Applying in silico Tools to the Discovery of Novel CXCR4 Inhibitors

International audienceThe process of HIV entry begins with the binding of the viral envelope glycoprotein gp120 to both the CD4 receptor and one of the CXCR4 or CCR5 chemokine coreceptors. There is currently considerable interest in developing novel ligands which can bind to these coreceptors and hence block virus-cell fusion. This article reviews the use of different in silico structure-based and ligand-based virtual screening (VS) tools for the discovery of potential HIV entry inhibitors for the CXCR4 receptor. More specifically, it discusses homology modelling, de novo design, docking, QSAR analyses, pharmacophore modelling, and similarity searches. Results from retrospective VS of a library of known CXCR4 inhibitors taken from the literature and from prospective VS of a combinatorial virtual library are reviewed. The structures of active compounds found by these approaches, as well as CXCR4 inhibitors currently in development are also discussed

An Agonist of the CXCR4 Receptor Strongly Promotes Regeneration of Degenerated Motor Axon Terminals

The activation of the G-protein coupled receptor CXCR4 by its ligand CXCL12\u3b1 is involved in a large variety of physiological and pathological processes, including the growth of B cells precursors and of motor axons, autoimmune diseases, stem cell migration, inflammation, and several neurodegenerative conditions. Recently, we demonstrated that CXCL12\u3b1 potently stimulates the functional recovery of damaged neuromuscular junctions via interaction with CXCR4. This result prompted us to test the neuroregeneration activity of small molecules acting as CXCR4 agonists, endowed with better pharmacokinetics with respect to the natural ligand. We focused on NUCC-390, recently shown to activate CXCR4 in a cellular system. We designed a novel and convenient chemical synthesis of NUCC-390, which is reported here. NUCC-390 was tested for its capability to induce the regeneration of motor axon terminals completely degenerated by the presynaptic neurotoxin \u3b1-Latrotoxin. NUCC-390 was found to strongly promote the functional recovery of the neuromuscular junction, as assayed by electrophysiology and imaging. This action is CXCR4 dependent, as it is completely prevented by AMD3100, a well-characterized CXCR4 antagonist. These data make NUCC-390 a strong candidate to be tested in human therapy to promote nerve recovery of function after different forms of neurodegeneratio

Modulators of CXCR4 and CXCR7/ACKR3 Function

Copyright © 2019 by The Author(s). The two G protein-coupled receptors (GPCRs) C-X-C chemokine receptor type 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) are part of the class A chemokine GPCR family and represent important drug targets for human immunodeficiency virus (HIV) infection, cancer, and inflammation diseases. CXCR4 is one of only three chemokine receptors with a US Food and Drug Administration approved therapeutic agent, the small-molecule modulator AMD3100. In this review, known modulators of the two receptors are discussed in detail. Initially, the structural relationship between receptors and ligands is reviewed on the basis of common structural motifs and available crystal structures. To date, no atypical chemokine receptor has been crystallized, which makes ligand design and predictions for these receptors more difficult. Next, the selectivity, receptor activation, and the resulting ligand-induced signaling output of chemokines and other peptide ligands are reviewed. Binding of pepducins, a class of lipid-peptides whose basis is the internal loop of a GPCR, to CXCR4 is also discussed. Finally, small-molecule modulators of CXCR4 and ACKR3 are reviewed. These modulators have led to the development of radio- and fluorescently labeled tool compounds, enabling the visualization of ligand binding and receptor characterization both in vitro and in vivo. SIGNIFICANCE STATEMENT: To investigate the pharmacological modulation of CXCR4 and ACKR3, significant effort has been focused on the discovery and development of a range of ligands, including small-molecule modulators, pepducins, and synthetic peptides. Imaging tools, such as fluorescent probes, also play a pivotal role in the field of drug discovery. This review aims to provide an overview of the aforementioned modulators that facilitate the study of CXCR4 and ACKR3 receptors

Molecular modeling studies on HIV-1 Reverse Transcriptase (RT) and Heat shock protein (Hsp) 90 as a potential anti-HIV-1 target.

Masters Degree. University of KwaZulu-Natal, Durban.Human immunodeficiency virus (HIV) infection is the leading cause of death globally. This dissertation addresses two HIV-1 target proteins namely, HIV-1 Reverse Transcriptase (RT) and Heat shock protein (Hsp) 90. More specifically for HIV-1 RT, a case study for the identification of potential inhibitors as anti-HIV agents was carried out. A more refined virtual screening (VS) approach was implemented, which was an improvement on work previously published by our group- “target-bound pharmacophore modeling approach”. This study generated a pharmacophore library based only on highly contributing amino acid residues (HCAAR), instead of arbitrary pharmacophores, most commonly used in the conventional approaches in literature. HCAAR were distinguished based on free binding energy (FBE) contributions, obtained using calculation from molecular dynamics (MD) simulations. Previous approaches have relied on the docking score (DS) to generate energy-based pharmacophore models. However, DS are reportedly unreliable. Thus we present a model for a per-residue energy decomposition (PRED), constructed from MD simulation ensembles generating a more trustworthy pharmacophore model which can be applied in drug discovery workflow. This approach was employed in screening for potential HIV-1 RT inhibitors using the pharmacophoric features of the compound GSK952. The complex was subjected to docking and thereafter MD simulations confirmed the stability of the system. Experimentally determined inhibitors with known HIV-RT inhibitory activity were used to validate the proposed protocol. Two potential hits ZINC46849657 and ZINC54359621 showed a significant potential with regards to FBE. Reported results obtained from this work confirm that this new approach is favourable to the future of drug design process. Hsp90 was recently discovered to play a vital role in HIV-1 replication. Thus has emerged, as a promising target for anti-HIV-1 drugs. The molecular mechanism of Hsp90 is poorly understood, thus the second study was aimed to address this issue and provide a clear insight to the inhibition mechanism of Hsp90. Reasonable continuous MD simulations were employed for both unbound and bound Hsp90 conformations, to understand the dimerization and inhibition mechanisms. Results demonstrated that coumermycin A1 (C-A1), a newly discovered Hsp90 inhibitor, binds at the CTD dimer of Hsp90 and lead to a significant separation between orthogonally opposed residues, such as Arg591.B, Lys594.A, Ser663.A, Thr653.B, Ala665.A, Thr649.B, Leu646.B and Asn669A. A Large difference in magnitudes was observed in the radius of gyration (Rg), per-residue fluctuation, root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) confirming a completely more flexible state for the unbound conformation associated with dimerization. Whereas, a less globally correlated motion in the case of the bound conformer of Hsp90 approved a reduction of the dimeric process. This undoubtedly underlines the inhibition process due to ligand binding. The detailed dynamic analyses of Hsp90 presented herein are believed to give a greater insight and understanding to the function and mechanisms of inhibition of Hsp90. The report on the inhibitor-binding mode would also be of great assistance in the design of prospective inhibitors against Hsp90 as potential HIV target

Estudio y cribado virtual de compuestos químicos antivirales (VIH). Estudio de la modulación alostérica de agonistas y antagonistas del receptor celular CXCR4

Els mètodes de descobriment de nous fàrmacs han evolucionat recentment gràcies a la resolució de les estructures proteiques que actuen com a dianes terapèutiques responsables de malalties o desregulacions biològiques. Aquestes estructures proteiques tridimensionals, juntament amb el desenvolupament de noves tècniques computacionals permeten el desenvolupament accelerat de nous compostos candidats a esdevenir fàrmacs. El present treball s’inicia proposant un nou mètode que millora l’elecció de compostos candidats a ser inhibidors d’una “diana difícil”, però ben coneguda com és el receptor VEGFR-2, a partir de la seva estructura tridimensional cristal·litzada, així com de compostos inhibidors coneguts de l’esmentada diana. La resolució tridimensional de l’estructura CXCR4 mitjançant cristal•lografia de raigs X a l’any 2010, ha esdevingut un avenç important a l’hora de millorar el disseny de compostos inhibidors del VIH, així com compostos antitumorals, malalties en les que intervé de forma determinant el receptor CXCR4. Així doncs, els models de cribratge virtual desenvolupats abans del 2010 dins el laboratori de disseny molecular de l’IQS (GEM) han estat generats a partir de models creats per homologia vers a altres proteïnes GPCRs i/o s’han basat solament en la forma de lligands coneguts. D’aquesta forma, a partir de les diferents estructures proteiques publicades de CXCR4, s’ha avaluat quina d’aquestes estructures presenta la conformació que distingeix millor els compostos antagonistes actius dels compostos inactius. A més, s’han avaluat múltiples mètodes de cribratge virtual de CXCR4 basats en l’estructura, en la forma del lligand i mitjançant models farmacofòrics. Una vegada obtinguda la millor estructura de CXCR4 i els millors mètodes de cribratge virtual retrospectiu, es realitzen cribratges virtuals prospectius d’una nova quimioteca generada de forma combinatòria, basada en estructures anàlogues prèviament desenvolupades al laboratori de disseny molecular de l’IQS. Addicionalment, s’ha estudiat el comportament al·lostèric del receptor CXCR4 davant de moduladors antagonistes petits i moduladors al·lostèrics agonistes de naturalesa pèptica. CXCR4 és qualificada com a una “diana difícil” per la gran mida del seu lloc actiu ortostèric, així com per l’ampli número de funcions reguladores en les que intervé el receptor. Per això la modulació al·lostèrica en CXCR4 s’ha estudiat utilitzant diferents aproximacions com són el docking cec, docking proteïna-proteïna, docking per subllocs d’unió i dinàmica molecular.Los métodos de descubrimiento de nuevos fármacos han evolucionado recientemente gracias a la resolución de las estructuras proteicas las cuales actúan como dianas terapéuticas responsables de enfermedades o desregulaciones biológicas. Estas estructuras proteicas tridimensionales, conjuntamente con el desarrollo de nuevas técnicas computacionales están permitiendo el desarrollo acelerado de nuevos compuestos candidatos a convertirse en fármacos. El presente trabajo se inicia proponiendo un nuevo método que permita mejorar la elección de compuestos candidatos a ser inhibidores de una “diana difícil” aunque bien conocida, como es el receptor VEGFR-2, partiendo de su estructura tridimensional cristalizada y de compuestos inhibidores conocidos de dicha diana. La resolución tridimensional de la estructura del receptor CXCR4 mediante cristalografía de rayos X, en el año 2010, ha supuesto un avance importante de cara a mejorar el diseño de compuestos inhibidores del VIH, así como de compuestos antitumorales, enfermedades en las que interviene de forma determinante el receptor CXCR4. Así pues, los modelos de cribado virtual desarrollados anteriormente al 2010 en el laboratorio de diseño molecular del IQS (GEM) han sido generados a partir de modelos creados por homología a otras proteínas GPCRs y/o basados únicamente en la forma de ligandos conocidos. De este modo, partiendo de las diferentes estructuras proteicas publicadas de CXCR4, se ha evaluado cuál de dichas estructuras presenta la conformación que distingue mejor los compuestos antagonistas activos de compuestos inactivos. Además, se han evaluado múltiples métodos de cribado virtual de CXCR4 basados en la estructura, en la forma del ligando y mediante modelos farmacofóricos. Una vez obtenida la mejor estructura de CXCR4 y los mejores métodos de cribado virtual retrospectivo, se realizan cribados virtuales prospectivos de una nueva quimioteca generada combinatoriamente, basada en análogos de estructuras previamente desarrolladas en el laboratorio de diseño molecular del IQS. Adicionalmente se ha estudiado el comportamiento alostérico del receptor CXCR4 frente a moduladores antagonistas de pequeño tamaño y moduladores alostéricos agonistas de naturaleza peptídica. CXCR4 se califica como “diana difícil” debido al gran tamaño del sitio activo ortostérico, juntamente con el amplio número de funciones reguladoras en las que interviene el receptor CXCR4. Por ello la modulación alostérica en CXCR4 se ha estudiado utilizando diferentes aproximaciones, como son: docking ciego, docking proteína-proteína, docking por subsitios y dinámica molecular.: Drug discovery methods have recently emerged thanks to the resolution of protein structures which act as therapeutic targets responsible for diseases or biological deregulations. These three dimensional structures in combination with the development of new computational techniques are accelerating the development of new candidates to become drug compounds. This work starts with the proposal of a new method that improves the selection of candidates to become inhibitors of a well-known “difficult target” such us VEGFR-2 receptor. This method is based on the crystal structure of the receptor and also by a number of inhibitors known for this target. CXCR4 crystal structure was solved in 2010 by X-ray crystallography and this has been an important event in order to improve the molecular design of HIV inhibitors, as well as anticancer compounds, diseases where CXCR4 receptor is involved. Therefore, virtual screening models developed in the laboratory of molecular design of IQS (GEM) were generated using homology models from other GPCRs and/or based on ligand shape techniques. In this sense, taking into consideration all published CXCR4 crystal structures, it has been evaluated which of them shows the most suitable conformation to distinguish antagonists actives from inactives. Moreover, different virtual screening methods have also been evaluated such us structure based methods, ligand based methods and pharmacophoric models. Once obtained the most suitable structure and the best retrospective virtual screening methods, a prospective virtual screening has been carried out using a new combinatorial library of chemical structures. This new library is based on analogous structures previously generated in the laboratory of molecular design of IQS (GEM). In addition, the allosteric behaviour of CXCR4 receptor has been studied versus small antagonist modulators and versus peptidomimetic agonist modulators. CXCR4 is classified as a “difficult target” due to the large size of its extracellular pocket that the orthosteric binding site is placed as well as the diverse number of biochemical regulations where the receptor mediates. Thus, the allosteric modulation of CXCR4 has been studied using different approaches such as blind docking, protein-protein docking, docking by subsites and molecular dynamics

Identification of Ligands with Tailored Selectivity: Strategies & Application

In the field of computer-aided drug design, docking is a computational tool, often used to evaluate the sterical and chemical complementarity between two molecules. This technique can be used to estimate the binding or non-binding of a small molecule to a protein binding site. The classical application of docking is to find those molecules within a large set of molecules that bind a certain target protein and modulate its biological activity. This setup can be considered as established for a single target protein. In contrast to this, the docking to multiple target structures offers new possible applications. It can be used, for example, to assess the binding profile of a ligand against a number of proteins. In this work, the applicability of docking is assessed in such a scenario where multiple target structures are used. The corresponding proteins mostly belong to the family of G protein-coupled receptors. This protein family is very large and numerous GPCRs have been identified as potential drug targets, explaining the their relevance in pharmaceutical research. The protein structures used herein have different relationships and thus represent different application scenarios. The first case study uses two structures belonging to different proteins. These proteins are CXCR3 and CXCR4, a pair of chemokine GPCRs. In this chapter, new ligands are identified that bind to these proteins and modulate their biological activity. More importantly, for each of these newly identified ligands it could be predicted using docking, whether this ligand binds only to one of the two target proteins or to both. This study proves the applicability of docking to identify ligands with tailored selectivity. In addition, these ligands show excellent binding affinities to their respective target or targets. In the following two studies, the docking to different structures of the same target protein is investigated. The first application aims at identifying ligands selective for either one of two isoforms of the zebrafish CXC receptor 4. Subsequently, multiple conformations of the chemokine receptor CCR5 are used to show that different starting structures can identify different ligands. Next to the plain identification of chemically new ligands, experimental hurdles to prove the biological activity of these molecules in a functional assay is discussed. These difficulties are based on the fact that docking evaluates the structural complementarity between molecules and protein structures rather than predicting the effect of these molecules on the proteins. In addition, GPCRs form a challenging set of target proteins, since their ligands can induce a variety of different effects. Finally, the general applicability of multi-target docking to a very large number of structures is investigated. For this evaluation, kinases are used as protein family since many more structures have been experimentally determined for these proteins compared to GPCRs as membrane proteins. First, using published experimental data, a dataset is created consisting of several hundred kinase structures and a set of small-molecule kinase inhibitors. This dataset is characterised by the availability of experimental binding data for each single kinase-inhibitor combination. These experimental data were subsequently compared to the docking results of each ligand into each single kinase structure. The results indicate that a reliable selectivity prediction for a ligand is highly demanding in such a large-scale setup and beyond current possibilities. However, it can be shown that the prediction accuracy of docking can be improved by normalising the docking scores over multiple ligands and proteins. Based on these findings, the idea of "protein decoys" is developed, which might in the future allow more accurate predictions of selectivity profiles using docking

Diverse computational tools towards the understanding of HIV targets and design of potential drug candidates.

Ph. D. University of KwaZulu-Natal, Durban 2014.HIV/AIDS still remains to be a challenging epidemic infecting millions of individuals worldwide. The morbidity and mortality rates of HIV-infected patients has been well documented over the years. Despite on-going HIV/AIDS research and access to antiretroviral therapy, to date still no cure exists for this deliberating disease. In recent years, computational approaches have emerged as close counterparts to experiments in modern drug discovery process and in understanding complex biological phenomena. An array of in-silico computational techniques were implemented ranging from molecular dynamic (MD) simulations, de-novo design, hybrid structure-based and pharmacophore-based virtual screening, quantitative structure-activity relationship (QSAR), homology modeling, principle component analysis (PCA), residue interaction network analysis (RIN), substrate envelope analysis (SEA), to molecular mechanics and quantum mechanics. The first report (Chapter 4), demonstrated a unique strategy for developing dual acting inhibitors against HIV-1 protease (PR) and reverse transcriptase (RT). The designed targets exhibited binding affinities and dual inhibiting activity comparable to, and in some cases better than, known active reference drugs. The second study (Chapter 5), reported the activity of flexible hydroquinone-based compounds as non-nucleoside reverse transcriptase inhibitors (NNRTIs), as proposed by Bruccoleri, where no experimental or computational work supported his proposal. Results concluded that the novel flexible hydroquinone-based compounds showed improved binding affinity as compared to FDA-approved prototype drugs and more specifically potent potential mutant-resistant NNRT inhibitor activity. The third report (Chapter 6), explored the activity of novel CCR5 antagonists as potential HIV- 1 entry inhibitors. Ten scaffolds were identified as novel CCR5 antagonists or potential HIV-1 entry inhibitors. Furthermore, from the generated atom-based 3D-QSAR model, all of the parameters showed certain reliability and feasible predictability to help us design new and high selectivity CCR5 inhibitors. The fourth study (Chapter 7), explored the atomistic basis of why the M184I single mutation renders complete resistance of HIV-1 RT to lamivudine. Multiple molecular dynamics simulations, binding free energy calculations, principle component analysis (PCA) and residue interaction network (RIN) analyses adequately clarified the effect of the M184I mutation on drug resistance to lamvudine. Results presented in this study verified that M184I mutation decreased drug binding affinity, distorted ligand optimum orientation in RT active site and affected the overall protein conformational landscape. The results also provided some potential clues for further design of novel inhibitors that are less susceptible to drug resistance. In the fifth study (Chapter 8), we identified potential HIV-Nef inhibitors by exploiting the structural features of B9 using an integrated computational tools framework. The top identified hit compounds demonstrated comparatively better binding affinities and relatable binding modes compared to the prototype antagonist, B9. Top identified hits were proposed as new potential novel leads targeting HIV-Nef with a detailed analysis of their respective binding modes. The sixth report (Chapter 9), aimed to reveal the dimer packing and unpacking phenomena of HIV-Nef in its apo and inhibitor bound conformations using molecular dynamic simulations. Results verified a more conformational flexible nature of HIV-Nef dimer in the absence of an inhibitor.as compared to B9 bound conformation of HIV-Nef, which was found to be more conformationally rigid with a lesser inter-dimeric association. We believe that the results obtained from these several studies could be of great benefit in the development of more effective therapeutic interventions for the treatment and cure of HIV/AIDS