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 (Mpro) 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 Mpro inhibitors towards low nM affinities.

Enhancing Paraoxon Binding to Organophosphorus Hydrolase Active Site.

Organophosphorus hydrolase (OPH) is a metalloenzyme that can hydrolyze organophosphorus agents resulting in products that are generally of reduced toxicity. The best OPH substrate found to date is diethyl p-nitrophenyl phosphate (paraoxon). Most structural and kinetic studies assume that the binding orientation of paraoxon is identical to that of diethyl 4-methylbenzylphosphonate, which is the only substrate analog co-crystallized with OPH. In the current work, we used a combined docking and molecular dynamics (MD) approach to predict the likely binding mode of paraoxon. Then, we used the predicted binding mode to run MD simulations on the wild type (WT) OPH complexed with paraoxon, and OPH mutants complexed with paraoxon. Additionally, we identified three hot-spot residues (D253, H254, and I255) involved in the stability of the OPH active site. We then experimentally assayed single and double mutants involving these residues for paraoxon binding affinity. The binding free energy calculations and the experimental kinetics of the reactions between each OPH mutant and paraoxon show that mutated forms D253E, D253E-H254R, and D253E-I255G exhibit enhanced substrate binding affinity over WT OPH. Interestingly, our experimental results show that the substrate binding affinity of the double mutant D253E-H254R increased by 19-fold compared to WT OPH.

Temperature artifacts in protein structures bias ligand-binding predictions.

X-ray crystallography is the gold standard to resolve conformational ensembles that are significant for protein function, ligand discovery, and computational methods development. However, relevant conformational states may be missed at common cryogenic (cryo) data-collection temperatures but can be populated at room temperature. To assess the impact of temperature on making structural and computational discoveries, we systematically investigated protein conformational changes in response to temperature and ligand binding in a structural and computational workhorse, the T4 lysozyme L99A cavity. Despite decades of work on this protein, shifting to RT reveals new global and local structural changes. These include uncovering an apo helix conformation that is hidden at cryo but relevant for ligand binding, and altered side chain and ligand conformations. To evaluate the impact of temperature-induced protein and ligand changes on the utility of structural information in computation, we evaluated how temperature can mislead computational methods that employ cryo structures for validation. We find that when comparing simulated structures just to experimental cryo structures, hidden successes and failures often go unnoticed. When using structural information in ligand binding predictions, both coarse docking and rigorous binding free energy calculations are influenced by temperature effects. The trend that cryo artifacts limit the utility of structures for computation holds across five distinct protein classes. Our results suggest caution when consulting cryogenic structural data alone, as temperature artifacts can conceal errors and prevent successful computational predictions, which can mislead the development and application of computational methods in discovering bioactive molecules.

B-Cell Lymphoblastic Lymphoma in Relapse Presenting as a Uterine Mass: A Case Report and Review of Literature.

B-cell lymphoblastic lymphoma (LBL) is a highly aggressive malignant proliferation of lymphoblasts of B-origin grouped with acute lymphoblastic leukemia. Multiple studies demonstrated the various sites of involvement in adult LBL. The involvement of the uterus as a site of relapse for such disease is rare. We herein report the case of relapsed B-cell LBL mimicking endometrial sarcoma. The patient is a 56-year-old female patient known to have B-cell LBL on chemotherapy. She presented with abdominal pain and fever. Positron emission tomodensitometry-computed tomography showed the presence of a uterine mass with bilateral iliac lymph node involvement. She underwent surgery with mass removal and pathology showed relapsed B-cell LBL.

A 58-Year-Old Woman with Acute Gastric Perforation Due to Metastatic Ductal Carcinoma 18 Years Following Bilateral Mastectomy for Invasive Ductal Carcinoma of the Breast.

BACKGROUND Invasive lobular carcinoma and ductal carcinoma of the breast can metastasize to all sites in the body, including the gastrointestinal tract. Late presentation of metastases of lobular carcinoma of the breast to the gastrointestinal tract have previously been reported, but late metastasis of ductal carcinoma of the breast to the gastric mucosa is rare. This report is of a 58-year-old Lebanese woman who presented with acute gastric perforation due to metastatic ductal carcinoma,18 years following bilateral mastectomy for invasive ductal carcinoma of the breast. CASE REPORT We present the case of a 58-year-old woman who underwent a right modified mastectomy for an invasive ductal carcinoma in 2002 combined with a contralateral prophylactic mastectomy for cosmetic purposes. She presented a secondary gastric lesion 18 years later. The clinical presentation resembled perforated ulcer. The choice of gastrectomy was denied due to retrogastric and pancreatic invasion by the tumor. A laparoscopic gastric closure failed to heal the perforation. A supraumbilical laparotomy incision was performed for the placement of a Pezzer tube in the gastric perforation and the installation of a feeding jejunostomy. CONCLUSIONS This report is of a rare presentation of metastatic ductal carcinoma of the breast to the gastric mucosa associated with gastric perforation that presented 18 years after bilateral mastectomy. This case highlights the importance of obtaining a full past medical history to identify previous primary malignancy, and also is a reminder that ductal carcinoma of the breast can present with metastatic involvement in the gastrointestinal tract several months, or even years, following mastectomy.

SAMPL7 Host-Guest Challenge Overview: assessing the reliability of polarizable and non-polarizable methods for binding free energy calculations.

The SAMPL challenges focus on testing and driving progress of computational methods to help guide pharmaceutical drug discovery. However, assessment of methods for predicting binding affinities is often hampered by computational challenges such as conformational sampling, protonation state uncertainties, variation in test sets selected, and even lack of high quality experimental data. SAMPL blind challenges have thus frequently included a component focusing on host-guest binding, which removes some of these challenges while still focusing on molecular recognition. Here, we report on the results of the SAMPL7 blind prediction challenge for host-guest affinity prediction. In this study, we focused on three different host-guest categories-a familiar deep cavity cavitand series which has been featured in several prior challenges (where we examine binding of a series of guests to two hosts), a new series of cyclodextrin derivatives which are monofunctionalized around the rim to add amino acid-like functionality (where we examine binding of two guests to a series of hosts), and binding of a series of guests to a new acyclic TrimerTrip host which is related to previous cucurbituril hosts. Many predictions used methods based on molecular simulations, and overall success was mixed, though several methods stood out. As in SAMPL6, we find that one strategy for achieving reasonable accuracy here was to make empirical corrections to binding predictions based on previous data for host categories which have been studied well before, though this can be of limited value when new systems are included. Additionally, we found that alchemical free energy methods using the AMOEBA polarizable force field had considerable success for the two host categories in which they participated. The new TrimerTrip system was also found to introduce some sampling problems, because multiple conformations may be relevant to binding and interconvert only slowly. Overall, results in this challenge tentatively suggest that further investigation of polarizable force fields for these challenges may be warranted.

Quantum-Chemistry Based Design of Halobenzene Derivatives With Augmented Affinities for the HIV-1 Viral G4/C16 Base-Pair.

The HIV-1 integrase (IN) is a major target for the design of novel anti-HIV inhibitors. Among these, three inhibitors which embody a halobenzene ring derivative (HR) in their structures are presently used in clinics. High-resolution X-ray crystallography of the complexes of the IN-viral DNA transient complex bound to each of the three inhibitors showed in all cases the HR ring to interact within a confined zone of the viral DNA, limited to the highly conserved 5'CpA 3'/5'TpG 3' step. The extension of its extracyclic CX bond is electron-depleted, owing to the existence of the "sigma-hole." It interacts favorably with the electron-rich rings of base G4. We have sought to increase the affinity of HR derivatives for the G4/C16 base pair. We thus designed thirteen novel derivatives and computed their Quantum Chemistry (QC) intermolecular interaction energies (ΔE) with this base-pair. Most compounds had ΔE values significantly more favorable than those of the HR of the most potent halobenzene drug presently used in clinics, Dolutegravir. This should enable the improvement in a modular piece-wise fashion, the affinities of halogenated inhibitors for viral DNA (vDNA). In view of large scale polarizable molecular dynamics simulations on the entirety of the IN-vDNA-inhibitor complexes, validations of the SIBFA polarizable method are also reported, in which the evolution of each ΔE(SIBFA) contribution is compared to its QC counterpart along this series of derivatives.

Pituitary Adenoma and Papillary Craniopharyngioma: A Rare Case of Collision Tumor and Review of the Literature.

BACKGROUND: Pituitary adenomas are the most common lesion of the sellar region. Very few cases in the literature have described their association with craniopharyngiomas in the same anatomic compartment, an entity defined as collision tumors of the sella. CASE DESCRIPTION: A 35-year-old man presented with headaches and progressive visual disturbances. Radiographic imaging initially highlighted the presence of a pituitary craniopharyngioma. An endoscopic transsphenoidal pituitary approach was performed, during which the tumor was partially resected. The pathology report was positive for 2 entities: a nonfunctioning pituitary adenoma and a papillary craniopharyngioma. This was an unexpected diagnosis based on the surgical and initial radiologic findings. CONCLUSIONS: To our knowledge, this is the first documented case of a collision tumor of the sella comprising a pituitary adenoma and a craniopharyngioma of the papillary type.

Reconciling NMR Structures of the HIV-1 Nucleocapsid Protein NCp7 Using Extensive Polarizable Force Field Free-Energy Simulations.

Using polarizable (AMOEBA) and nonpolarizable (CHARMM) force fields, we compare the relative free energy stability of two extreme conformations of the HIV-1 nucleocapsid protein NCp7 that had been previously experimentally advocated to prevail in solution. Using accelerated sampling techniques, we show that they differ in stability by no more than 0.75-1.9 kcal/mol depending on the reference protein sequence. While the extended form appears to be the most probable structure, both forms should thus coexist in water explaining the differing NMR findings.

D3R Grand Challenge 4: ligand similarity and MM-GBSA-based pose prediction and affinity ranking for BACE-1 inhibitors.

The Drug Design Data Resource (D3R) Grand Challenges present an opportunity to assess, in the context of a blind predictive challenge, the accuracy and the limits of tools and methodologies designed to help guide pharmaceutical drug discovery projects. Here, we report the results of our participation in the D3R Grand Challenge 4 (GC4), which focused on predicting the binding poses and affinity ranking for compounds targeting the [Formula: see text]-amyloid precursor protein (BACE-1). Our ligand similarity-based protocol using HYBRID (OpenEye Scientific Software) successfully identified poses close to the native binding mode for most of the ligands with less than 2 Å RMSD accuracy. Furthermore, we compared the performance of our HYBRID-based approach to that of AutoDock Vina and DOCK 6 and found that using a reference ligand to guide the docking process is a better strategy for pose prediction and helped HYBRID to perform better here. We also conducted end-point free energy estimates on molecules dynamics based ensembles of protein-ligand complexes using molecular mechanics combined with generalized Born surface area method (MM-GBSA). We found that the binding affinity ranking based on MM-GBSA scores have poor correlation with the experimental values. Finally, the main lessons from our participation in D3R GC4 are: (i) the generation of the macrocyclic conformers is a key step for successful pose prediction, (ii) the protonation states of the BACE-1 binding site should be treated carefully, (iii) the MM-GBSA method could not discriminate well between different predicted binding poses, and (iv) the MM-GBSA method does not perform well at predicting protein-ligand binding affinities here.

Comparison of affinity ranking using AutoDock-GPU and MM-GBSA scores for BACE-1 inhibitors in the D3R Grand Challenge 4.

Molecular docking has been successfully used in computer-aided molecular design projects for the identification of ligand poses within protein binding sites. However, relying on docking scores to rank different ligands with respect to their experimental affinities might not be sufficient. It is believed that the binding scores calculated using molecular mechanics combined with the Poisson-Boltzman surface area (MM-PBSA) or generalized Born surface area (MM-GBSA) can predict binding affinities more accurately. In this perspective, we decided to take part in Stage 2 of the Drug Design Data Resource (D3R) Grand Challenge 4 (GC4) to compare the performance of a quick scoring function, AutoDock4, to that of MM-GBSA in predicting the binding affinities of a set of [Formula: see text]-Amyloid Cleaving Enzyme 1 (BACE-1) ligands. Our results show that re-scoring docking poses using MM-GBSA did not improve the correlation with experimental affinities. We further did a retrospective analysis of the results and found that our MM-GBSA protocol is sensitive to details in the protein-ligand system: (i) neutral ligands are more adapted to MM-GBSA calculations than charged ligands, (ii) predicted binding affinities depend on the initial conformation of the BACE-1 receptor, (iii) protonating the aspartyl dyad of BACE-1 correctly results in more accurate binding affinity predictions.

Calibration of 1,2,4-Triazole-3-Thione, an Original Zn-Binding Group of Metallo-ß-Lactamase Inhibitors. Validation of a Polarizable MM/MD Potential by Quantum Chemistry.

In the context of the SIBFA polarizable molecular mechanics/dynamics (PMM/PMD) procedure, we report the calibration and a series of validation tests for the 1,2,4-triazole-3-thione (TZT) heterocycle. TZT acts as the chelating group of inhibitors of dizinc metallo-ß-lactamases (MBL), an emerging class of Zn-dependent bacterial enzymes, which by cleaving the ß-lactam bond of most ß-lactam antibiotics are responsible for the acquired resistance of bacteria to these drugs. Such a study is indispensable prior to performing PMD simulations of complexes of TZT-based inhibitors with MBL's, on account of the anchoring role of TZT in the dizinc MBL recognition site. Calibration was done by comparisons to energy decomposition analyses (EDA) of high-level ab initio QC computations of the TZT complexes with two probes: Zn(II), representative of "soft" dications, and water, representative of dipolar molecules. We performed distance variations of the approach of each probe to each of the two TZT atoms involved in Zn ligation, the S atom and the N atom ortho to it, so that each SIBFA contribution matches its QC counterpart. Validations were obtained by performing in- and out-of-plane angular variations of Zn(II) binding in monoligated Zn(II)-TZT complexes. The most demanding part of this study was then addressed. How well does ΔE(SIBFA) and its individual contributions compare to their QC counterparts in the dizinc binding site of one MBL, L1, whose structure is known from high-resolution X-ray crystallography? Six distinct complexes were considered, namely each separate monozinc site, and the dizinc site, whether ligated or unligated by TZT. Despite the large magnitude of the interaction energies, in all six complexes ΔE(SIBFA) can match ΔE(QC) with relative errors <2% and the proper balance of individual energy contributions. The computations were extended to the dizinc site of another MBL, VIM-2, and its complexes with two other TZT analogues. ΔE(SIBFA) faithfully reproduced ΔE(QC) in terms of magnitude, ranking of the three ligands, and trends of the separate energy contributions. A preliminary extension to correlated calculations is finally presented. All these validations should enable a secure design of a diversity of TZT-containing MBL inhibitors: a structurally and energetically correct anchoring of TZT should enable all other inhibitor groups to in turn optimize their interactions with the other target MBL residues.

The inhibition process of HIV-1 integrase by diketoacids molecules: Understanding the factors governing the better efficiency of dolutegravir.

The Human Immunodeficiency Virus-1 integrase is responsible for the covalent insertion of a newly synthesized double-stranded viral DNA into the host cells, and is an emerging target for antivirus drug design. Raltegravir (RAL) and elvitegravir (EVG) are the first two integrase strand transfer inhibitors used in therapy. However, treated patients eventually develop detrimental resistance mutations. By contrast, a recently approved drug, dolutegravir (DTG), presents a high barrier to resistance. This study aims to understand the increased efficiency of DTG upon focusing on its interaction properties with viral DNA. The results showed DTG to be involved in more extended interactions with viral DNA than EVG. Such interactions involve the halobenzene and scaffold of DTG and EVG and bases 5'G-43', 3'A35'and 3'C45'.

Importance of explicit smeared lone-pairs in anisotropic polarizable molecular mechanics. Torture track angular tests for exchange-repulsion and charge transfer contributions.

A correct representation of the short-range contributions such as exchange-repulsion (Erep ) and charge-transfer (Ect ) is essential for the soundness of separable, anisotropic polarizable molecular mechanics potentials. Within the context of the SIBFA procedure, this is aimed at by explicit representations of lone pairs in their expressions. It is necessary to account for their anisotropic behaviors upon performing not only in-plane, but also out-of-plane, variations of a probe molecule or cation interacting with a target molecule or molecular fragment. Thus, Erep and Ect have to reproduce satisfactorily the corresponding anisotropies of their quantum chemical (QC) counterparts. A significant improvement of the out-of-plane dependencies was enabled when the sp2 and sp localized lone-pairs are, even though to a limited extent, delocalized on both sides of the plane, above and below the atom bearer but at the closely similar angles as the in-plane lone pair. We report calibration and validation tests on a series of monoligated complexes of a probe Zn(II) cation with several biochemically relevant ligands. Validations are then performed on several polyligated Zn(II) complexes found in the recognition sites of Zn-metalloproteins. Such calibrations and validations are extended to representative monoligated and polyligated complexes of Mg(II) and Ca(II). It is emphasized that the calibration of all three cations was for each ΔE contribution done on a small training set bearing on a limited number of representative N, O, and S monoligated complexes. Owing to the separable nature of ΔE, a secure transferability is enabled to a diversity of polyligated complexes. For these the relative errors with respect to the target ΔE(QC) values are generally < 3%. Overall, the article proposes a full set of benchmarks that could be useful for force field developers. © 2017 Wiley Periodicals, Inc.

Complexes of a Zn-metalloenzyme binding site with hydroxamate-containing ligands. A case for detailed benchmarkings of polarizable molecular mechanics/dynamics potentials when the experimental binding structure is unknown.

Zn-metalloproteins are a major class of targets for drug design. They constitute a demanding testing ground for polarizable molecular mechanics/dynamics aimed at extending the realm of quantum chemistry (QC) to very long-duration molecular dynamics (MD). The reliability of such procedures needs to be demonstrated upon comparing the relative stabilities of competing candidate complexes of inhibitors with the recognition site stabilized in the course of MD. This could be necessary when no information is available regarding the experimental structure of the inhibitor-protein complex. Thus, this study bears on the phosphomannose isomerase (PMI) enzyme, considered as a potential therapeutic target for the treatment of several bacterial and parasitic diseases. We consider its complexes with 5-phospho-d-arabinonohydroxamate and three analog ligands differing by the number and location of their hydroxyl groups. We evaluate the energy accuracy expectable from a polarizable molecular mechanics procedure, SIBFA. This is done by comparisons with ab initio quantum-chemistry (QC) calculations in the following cases: (a) the complexes of the four ligands in three distinct structures extracted from the entire PMI-ligand energy-minimized structures, and totaling up to 264 atoms; (b) the solvation energies of several energy-minimized complexes of each ligand with a shell of 64 water molecules; (c) the conformational energy differences of each ligand in different conformations characterized in the course of energy-minimizations; and (d) the continuum solvation energies of the ligands in different conformations. The agreements with the QC results appear convincing. On these bases, we discuss the prospects of applying the procedure to ligand-macromolecule recognition problems. © 2016 Wiley Periodicals, Inc.