of 5-HT and antidepressants suggest that MDMA may have a distinctive chemotype. Thus, our models may provide a framework for future virtual screening studies and pharmaceutical design and to develop hSERT allosteric compounds with a unique psychoactive MDMA-like profile.     

Synthesis, characterization and development of a high-throughput methodology for the discovery of botulinum neurotoxin a inhibitors

Botulinum neurotoxins (BoNTs), etiological agents of the deadly food poisoning disease botulism, are the most toxic proteins currently known. Although only a few hundred cases of botulism are reported in the United States annually, there is growing interest in BoNTs attributable to their potential use as biological warfare agents. Neurotoxicity results from cleavage of the soluble NSF-attachment protein receptor complex proteins of the presynaptic vesicles by the BoNT light chain subunit, a Zn endopeptidase. Few effective inhibitors of BoNT/A LC (light chain) activity are known, and the discovery process is hampered by the lack of an efficient high-throughput assay for screening compound libraries. To alleviate this bottleneck, we have synthesized the peptide SNAPtide and have developed a robust assay for the high-throughput evaluation of BoNT/A LC inhibitors. Key aspects for the development of this optimized assay include the addition of a series of detergents, cosolvents, and salts, including 0.01% w/v Tween 20 to increase BoNT/A LC catalysis, stability, and ease of small molecule screening. To evaluate the effectiveness of the assay, a series of hydroxamate-based small molecules were synthesized and examined with BoNT/A LC. The methodology described is superior to other assays reported to date for the high-throughput identification of BoNT/A inhibitors.     

Docking studies of novel pyrazinopyridoindoles class of antihistamines with the homology modelled H(1)-receptor

Histamine is an important neurotransmitter as it controls a multitude of physiological functions by activating specific receptors on target cells. It exerts its effects by binding to four different histamine receptors (H(1)-H(4)), which all belong to the large family of G protein-coupled receptors (GPCRs). Research and development of H(1) ligand has largely focused on antagonists which are used for their anti-allergy effects in the periphery. Recent understanding of the clinical importance of H(1) receptors in brain, however, suggests the pharmacotherapeutic potential of H(1) agonists in neurodegenerative and neuropsychiatric disorders. Despite the therapeutic importance of the H(1) receptor, for many years the molecular features of the H(1) receptor protein had been unknown. In view of the recently reported crystal structure of human H(1) receptor and in continuation of our work on 3D-pharmacophore on antihistamine H(1) and homology model of histamine H(1) receptor, docking studies have been carried out on some promising pyrazinopyridoindole class of antihistamine H(1), including two outliers, to validate our earlier reported models/hypotheses on H(1)-receptor, where a good explanation between estimated and observed activities has been obtained. In addition, the docking study also provided insights about the optimal activity of the outliers, for which no explanation was reported previously.     

Computational discovery of picomolar Q(o) site inhibitors of cytochrome bc1 complex

A critical challenge to the fragment-based drug discovery (FBDD) is its low-throughput nature due to the necessity of biophysical method-based fragment screening. Herein, a method of pharmacophore-linked fragment virtual screening (PFVS) was successfully developed. Its application yielded the first picomolar-range Q(o) site inhibitors of the cytochrome bc(1) complex, an important membrane protein for drug and fungicide discovery. Compared with the original hit compound 4 (K(i) = 881.80 nM, porcine bc(1)), the most potent compound 4f displayed 20?507-fold improved binding affinity (K(i) = 43.00 pM). Compound 4f was proved to be a noncompetitive inhibitor with respect to the substrate cytochrome c, but a competitive inhibitor with respect to the substrate ubiquinol. Additionally, we determined the crystal structure of compound 4e (K(i) = 83.00 pM) bound to the chicken bc(1) at 2.70 ? resolution, providing a molecular basis for understanding its ultrapotency. To our knowledge, this study is the first application of the FBDD method in the discovery of picomolar inhibitors of a membrane protein. This work demonstrates that the novel PFVS approach is a high-throughput drug discovery method, independent of biophysical screening techniques.     

A novel methodology for the synthesis of 1-desoxy-Δ-tetrahydrocannabinol (THC) analogues

A versatile and efficient sequence was developed for the synthesis of 1-desoxy-Δ⁸-THC analogues and is demonstrated by the synthesis of sulfonamide analogues with an acetylene group at the C-2^′ position in the side chain. In this procedure the 1-desoxy-Δ⁸- THC ring structure is built first and the synthesis of the side chain is then developed.     

Design, synthesis and bioactivity of novel ALS inhibitors (V)

By means of the X-ray diffraction method, quantum pharmacology analysis and quantitative structure-activity relationships (QSAR) analysis, the structure-activity relationships of the herbicidal sulfonylureas and fused heterocyclic sulfonamides were systematically studied. The results showed that the structure-activity relationships of these two kinds of herbicides were identical and that the heterocyclic ring and sulfonyl moiety in the molecules were their pharmacophores which likely bind in the same receptor sites of acetolactate synthase (ALS). According to these results, the initial model of these two kinds of herbicides binding with the receptor was put forward. Project supported by the National Natural Science Foundation of China (Grant No.29472056).     

Toward the discovery of functional transthyretin amyloid inhibitors: application of virtual screening methods

Inhibition of amyloid fibril formation by stabilization of the native form of the protein transthyretin (TTR) is a viable approach for the treatment of familial amyloid polyneuropathy that has been gaining momentum in the field of amyloid research. The TTR stabilizer molecules discovered to date have shown efficacy at inhibiting fibrilization in vitro but display impairing issues of solubility, affinity for TTR in the blood plasma and/or adverse effects. In this study we present a benchmark of four protein- and ligand-based virtual screening (VS) methods for identifying novel TTR stabilizers: (i) two-dimensional (2D) similarity searches with chemical hashed, pharmacophore, and UNITY fingerprints, (ii) 3D searches based on shape, chemical, and electrostatic similarity, (iii) LigMatch, a new ligand-based method which uses multiple templates and combines 3D geometric hashing with a 2D preselection process, and (iv) molecular docking to consensus X-ray crystal structures of TTR. We illustrate the potential of the best-performing VS protocols to retrieve promising new leads by ranking a tailored library of 2.3 million commercially available compounds. Our predictions show that the top-scoring molecules possess distinctive features from the known TTR binders, holding better solubility, fraction of halogen atoms, and binding affinity profiles. To the best of our knowledge, this is the first attempt to rationalize the utilization of a large battery of in silico screening techniques toward the identification of a new generation of TTR amyloid inhibitors.     

Structure-based drug design: the discovery of novel nonpeptide orally active inhibitors of human renin

BACKGROUND: The aspartic proteinase renin plays an important physiological role in the regulation of blood pressure. It catalyses the first step in the conversion of angiotensinogen to the hormone angiotensin II. In the past, potent peptide inhibitors of renin have been developed, but none of these compounds has made it to the end of clinical trials. Our primary aim was to develop novel nonpeptide inhibitors. Based on the available structural information concerning renin-substrate interactions, we synthesized inhibitors in which the peptide portion was replaced by lipophilic moieties that interact with the large hydrophobic S1/S3-binding pocket in renin. RESULTS: Crystal structure analysis of renin-inhibitor complexes combined with computational methods were employed in the medicinal-chemistry optimisation process. Structure analysis revealed that the newly designed inhibitors bind as predicted to the S1/S3 pocket. In addition, however, these compounds interact with a hitherto unrecognised large, distinct, sub-pocket of the enzyme that extends from the S3-binding site towards the hydrophobic core of the enzyme. Binding to this S3(sp) sub-pocket was essential for high binding affinity. This unprecedented binding mode guided the drug-design process in which the mostly hydrophobic interactions within subsite S3(sp) were optimised. CONCLUSIONS: Our design approach led to compounds with high in vitro affinity and specificity for renin, favourable bioavailability and excellent oral efficacy in lowering blood pressure in primates. These renin inhibitors are therefore potential therapeutic agents for the treatment of hypertension and related cardiovascular diseases.     

Discovery of novel indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors by virtual screening

In this study, a combination of virtual screening methods were utilized to identify novel potential indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors. A series of IDO1 potential inhibitors were identified by a combination of following steps: Lipinski's Rule of Five, Veber rules filter, molecular docking, HipHop pharmacophores, 3D-Quantitative structure activity relationship (3D-QSAR) studies and Pan-assay Interference Compounds (PAINS) filter. Three known categories of IDO1 inhibitors were used to constructed pharmacophores and 3D-QSAR models. Four point pharmacophores (RHDA) of IDO1 inhibitors were generated from the training set. The 3D-QSAR models were obtained using partial least squares (PLS) analyze based on the docking conformation alignment from the training set. The leave-one-out correlation (q²) and non-cross-validated correlation coefficient (r²_pred) of the best CoMFA model were 0.601 and 0.546, and the ones from the best CoMSIA model were 0.506 and 0.541, respectively. Six hits from Specs database were identified and analyzed to confirm their binding modes and key interactions to the amino acid residues in the protein. This work may provide novel backbones for new generation of inhibitors of IDO1.     

Design, syntheses and biological activity of novel ALS inhibitors (IX)

Quantitative structure-activity relationships for herbicidal activity against rape of sulfonylurea and triazolopyrimidine-2-sulfonamide derivatives were examined three-dimensionally using comparative molecular field analysis (CoMFA). The CoMFA results show that the slopes in steric and electrostatic fields around the molecule were significant for both series in governing the potency variations in herbicidal activity. Based upon the successful superposition between the two series, the herbicidal activity was analyzable with a single equation for the combined set of compounds, which suggested that the two different series of compounds have a common region of the receptor site. Project supported by the National Natural Science Foundation of China (Grant No. 29832050), the Special Key Fund of Natural Science of Tianjin (Grant No. 983801011) and the Doctoral Fund of the Education Ministry of China.     

Synthesis and stability evaluation of novel peptidomimetic Caspase-1 inhibitors for topical application

During our search for topically-active Caspase-1 inhibitors, we identified a novel class of potent inhibitors based on a 1,3,5-trisubstituted uracil motif equipped with an l-aspartate semi-aldehyde derived warhead. In the literature, the majority of Caspase-1 inhibitors possessing the same warhead have been designed and evaluated for oral administration as the ethyl acetal pro-drug form. For our topical program, the pro-drug acetal form was not fully hydrolysed in the skin and was unstable in many of our standard topical excipients, therefore, we were obliged to focus on the actual hemiacetal drug form of the molecule during our drug discovery program. Our work focuses on both the synthesis and achiral and chiral stability of the final drug molecules in topical excipients.     

Synthesis of novel curcumin analogues and their evaluation as selective cyclooxygenase-1 (COX-1) inhibitors

Curcumin, a major yellow pigment and active component of turmeric, has been shown to possess anti-inflammatory and anti-cancer activities. Recent studies have indicated that cyclooxygenase-1 (COX-1) plays an important role in inflammation and carcinogenesis. In order to find more selective COX-1 inhibitors a series of novel curcumin derivatives was synthesized and evaluated for their ability to inhibit this enzyme using in vitro inhibition assays for COX-1 and COX-2 by measuring PGE(2) production. All curcumin analogues showed a higher rate of COX-1 inhibition. The most potent curcumin compounds were (1E,6E)-1,7-di-(2,3,4-trimethoxyphenyl)-1,6-heptadien-3,5-dione (4) (COX-1: IC(50) = 0.06 microM, COX-2: IC(50) > 100 microM, selectivity index>1666) and (1E,6E)-methyl 4-[7-(4-methoxycarbonyl)phenyl]-3,5-dioxo-1,6-heptadienyl]benzoate (6) (COX-1: IC(50) = 0.05 microM, COX-2: IC(50) > 100 microM, selectivity index > 2000). Curcumin analogues therefore represent a novel class of highly selective COX-1 inhibitors and promising candidates for in vivo studies.     

Developmental validation of the AGCU 21+1 STR kit: A novel multiplex assay for forensic application

In this study, we describe the developmental validation assay performed on a novel designed STR multiplex system, AGCU 21+1 STR kit. This kit contains a sex‐determining locus amelogenin and 21 noncombined DNA index system STR loci, that are, D6S474, D12ATA63, D22S1045, D10S1248, D1S1677, D11S4463, D1S1627, D3S4529, D2S441, D6S1017, D4S2408, D19S433, D17S1301, D1GATA113, D18S853, D20S482, D14S1434, D9S1122, D2S1776, D10S1435, and D5S2500. The 21+1 kit was validated by a series of tests including optimized PCR conditions, sensitivity, precision and accuracy, stutter ratio, DNA mixture, inhibitors, and species specificity according to the revised validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Our results in this study show that the kit is a useful tool for forensic application.     

A structure-based design approach for the identification of novel inhibitors: application to an alanine racemase

We report a new structure-based strategy for the identification of novel inhibitors. This approach has been applied to Bacillus stearothermophilus alanine racemase (AlaR), an enzyme implicated in the biosynthesis of the bacterial cell wall. The enzyme catalyzes the racemization of l- and d-alanine using pyridoxal 5-phosphate (PLP) as a cofactor. The restriction of AlaR to bacteria and some fungi and the absolute requirement for d-alanine in peptidoglycan biosynthesis make alanine racemase a suitable target for drug design. Unfortunately, known inhibitors of alanine racemase are not specific and inhibit the activity of other PLP-dependent enzymes, leading to neurological and other side effects.This article describes the development of a receptor-based pharmacophore model for AlaR, taking into account receptor flexibility (i.e. a `dynamic' pharmacophore model). In order to accomplish this, molecular dynamics (MD) simulations were performed on the full AlaR dimer from Bacillus stearothermophilus (PDB entry, 1sft) with a d-alanine molecule in one active site and the non-covalent inhibitor, propionate, in the second active site of this homodimer. The basic strategy followed in this study was to utilize conformations of the protein obtained during MD simulations to generate a dynamic pharmacophore model using the property mapping capability of the LigBuilder program. Compounds from the Available Chemicals Directory that fit the pharmacophore model were identified and have been submitted for experimental testing.The approach described here can be used as a valuable tool for the design of novel inhibitors of other biomolecular targets.     

First generation of pentazole (HN5, pentazolic acid), the final azole,and a zinc pentazolate salt in solution: A new N-dearylation of 1-(p-methoxyphenyl) pyrazoles,a 2-(p-methoxyphenyl) tetrazole and application of the methodology to 1-(p-methoxyphenyl) pentazole

Ceric ammonium nitrate (CAN) in methanol-water gave a new N-dearylation of a series of substituted 1-(p-methoxyphenyl) pyrazoles and a 2-(p-methoxyphenyl)tetrazole producing p-benzoquinone and the parent azole in a mole for mole ratio. Application of this reaction to 1-(p-methoxyphenyl) pentazole at -40 degrees C produced p-benzoquinone. 15N NMR spectra suggest that pentazole, HN5, was also produced and held in solution as N5- with Zn2+ ion. The 15N signal from N5- was -10.0 +/- 2.0 ppm in agreement with calculated values.     

Successful screening of large encoded combinatorial libraries leading to the discovery of novel p38 MAP kinase inhibitors

Screening of more than 2 million compounds comprising 41 distinct encoded combinatorial libraries revealed a novel structural class of p38 mitogen-activated protein (MAP) kinase inhibitors. The methodology used for screening large encoded combinatorial libraries combined with the statistical interpretation of screening results is described. A strong preference for a particular triaminotriazine aniline amide was discovered based on biological activity observed in the screening campaign. Additional screening of a focused follow-up combinatorial library yielded data expanding the unique combinatorial SAR and emphasizing an extraordinary preference for this particular building block and structural class. The preference is further highlighted when the p38 inhibitor data set is compared to data obtained for a panel of other kinases.     

Application of acyl cyanophosphorane methodology to the synthesis of protease inhibitors: poststatin, eurystatin, phebestin, probestin and bestatin

Full details are given for the syntheses of the protease inhibitors, poststatin and eurystatin by the acyl cyanophosphorane coupling procedure used for the formation of α-keto amides. We have also extended this methodology to the syntheses of the related α-hydroxy amide natural products, phebestin, probestin and bestatin. The key step in the latter synthetic sequences involved diastereomeric selectivity in the reduction of the α-keto precursor to the corresponding α-hydroxy amide by the use of zinc borohydride.     

Efficient synthetic methodology for 1-(phenylthio)cyclopropylsilanes,precursors of 1-(lithio)cyclopropylsilanes

Two one flask connective methods for preparing 1-(phenylthio)-cyclopropylsilanes, precursors of 1-(lithio)cyclopropylsilanes, are described.