Virtual Fragment Screening Research Articles

Molecular Dynamics of Outer Membrane-Embedded Polysaccharide Secretion Porins Reveals Closed Resting-State Surface Gates Targetable by Virtual Fragment Screening for Drug Hotspot Identification.

Recent advances in iterative neural network analyses (e.g., AlphaFold2 and RoseTTA fold) have been revolutionary for protein 3D structure prediction, especially for difficult-to-manipulate α-helical/β-barrel integral membrane proteins. These model structures are calculated based on the coevolution of amino acids within the protein of interest and similarities to existing protein structures; the local effects of the membrane on folding and stability of the calculated model structures are not considered. We recently reported the discovery, 3D modeling, and characterization of 18-β-stranded outer-membrane (OM) WzpX, WzpS, and WzpB β-barrel secretion porins for the exopolysaccharide (EPS), major spore coat polysaccharide (MASC), and biosurfactant polysaccharide (BPS) pathways (respectively) in the Gram-negative social predatory bacterium Myxococcus xanthus DZ2. However, information was not obtained regarding the dynamic behavior of surface-gating WzpX/S/B loop domains or on potential treatments to inactivate these porins. Herein, we developed a molecular dynamics (MD) protocol to study the core stability and loop dynamism of neural network-based integral membrane protein structure models embedded in an asymmetric OM bilayer, using the M. xanthus WzpX, WzpS, and WzpB proteins as test candidates. This was accomplished through integration of the CHARMM-graphical user interface (GUI) and Molecular Operating Environment (MOE) workflows to allow for a rapid simulation system setup and facilitate data analysis. In addition to serving as a method of model structure validation, our molecular dynamics simulations revealed a minimal movement of extracellular WzpX/S/B loops in the absence of an external stimulus as well as druggable cavities between the loops. Virtual screening of a commercial fragment library against these cavities revealed putative fragment-binding hotspots on the cell-surface face of each β-barrel, along with key interacting residues, and identified promising hits for the design of potential binders capable of plugging the β-barrels and inhibiting polysaccharide secretion.

Structural studies identify angiotensin II receptor blocker-like compounds as branched-chain ketoacid dehydrogenase kinase inhibitors

The mammalian mitochondrial branched-chain ketoacid dehydrogenase (BCKD) complex is a multienzyme complex involved in the catabolism of branched-chain amino acids. BCKD is regulated by the BCKD kinase, or BCKDK, which binds to the E2 subunit of BCKD, phosphorylates its E1 subunit, and inhibits enzymatic activity. Inhibition of the BCKD complex results in increased levels of branched-chain amino acids and branched-chain ketoacids, and this buildup has been associated with heart failure, type 2 diabetes mellitus, and nonalcoholic fatty liver disease. To find BCKDK inhibitors for potential treatment of these diseases, we performed both NMR and virtual fragment screening and identified tetrazole-bearing fragments that bind BCKDK at multiple sites. Through structure-based virtual screening expanding from these fragments, the angiotensin receptor blocker class antihypertension drugs and angiotensin receptor blocker-like compounds were discovered to be potent BCKDK inhibitors, suggesting potential new avenues for heart failure treatment combining BCKDK inhibition and antihypertension.

Fragment-Based and Structural Investigation for Discovery of JNK3 Inhibitors.

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family and are related to cell proliferation, gene expression, and cell death. JNK isoform 3 (JNK3) is an important therapeutic target in varieties of pathological conditions including cancers and neuronal death. There is no approved drug targeting JNKs. To discover chemical inhibitors of JNK3, virtual fragment screening, the saturation transfer difference (STD) NMR, in vitro kinase assay, and X-ray crystallography were employed. A total of 27 fragments from the virtually selected 494 compounds were identified as initial hits via STD NMR and some compounds showed the inhibition of the activity of JNK3 in vitro. The structures of JNK3 with a fragment and a potent inhibitor were determined by X-ray crystallography. The fragment and inhibitor shared a common JNK3-binding feature. The result shows that fragment screening by NMR spectroscopy is a very efficient method to screen JNK3 binders and the structure of JNK3-inhibitor complex can be used to design and develop more potent inhibitors.

Identification of 2-(thiophen-2-yl)acetic Acid-Based Lead Compound for mPGES-1 Inhibition.

We report the implementation of our in silico/synthesis pipeline by targeting the glutathione-dependent enzyme mPGES-1, a valuable macromolecular target in both cancer therapy and inflammation therapy. Specifically, by using a virtual fragment screening approach of aromatic bromides, straightforwardly modifiable by the Suzuki-Miyaura reaction, we identified 3-phenylpropanoic acid and 2-(thiophen-2-yl)acetic acid to be suitable chemical platforms to develop tighter mPGES-1 inhibitors. Among these, compounds 1c and 2c showed selective inhibitory activity against mPGES-1 in the low micromolar range in accordance with molecular modeling calculations. Moreover, 1c and 2c exhibited interesting IC50 values on A549 cell lines compared to CAY10526, selected as reference compound. The most promising compound 2c induced the cycle arrest in the G0/G1 phase at 24 h of exposure, whereas at 48 and 72 h, it caused an increase of subG0/G1 fraction, suggesting an apoptosis/necrosis effect.

Mapping major SARS-CoV-2 drug targets and assessment of druggability using computational fragment screening: Identification of an allosteric small-molecule binding site on the Nsp13 helicase.

The 2019 emergence of, SARS-CoV-2 has tragically taken an immense toll on human life and far reaching impacts on society. There is a need to identify effective antivirals with diverse mechanisms of action in order to accelerate preclinical development. This study focused on five of the most established drug target proteins for direct acting small molecule antivirals: Nsp5 Main Protease, Nsp12 RNA-dependent RNA polymerase, Nsp13 Helicase, Nsp16 2’-O methyltransferase and the S2 subunit of the Spike protein. A workflow of solvent mapping and free energy calculations was used to identify and characterize favorable small-molecule binding sites for an aromatic pharmacophore (benzene). After identifying the most favorable sites, calculated ligand efficiencies were compared utilizing computational fragment screening. The most favorable sites overall were located on Nsp12 and Nsp16, whereas the most favorable sites for Nsp13 and S2 Spike had comparatively lower ligand efficiencies relative to Nsp12 and Nsp16. Utilizing fragment screening on numerous possible sites on Nsp13 helicase, we identified a favorable allosteric site on the N-terminal zinc binding domain (ZBD) that may be amenable to virtual or biophysical fragment screening efforts. Recent structural studies of the Nsp12:Nsp13 replication-transcription complex experimentally corroborates ligand binding at this site, which is revealed to be a functional Nsp8:Nsp13 protein-protein interaction site in the complex. Detailed structural analysis of Nsp13 ZBD conformations show the role of induced-fit flexibility in this ligand binding site and identify which conformational states are associated with efficient ligand binding. We hope that this map of over 200 possible small-molecule binding sites for these drug targets may be of use for ongoing discovery, design, and drug repurposing efforts. This information may be used to prioritize screening efforts or aid in the process of deciphering how a screening hit may bind to a specific target protein.

Identification of the 2-Benzoxazol-2-yl-phenol Scaffold as New Hit for JMJD3 Inhibition.

JMJD3 is a member of the KDM6 subfamily and catalyzes the demethylation of lysine 27 on histone H3 (H3K27). This protein was identified as a useful tool in understanding the role of epigenetics in inflammatory conditions and in cancer as well. Guided by a virtual fragment screening approach, we identified the benzoxazole scaffold as a new hit suitable for the development of tighter JMJD3 inhibitors. Compounds were synthesized by a microwave-assisted one-pot reaction under catalyst and solvent-free conditions. Among these, compound 8 presented the highest inhibitory activity (IC50 = 1.22 ± 0.22 μM) in accordance with molecular modeling calculations. Moreover, 8 induced the cycle arrest in S-phase on A375 melanoma cells.

Structure-based exploration and pharmacological evaluation of N-substituted piperidin-4-yl-methanamine CXCR4 chemokine receptor antagonists

Using the available structural information of the chemokine receptor CXCR4, we present hit finding and hit exploration studies that make use of virtual fragment screening, design, synthesis and structure-activity relationship (SAR) studies. Fragment 2 was identified as virtual screening hit and used as a starting point for the exploration of 31 N-substituted piperidin-4-yl-methanamine derivatives to investigate and improve the interactions with the CXCR4 binding site. Additionally, subtle structural ligand changes lead to distinct interactions with CXCR4 resulting in a full to partial displacement of CXCL12 binding and competitive and/or non-competitive antagonism. Three-dimensional quantitative structure-activity relationship (3D-QSAR) and binding model studies were used to identify important hydrophobic interactions that determine binding affinity and indicate key ligand-receptor interactions.

Virtual Fragment Screening Identification of a Quinoline-5,8-dicarboxylic Acid Derivative as a Selective JMJD3 Inhibitor.

The quinoline‐5,8 dicarboxylic acid scaffold has been identified by a fragment‐based approach as new potential lead compound for the development of JMJD3 inhibitors. Among them, 3‐(2,4‐dimethoxypyrimidin‐5‐yl)quinoline‐5,8‐dicarboxylic acid (compound 3) shows low micromolar inhibitory activity against Jumonji domain‐containing protein 3 (JMJD3). The experimental evaluation of inhibitory activity against seven related isoforms of JMJD3 highlighted an unprecedented selectivity toward the biological target of interest.

Spiro[pyrrolidine-3,3'-oxindoles] and Their Indoline Analogues as New 5-HT6 Receptor Chemotypes.

Synthetic derivatives of spiro[pyrrolidinyl-3,3′-oxindole] alkaloids (coerulescine analogues) were investigated as new ligands for aminergic G-protein coupled receptors (GPCRs). The chemical starting point 2′-phenylspiro[indoline-3,3′-pyrrolidin]-2-one scaffold was identified by virtual fragment screening utilizing ligand- and structure based methods. As a part of the hit-to-lead optimization a structure-activity relationship analysis was performed to explore the differently substituted 2′-phenyl-derivatives, introducing the phenylsulphonyl pharmacophore and examining the corresponding reduced spiro[pyrrolidine-3,3′-indoline] scaffold. The optimization process led to ligands with submicromolar affinities towards the 5-HT6 receptor that might serve as viable leads for further optimization.

Design and synthesis of piperidine derivatives as novel human heat shock protein 70 inhibitors for the treatment of drug-resistant tumors

HSP70 is a potential target for tumour treatment. HSP70 plays significant roles in several biological processes, including the regulation of apoptosis. In this study, piperidine derivatives were designed as novel HSP70 inhibitors based on virtual fragment screening performed in Dock 4.0, Discovery Studio 2.5 and SYBYL 6.9. A total of 67 novel piperidine derivatives were synthesized. Cell viability assays were performed in 16 cancer cell lines. The emphasis was placed on lapatinib-resistant breast cancer cells (BT/LapR1.0, MDA-MB-361, SK/LapR1.0, and MDA-MB-453). The compounds HSP70-36/37/40/43/46 significantly inhibited the proliferation of human breast cancer cells. Compound HSP70-36 inhibited the growth of BT474 and BT/LapR1.0 cells with IC50 values of 1.41 μM and 1.47 μM, respectively. The binding affinity of HSP70-36/HSP70 was evaluated by surface plasmon resonance and yielded Kd values of 2.46 μM. The LD50 was 869.0 mgkg−1. These data suggest that HSP70-36 may be a potential candidate compound for tumour treatment.

A combination of in silico and SAR studies to identify binding hot spots of Bcl-xL inhibitors

Inhibition of Bcl-2 family protein–protein interactions (PPI) is a very promising direction in cancer chemotherapy. Hence over the last decade, many medicinal chemistry studies endeavoured to discover drug candidates, and a wealth of chemical scaffolds with striking chemical diversity was reported as Bcl-xL inhibitors. This raises the question of whether all these molecules could occupy a unique binding site, or rather discrete pockets of the protein surface.To test if small and chemically diverse Bcl-xL inhibitors are likely to bind a single pocket, and to identify which pocket, we used a battery of computational and modeling approaches. We first checked that the large dataset of Bcl-xL inhibitors we built can actually fit to a universal pharmacophore. Then we defined the probable binding hot spots of interaction through comparison of crystal structures, as well as virtual fragment screening. Finally, new analogues of small polyphenol derivatives were synthesized to precisely probe a hydrogen bond suggested by docking experiments. Bcl-xL inhibition potency of these products confirmed the predicted binding mode.This combination of X-ray structure exploration, molecular modeling studies and medicinal chemistry supports that all these small Bcl-xL inhibitors occupy the same hot spot of interaction. The identification of this binding site should help the design and optimization of small PPI Bcl-xL inhibitors.

Cell-based and virtual fragment screening for adrenergic α2C receptor agonists

Fragment-based drug discovery has emerged as an alternative to conventional lead identification and optimization strategies generally supported by biophysical detection techniques. Membrane targets like G protein-coupled receptors (GPCRs), however, offer challenges in lack of generic immobilization or stabilization methods for the dynamic, membrane-bound supramolecular complexes. Also modeling of different functional states of GPCRs proved to be a challenging task. Here we report a functional cell-based high concentration screening campaign for the identification of adrenergic α2C receptor agonists compared with the virtual screening of the same ligand set against an active-like homology model of the α2C receptor. The conventional calcium mobilization-based assay identified active fragments with a similar incidence to several other reported fragment screens on GPCRs. 16 out of 3071 screened fragments turned out as specific ligands of α2C, two of which were identified by virtual screening as well and several of the hits possessed surprisingly high affinity and ligand efficiency. Our results indicate that in vitro biological assays can be utilized in the fragment hit identification process for GPCR targets.

Virtual fragment screening on GPCRs: A case study on dopamine D3 and histamine H4 receptors

Prospective structure based virtual fragment screening methodologies on two GPCR targets namely the dopamine D3 and the histamine H4 receptors with a library of 12,905 fragments were evaluated. Fragments were docked to the X-ray structure and the homology model of the D3 and H4 receptors, respectively. Representative receptor conformations for ensemble docking were obtained from molecular dynamics trajectories. In vitro confirmed hit rates ranged from 16% to 32%. Hits had high ligand efficiency (LE) values in the range of 0.31–0.74 and also acceptable lipophilic efficiency. The X-ray structure, the homology model and structural ensembles were all found suitable for docking based virtual screening of fragments against these GPCRs. However, there was little overlap among different hit sets and methodologies were thus complementary to each other.

Virtual Fragment Screening: Discovery of Histamine H3 Receptor Ligands Using Ligand-Based and Protein-Based Molecular Fingerprints

Virtual fragment screening (VFS) is a promising new method that uses computer models to identify small, fragment-like biologically active molecules as useful starting points for fragment-based drug discovery (FBDD). Training sets of true active and inactive fragment-like molecules to construct and validate target customized VFS methods are however lacking. We have for the first time explored the possibilities and challenges of VFS using molecular fingerprints derived from a unique set of fragment affinity data for the histamine H(3) receptor (H(3)R), a pharmaceutically relevant G protein-coupled receptor (GPCR). Optimized FLAP (Fingerprints of Ligands and Proteins) models containing essential molecular interaction fields that discriminate known H(3)R binders from inactive molecules were successfully used for the identification of new H(3)R ligands. Prospective virtual screening of 156,090 molecules yielded a high hit rate of 62% (18 of the 29 tested) experimentally confirmed novel fragment-like H(3)R ligands that offer new potential starting points for the design of H(3)R targeting drugs. The first construction and application of customized FLAP models for the discovery of fragment-like biologically active molecules demonstrates that VFS is an efficient way to explore protein-fragment interaction space in silico.

Virtual fragment screening: exploration of MM-PBSA re-scoring

An NMR fragment screening dataset with known binders and decoys was used to evaluate the ability of docking and re-scoring methods to identify fragment binders. Re-scoring docked poses using the Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) implicit solvent model identifies additional active fragments relative to either docking or random fragment screening alone. Early enrichment, which is clearly most important in practice for selecting relatively small sets of compounds for experimental testing, is improved by MM-PBSA re-scoring. In addition, the value in MM-PBSA re-scoring of docked poses for virtual screening may be in lessening the effect of the variation in the protein complex structure used.

Computational fragment-based screening using RosettaLigand: the SAMPL3 challenge

SAMPL3 fragment based virtual screening challenge provides a valuable opportunity for researchers to test their programs, methods and screening protocols in a blind testing environment. We participated in SAMPL3 challenge and evaluated our virtual fragment screening protocol, which involves RosettaLigand as the core component by screening a 500 fragments Maybridge library against bovine pancreatic trypsin. Our study reaffirmed that the real test for any virtual screening approach would be in a blind testing environment. The analyses presented in this paper also showed that virtual screening performance can be improved, if a set of known active compounds is available and parameters and methods that yield better enrichment are selected. Our study also highlighted that to achieve accurate orientation and conformation of ligands within a binding site, selecting an appropriate method to calculate partial charges is important. Another finding is that using multiple receptor ensembles in docking does not always yield better enrichment than individual receptors. On the basis of our results and retrospective analyses from SAMPL3 fragment screening challenge we anticipate that chances of success in a fragment screening process could be increased significantly with careful selection of receptor structures, protein flexibility, sufficient conformational sampling within binding pocket and accurate assignment of ligand and protein partial charges.

Hit clustering can improve virtual fragment screening: CDK2 and PARP1 case studies

Virtual fragment screening could be a promising alternative to existing experimental screening techniques. However, reliable methods of in silico fragment screening are yet to be established and validated. In order to develop such an approach we first checked how successful the existing molecular docking methods can be in predicting fragment binding affinities and poses. Using our Lead Finder docking software the RMSD of the binding energy prediction was observed to be 1.35 kcal/mol(-1) on a set of 26 experimentally characterized fragment inhibitors, and the RMSD of the predicted binding pose from the experimental one was <1.5 Å. Then, we explored docking of 68 fragments obtained from 39 drug molecules for which co-crystal structures were available from the PDB. It appeared that fragments that participate in oriented non-covalent interactions, such as hydrogen bonds and metal coordination, could be correctly docked in 70-80% of cases suggesting the potential success of rediscovering of corresponding drugs by in silico fragment approach. Based on these findings we've developed a virtual fragment screening technique which involved structural filtration of protein-ligand complexes for specific interactions and subsequent clustering in order to minimize the number of preferable starting fragment candidates. Application of this method led to 2 millimolar-scale fragment PARP1 inhibitors with a new scaffold.

Crystal Structure-Based Virtual Screening for Fragment-like Ligands of the Human Histamine H1 Receptor

The recent crystal structure determinations of druggable class A G protein-coupled receptors (GPCRs) have opened up excellent opportunities in structure-based ligand discovery for this pharmaceutically important protein family. We have developed and validated a customized structure-based virtual fragment screening protocol against the recently determined human histamine H(1) receptor (H(1)R) crystal structure. The method combines molecular docking simulations with a protein-ligand interaction fingerprint (IFP) scoring method. The optimized in silico screening approach was successfully applied to identify a chemically diverse set of novel fragment-like (≤22 heavy atoms) H(1)R ligands with an exceptionally high hit rate of 73%. Of the 26 tested fragments, 19 compounds had affinities ranging from 10 μM to 6 nM. The current study shows the potential of in silico screening against GPCR crystal structures to explore novel, fragment-like GPCR ligand space.

Virtual fragment screening for novel inhibitors of 6-phosphogluconate dehydrogenase

The enzyme 6-phosphogluconate dehydrogenase is a potential drug target for the parasitic protozoan Trypanosoma brucei, the causative organism of human African trypanosomiasis. This enzyme has a polar active site to accommodate the phosphate, hydroxyl and carboxylate groups of the substrate, 6-phosphogluconate. A virtual fragment screen was undertaken of the enzyme to discover starting points for the development of inhibitors which are likely to have appropriate physicochemical properties for an orally bioavailable compound. A virtual screening library was developed, consisting of compounds with functional groups that could mimic the phosphate group of the substrate, but which have a higher pKa. Following docking, hits were clustered and appropriate compounds purchased and assayed against the enzyme. Three fragments were identified that had IC50 values in the low micromolar range and good ligand efficiencies. Based on these initial hits, analogues were procured and further active compounds were identified. Some of the fragments identified represent potential starting points for a medicinal chemistry programme to develop potent drug-like inhibitors of the enzyme.

Virtual Fragment Docking by Glide: a Validation Study on 190 Protein−Fragment Complexes

The docking accuracy of Glide was evaluated using 16 different docking protocols on 190 protein-fragment complexes representing 78 targets. Standard precision docking (Glide SP) based protocols showed the best performance. The average root-mean-square deviation (rmsd) between the docked and cocrystallized poses achieved by Glide SP with pre- and postprocessing was 1.17 A, and an acceptable binding mode with rmsd < 2 A could be found in 80% of the cases. Comparison of the docking results produced by different protocols suggests that the sampling efficacy of Glide is adequate for fragment docking. The docking accuracy seems to be limited by the performance of scoring schemes, which is supported by the weak correlation between experimental binding affinities and GlideScores. Cross-docking experiments performed on 8 targets represented by 63 complexes revealed that Glide SP gave similar results to that of the computationally more intensive Glide XP. The average rmsd achieved by Glide SP with pre- and postprocessing was 2.06 A, and an acceptable binding mode with rmsd < 2 A could be found in 63% of the cases. These cross-docking results were improved significantly selecting the optimal X-ray structure for each target (average rmsd = 1.3 A, success rate = 77%), indicating the importance of enrichment studies and the use of multiple X-ray structures in virtual fragment screening.