Drug Repurposing and Polypharmacology to Fight SARS-CoV-2 Through Inhibition of the Main Protease.

The outbreak of a new coronavirus (SARS-CoV-2), which is responsible for the COVID-19 disease and is spreading rapidly around the world, urgently requires effective therapeutic treatments. In this context, drug repurposing represents a valuable strategy, as it enables accelerating the identification of drug candidates with already known safety profiles, possibly aiding in the late stages of clinical evaluation. Moreover, therapeutic treatments based on drugs with beneficial multi-target activities (polypharmacology) may show an increased antiviral activity or help to counteract severe complications concurrently affecting COVID-19 patients. In this study, we present the results of a computational drug repurposing campaign that aimed at identifying potential inhibitors of the main protease (Mpro) of the SARS-CoV-2. The performed in silico screening allowed the identification of 22 candidates with putative SARS-CoV-2 Mpro inhibitory activity. Interestingly, some of the identified compounds have recently entered clinical trials for COVID-19 treatment, albeit not being assayed for their SARS-CoV-2 antiviral activity. Some candidates present a polypharmacology profile that may be beneficial for COVID-19 treatment and, to the best of our knowledge, have never been considered in clinical trials. For each repurposed compound, its therapeutic relevance and potential beneficial polypharmacological effects that may arise due to its original therapeutic indication are thoroughly discussed.

Repurposing Known Drugs as Covalent and Non-covalent Inhibitors of the SARS-CoV-2 Papain-Like Protease.

In the absence of an approved vaccine, developing effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antivirals is essential to tackle the current pandemic health crisis due to the coronavirus disease 2019 (COVID-19) spread. As any traditional drug discovery program is a time-consuming and costly process requiring more than one decade to be completed, in silico repurposing of existing drugs is the preferred way for rapidly selecting promising clinical candidates. We present a virtual screening campaign to identify covalent and non-covalent inhibitors of the SARS-CoV-2 papain-like protease (PLpro) showing potential multitarget activities (i.e., a desirable polypharmacology profile) for the COVID-19 treatment. A dataset including 688 phase III and 1,702 phase IV clinical trial drugs was downloaded from ChEMBL (version 27.1) and docked to the recently released crystal structure of PLpro in complex with a covalently bound peptide inhibitor. The obtained results were analyzed by combining protein-ligand interaction fingerprint similarities, conventional docking scores, and MM-GBSA-binding free energies and allowed the identification of some interesting candidates for further in vitro testing. To the best of our knowledge, this study represents the first attempt to repurpose drugs for a covalent inhibition of PLpro and could pave the way for new therapeutic strategies against COVID-19.

Structure-Activity Relationships of Hexahydrocyclopenta[c]quinoline Derivatives as Allosteric Inhibitors of CDK2 and EGFR.

Following the discovery of a type III allosteric modulator of cyclin-dependent kinase 2 (CDK2) characterized by a hexahydrocyclopenta[c]quinolone scaffold, three different series of its derivatives were synthesized and biologically evaluated. Docking of the synthesized compounds into the allosteric pocket of CDK2 allowed the elucidation of structure-activity relationships (SARs). Moreover, the compounds were tested on the wild-type epidermal growth factor receptor (EGFR) kinase domain (KD) and its clinically relevant T790M/L858R mutant form. Herein we describe the first SAR investigation of allosteric ligands that bind to the type III inhibitor pocket of CDK2 and EGFR-KD. Although the activity of the synthesized inhibitors needs to be improved, the obtained results provide clear-cut indications about pharmacophore requirements and selectivity determinants. Remarkably, this study led to the identification of a selective T790M/L858R EGFR allosteric inhibitor that is inactive toward both wild-type EGFR and CDK2. Finally, docking into the T790M/L858R EGFR-KD led us to hypothesize that the compounds bind to the double-mutant EGFR-KD by adopting a binding mode different from that in CDK2, thus rationalizing the observed selectivity profile.

Selection of protein conformations for structure-based polypharmacology studies.

Several drugs exert their therapeutic effect through the modulation of multiple targets. Structure-based approaches hold great promise for identifying compounds with the desired polypharmacological profiles. These methods use knowledge of the protein binding sites to identify stereoelectronically complementary ligands. The selection of the most suitable protein conformations to be used in the design process is vital, especially for multitarget drug design in which the same ligand has to be accommodated in multiple binding pockets. Herein, we focus on currently available techniques for the selection of the most suitable protein conformations for multitarget drug design, compare the potential advantages and limitations of each method, and comment on how their combination could help in polypharmacology drug design.

Identification of small-molecule EGFR allosteric inhibitors by high-throughput docking.

AIM: The EGFR inhibitors represent the first-line treatment of non-small-cell lung cancer. However, the emergence of resistance urgently requires the development of new inhibitors targeting drug-resistant mutants. METHODOLOGY: A recently released structure of an EGFR kinase domain in complex with an allosteric inhibitor and a mutant protein model derived from it were used to set up a low-cost high-throughput docking protocol for the fast identification of EGFR allosteric inhibitors. CONCLUSION: The virtual screening of commercially available compounds led to the identification of interesting new hit compounds. The most promising hit was confirmed to be a new allosteric inhibitor of wild-type and T790M/L858R double mutant EGFR which was able to inhibit the growth of  non-small-cell lung cancer cell lines.

Contribution of the residue at position 4 within classical nuclear localization signals to modulating interaction with importins and nuclear targeting.

Nuclear import involves the recognition by importin (IMP) superfamily members of nuclear localization signals (NLSs) within protein cargoes destined for the nucleus, the best understood being recognition of classical NLSs (cNLSs) by the IMPα/ß1 heterodimer. Although the cNLS consensus [K-(K/R)-X-(K/R) for positions P2-P5] is generally accepted, recent studies indicated that the contribution made by different residues at the P4 position can vary. Here, we apply a combination of microscopy, molecular dynamics, crystallography, in vitro binding, and bioinformatics approaches to show that the nature of residues at P4 indeed modulates cNLS function in the context of a prototypical Simian Virus 40 large tumor antigen-derived cNLS (KKRK, P2-5). Indeed, all hydrophobic substitutions in place of R impaired binding to IMPα and nuclear targeting, with the largest effect exerted by a G residue at P4. Substitution of R with neutral hydrophobic residues caused the loss of electrostatic and van der Waals interactions between the P4 residue side chains and IMPα. Detailed bioinformatics analysis confirmed the importance of the P4 residue for cNLS function across the human proteome, with specific residues such as G being associated with low activity. Furthermore, we validate our findings for two additional cNLSs from human cytomegalovirus (HCMV) DNA polymerase catalytic subunit UL54 and processivity factor UL44, where a G residue at P4 results in a 2-3-fold decrease in NLS activity. Our results thus showed that the P4 residue makes a hitherto poorly appreciated contribution to nuclear import efficiency, which is essential to determining the precise nuclear levels of cargoes.

Probing an Allosteric Pocket of CDK2 with Small Molecules.

The availability of well-characterized allosteric modulators is crucial for investigating the allosteric regulation of protein function. In a recently identified inactive conformation of cyclin-dependent kinase 2 (CDK2), an open allosteric pocket was detected and proposed as a site to accommodate allosteric inhibitors. Previous structure-based approaches allowed the identification of a hit compound expected to bind to this pocket. Herein we report the characterization of this compound by X-ray crystallography, which surprisingly provided a chemical structure different from that previously reported. Therefore, the compound was synthesized and completely characterized. X-ray structures of the synthesized and purchased compounds were found to be superimposable. A reaction mechanism was proposed to explain the formation of the structure indicated by crystallography. Moreover, a stereoselective synthesis was developed to evaluate the biological activity of the pure stereoisomers. Modeling studies were performed to unveil the details of the interaction with CDK2. The activity of the obtained compounds was evaluated with various biological assays. Mutagenesis experiments confirmed binding to the allosteric pocket. Finally, the allosteric ligands were shown to inhibit the growth of lung (A549) and ovarian (SKOV3) cancer cell lines. Therefore, this report presents a thorough chemical and biological characterization of the first small-molecule ligands to be used as probes to study the allosteric modulation of CDK2 activity.

Exploiting computationally derived out-of-the-box protein conformations for drug design.

Structural plasticity is an intrinsic property of proteins that allows each gene product to accomplish its tasks in a strictly regulated manner at a precise time and cellular location. Moreover, protein motions allow protein-ligand and protein-protein recognition. The knowledge of the conformational ensemble that a drug target populates may be crucial for the design of small molecules that can differently modulate its function. X-ray crystallography and NMR have endlessly provided snapshots of protein states. However, experimental structure determination is not always straightforward. Therefore, attempts have been made to depict protein conformational landscapes through molecular dynamics and enhanced sampling methods. Here, we review how accounting for protein dynamics through in silico generated out-of-the-box protein conformations has started to impact on drug discovery.

Molecular Dynamics Simulations and Classical Multidimensional Scaling Unveil New Metastable States in the Conformational Landscape of CDK2.

Protein kinases are key regulatory nodes in cellular networks and their function has been shown to be intimately coupled with their structural flexibility. However, understanding the key structural mechanisms of large conformational transitions remains a difficult task. CDK2 is a crucial regulator of cell cycle. Its activity is finely tuned by Cyclin E/A and the catalytic segment phosphorylation, whereas its deregulation occurs in many types of cancer. ATP competitive inhibitors have failed to be approved for clinical use due to toxicity issues raised by a lack of selectivity. However, in the last few years type III allosteric inhibitors have emerged as an alternative strategy to selectively modulate CDK2 activity. In this study we have investigated the conformational variability of CDK2. A low dimensional conformational landscape of CDK2 was modeled using classical multidimensional scaling on a set of 255 crystal structures. Microsecond-scale plain and accelerated MD simulations were used to populate this landscape by using an out-of-sample extension of multidimensional scaling. CDK2 was simulated in the apo-form and in complex with the allosteric inhibitor 8-anilino-1-napthalenesulfonic acid (ANS). The apo-CDK2 landscape analysis showed a conformational equilibrium between an Src-like inactive conformation and an active-like form. These two states are separated by different metastable states that share hybrid structural features with both forms of the kinase. In contrast, the CDK2/ANS complex landscape is compatible with a conformational selection picture where the binding of ANS in proximity of the αC helix causes a population shift toward the inactive conformation. Interestingly, the new metastable states could enlarge the pool of candidate structures for the development of selective allosteric CDK2 inhibitors. The method here presented should not be limited to the CDK2 case but could be used to systematically unmask similar mechanisms throughout the human kinome.

Discovery of Multitarget Antivirals Acting on Both the Dengue Virus NS5-NS3 Interaction and the Host Src/Fyn Kinases.

This study describes the discovery of novel dengue virus inhibitors targeting both a crucial viral protein-protein interaction and an essential host cell factor as a strategy to reduce the emergence of drug resistance. Starting from known c-Src inhibitors, a virtual screening was performed to identify molecules able to interact with a recently discovered allosteric pocket on the dengue virus NS5 polymerase. The selection of cheap-to-produce scaffolds and the exploration of the biologically relevant chemical space around them suggested promising candidates for chemical synthesis. A series of purines emerged as the most interesting candidates able to inhibit virus replication at low micromolar concentrations with no significant toxicity to the host cell. Among the identified antivirals, compound 16i proved to be 10 times more potent than ribavirin, showed a better selectivity index and represents the first-in-class DENV-NS5 allosteric inhibitor able to target both the virus NS5-NS3 interaction and the host kinases c-Src/Fyn.

Structure-based design of small-molecule protein-protein interaction modulators: the story so far.

As the pivotal role of protein-protein interactions in cell growth, transcriptional activity, intracellular trafficking, signal transduction and pathological conditions has been assessed, experimental and in silico strategies have been developed to design protein-protein interaction modulators. State-of-the-art structure-based design methods, mainly pharmacophore modeling and docking, which have succeeded in the identification of enzyme inhibitors, receptor agonists and antagonists, and new tools specifically conceived to target protein-protein interfaces (e.g., hot-spot and druggable pocket prediction methods) have been applied in the search for small-molecule protein-protein interaction modulators. Many successful applications of structure-based design approaches that, despite the challenge of targeting protein-protein interfaces with small molecules, have led to the identification of micromolar and submicromolar hits are reviewed here.

Application of a post-docking procedure based on MM-PBSA and MM-GBSA on single and multiple protein conformations.

In the last decades, molecular docking has emerged as an increasingly useful tool in the modern drug discovery process, but it still needs to overcome many hurdles and limitations such as how to account for protein flexibility and poor scoring function performance. For this reason, it has been recognized that in many cases docking results need to be post-processed to achieve a significant agreement with experimental activities. In this study, we have evaluated the performance of MM-PBSA and MM-GBSA scoring functions, implemented in our post-docking procedure BEAR, in rescoring docking solutions. For the first time, the performance of this post-docking procedure has been evaluated on six different biological targets (namely estrogen receptor, thymidine kinase, factor Xa, adenosine deaminase, aldose reductase, and enoyl ACP reductase) by using i) both a single and a multiple protein conformation approach, and ii) two different software, namely AutoDock and LibDock. The assessment has been based on two of the most important criteria for the evaluation of docking methods, i.e., the ability of known ligands to enrich the top positions of a ranked database with respect to molecular decoys, and the consistency of the docking poses with crystallographic binding modes. We found that, in many cases, MM-PBSA and MM-GBSA are able to yield higher enrichment factors compared to those obtained with the docking scoring functions alone. However, for only a minority of the cases, the enrichment factors obtained by using multiple protein conformations were higher than those obtained by using only one protein conformation.

Structure-based design of potent aromatase inhibitors by high-throughput docking.

Cytochrome P450 aromatase catalyzes the conversion of androgen substrates into estrogens. Aromatase inhibitors (AIs) have been used as first-line drugs in the treatment of estrogen-dependent breast cancer in postmenopausal women. However, the search for new, more potent, and selective AIs still remains necessary to avoid the risk of possible resistances and reduce toxicity and side effects of current available drugs. The publication of a high resolution X-ray structure of human aromatase has opened the way to structure-based virtual screening to identify new small-molecule inhibitors with structural motifs different from all known AIs. In this context, a high-throughput docking protocol was set up and led to the identification of nanomolar AIs with new core structures.

CoCoCo: a free suite of multiconformational chemical databases for high-throughput virtual screening purposes.

In the last few decades, virtual screening has proved to be able to guide the selection of new hit compounds with predefined biological activity. However, the usage of these computational techniques is often associated with resource- and time-consuming preparation protocols. In this work we present Commercial Compound Collection (CoCoCo), a suite of free and ready-to-use chemical databases to help setting up in silico screening projects. CoCoCo collects molecular structural information of commercial compounds from various chemical vendors by merging them in a unique, non-redundant format. CoCoCo databases are prepared with transparent and straightforward routines based on state-of-the-art computational tools that introduce comprehensive structural information about tautomers, stereoisomers and conformational states of each compound. CoCoCo suite is especially conceived as a set of valuable tools that may help a wide range of researchers who wish to initiate their own project in the field of computational drug design. CoCoCo suite is available free of charge at the website .

A dynamic target-based pharmacophoric model mapping the CD4 binding site on HIV-1 gp120 to identify new inhibitors of gp120-CD4 protein-protein interactions.

A dynamic target-based pharmacophoric model mapping the CD4 binding site on HIV-1 gp120 was built and used to identify new hits able to inhibit gp120-CD4 protein-protein interactions. Two compounds showed micromolar inhibition of HIV-1 replication in cells attributable to an interference with the entry step of infection, by direct interaction with gp120. Inactivity of compounds toward a M475I strain suggested specific contacts with the Phe43 cavity of gp120.

1-[(3-Aryloxy-3-aryl)propyl]-1H-imidazoles, new imidazoles with potent activity against Candida albicans and dermatophytes. Synthesis, structure-activity relationship, and molecular modeling studies.

New 1-[(3-aryloxy-3-aryl)propyl]-1 H-imidazoles were synthesized and evaluated against Candida albicans and dermatophytes in order to develop structure-activity relationships (SARs). Against C. albicans the new imidazoles showed minimal inhibitory concentrations (MICs) comparable to those of ketoconazole, miconazole, and econazole, and were more potent than fluconazole. Several derivatives ( 10, 12, 14, 18- 20, 24, 28, 29, 30, and 34) turned out to be potent inhibitors of C. albicans strains resistant to fluconazole, with MIC values less than 10 microg/mL. Against dermatophytes strains, compounds 20, 25, and 33 (MIC

Modelling amphetamine/receptor interactions: a gas-phase study of complexes formed between amphetamine and Some chiral amido[4]resorcinarenes.

Diastereomeric proton-bound complexes formed between (R)- and (S)-amphetamine and some chiral amido[4]resorcinarene receptors display significant enantioselectivities when reacting with the enantiomers of 2-aminobutane in the gas phase. The origins of the measured enantioselectivities are discussed in the light of molecular mechanics calculations and molecular dynamics simulations and are ascribed to a combination of structural and dynamic factors, including the lengths and the isomeric structures of the host asymmetric pendants and the frequencies and amplitudes of their motion, as well as those of the proton-bonded amphetamine guests. The emerging picture may represent a starting point for deeper comprehension of the factors determining the different affinities of (R)- and (S)-amphetamine towards various chiral receptors, their selective binding to the monoamine transporters, and their sensitivity to specific inorganic ions.

Gas-phase enantioselectivity of chiral amido[4]resorcinarene receptors.

Diastereomeric proton-bound [1(L)HA]+ complexes between selected amino acids (A=phenylglycine (Phg), tryptophan (Trp), tyrosine methyl ester (TyrOMe), threonine (Thr), and allothreonine (AThr)) and a chiral amido[4]resorcinarene receptor (1(L)) display a significant enantioselectivity when undergoing loss of the amino acid guest A by way of the enantiomers of 2-aminobutanes (B) in the gas phase. The enantioselectivity of the B-to-A displacement is ascribed to a combination of thermodynamic and kinetic factors related to the structure and the stability of the diastereomeric [1(L)HA]+ complexes and of the reaction transition states. The results of the present and previous studies allow classification of the [1(L)HA]+ complexes in three main categories wherein: i) guest A does not present any additional functionalities besides the amino acid one (alanine (Ala), Phg, and phenylalanine (Phe)); ii) guest A presents an additional alcohol function (serine (Ser), Thr, and AThr); and iii) guest A contains several additional functionalities on its aromatic ring (tyrosine (Tyr), TyrOMe, Trp, and 3,4-dihydroxyphenylalanine (DOPA)). Each category exhibits a specific enantioselectivity depending upon the predominant [1(L)HA]+ structures and the orientation of the 2-aminobutane reactant in the relevant adducts observed. The results may contribute to the understanding of the exceptional selectivity and catalytic properties of enzyme mimics towards unsolvated biomolecules.

Antifungal agents. 11. N-substituted derivatives of 1-[(aryl)(4-aryl-1H-pyrrol-3-yl)methyl]-1H-imidazole: synthesis, anti-Candida activity, and QSAR studies.

1-[(Aryl)(4-aryl-1H-pyrrol-3-yl)methyl]-1H-imidazoles were recently reported by our group as potent anti-Candida agents belonging to the antifungal azole class. In the present paper the synthesis, anti-Candida activities, and QSAR studies on a novel series of N-substituted 1-[(aryl)(4-aryl-1H-pyrrol-3-yl)methyl]-1H-imidazole derivatives are reported. The newly synthesized azoles were tested against 12 strains of Candida albicans together with bifonazole, miconazole, itraconazole, fluconazole, and compounds 1a, 1b, 3a, 3b, and 3c used as reference drugs. In general, tested derivatives showed good antifungal activities, and the most potent compound was 1d (MIC(90) = 0.032 microg/mL), which was from 4- to 250-fold more potent than reference drugs. Catalyst software was applied to develop a quantitative pharmacophore model to be used for the rational design of new antifungal azoles. Some key interactions, as well as excluded volumes, further to the coordination bond of azole antifungals with the demethylase enzyme, are highlighted.