Identification of G-quadruplex DNA/RNA binders: Structure-based virtual screening and biophysical characterization

Background Recent findings demonstrated that, in mammalian cells, telomere DNA (Tel) is transcribed into telomeric repeat-containing RNA (TERRA), which is involved in fundamental biological processes, thus representing a promising anticancer target. For this reason, the discovery of dual (as well as selective) Tel/TERRA G-quadruplex (G4) binders could represent an innovative strategy to enhance telomerase inhibition. Methods Initially, docking simulations of known Tel and TERRA active ligands were performed on the 3D coordinates of bimolecular G4 Tel DNA (Tel2) and TERRA (TERRA2). Structure-based pharmacophore models were generated on the best complexes and employed for the virtual screening of ~ 257,000 natural compounds. The 20 best candidates were submitted to biophysical assays, which included circular dichroism and mass spectrometry at different K+ concentrations. Results Three hits were here identified and characterized by biophysical assays. Compound 7 acts as dual Tel2/TERRA2 G4-ligand at physiological KCl concentration, while hits 15 and 17 show preferential thermal stabilization for Tel2 DNA. The different molecular recognition against the two targets was also discussed. Conclusions Our successful results pave the way to further lead optimization to achieve both increased selectivity and stabilizing effect against TERRA and Tel DNA G4s. General significance The current study combines for the first time molecular modelling and biophysical assays applied to bimolecular DNA and RNA G4s, leading to the identification of innovative ligand chemical scaffolds with a promising anticancer profile. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio

Structural characterization and selective drug targeting of higher-order DNA G-quadruplex systems.

There is now substantial evidence that guanine-rich regions of DNA form non-B DNA structures known as G-quadruplexes in cells. G-quadruplexes (G4s) are tetraplex DNA structures that form amid four runs of guanines which are stabilized via Hoogsteen hydrogen bonding to form stacked tetrads. DNA G4s have roles in key genomic functions such as regulating gene expression, replication, and telomere homeostasis. Because of their apparent role in disease, G4s are now viewed as important molecular targets for anticancer therapeutics. To date, the structures of many important G4 systems have been solved by NMR or X-ray crystallographic techniques. Small molecules developed to target these structures have shown promising results in treating cancer in vitro and in vivo, however, these compounds commonly lack the selectivity required for clinical success. There is now evidence that long single-stranded G-rich regions can stack or otherwise interact intramolecularly to form G4-multimers, opening a new avenue for rational drug design. For a variety of reasons, G4 multimers are not amenable to NMR or X-ray crystallography. In the current dissertation, I apply a variety of biophysical techniques in an integrative structural biology (ISB) approach to determine the primary conformation of two disputed higher-order G4 systems: (1) the extended human telomere G-quadruplex and (2) the G4-multimer formed within the human telomerase reverse transcriptase (hTERT) gene core promoter. Using the higher-order human telomere structure in virtual drug discovery approaches I demonstrate that novel small molecule scaffolds can be identified which bind to this sequence in vitro. I subsequently summarize the current state of G-quadruplex focused virtual drug discovery in a review that highlights successes and pitfalls of in silico drug screens. I then present the results of a massive virtual drug discovery campaign targeting the hTERT core promoter G4 multimer and show that discovering selective small molecules that target its loops and grooves is feasible. Lastly, I demonstrate that one of these small molecules is effective in down-regulating hTERT transcription in breast cancer cells. Taken together, I present here a rigorous ISB platform that allows for the characterization of higher-order DNA G-quadruplex structures as unique targets for anticancer therapeutic discovery

Insights into Ebola Virus VP35 and VP24 Interferon inhibitory functions and their initial exploitation as drug targets

Upon viral infection, the interferon (IFN) system triggers potent antiviral mechanisms limiting viral growth and spread. Hence, to sustain their infection, viruses evolved efficient counteracting strategies to evade IFN control. Ebola virus (EBOV), member of the family Filoviridae, is one of the most virulent and deadly pathogen ever faced by humans. Etiological agent of the Ebola virus disease (EVD), EBOV can be undoubtedly considered the perfect example of a powerful inhibitor of the host organism immune response activation. Particularly, the efficacious suppression of the IFN cascade contributes to disease progression and severity. Among the EBOV-encoded proteins, the viral proteins 35 (VP35) and 24 (VP24) are responsible for the EBOV extreme virulence, representing the core of such inhibitory function through which EBOV determines its very effective shield to the cellular immune defenses. VP35 inhibits the activation of the cascade leading to IFN production, while VP24 inhibits the activation of the IFN-stimulated genes. A number of studies demonstrated that both VP35 and VP24 are validated target for drug development. Insights of the structural characteristics of VP35 and VP24 domains revealed crucial pockets exploitable for drug development. Considered the lack of therapy for EVD, restoring the immune activation is a promising approach for drug development. In the present review we summarize the importance of VP35 and VP24 proteins in counteracting the host IFN cellular response and discuss their potential as druggable viral targets as a promising approach toward attenuation of EBOV virulence

Mechanism of MicroRNA-Target Interaction: Molecular Dynamics Simulations and Thermodynamics Analysis

MicroRNAs (miRNAs) are endogenously produced ∼21-nt riboregulators that associate with Argonaute (Ago) proteins to direct mRNA cleavage or repress the translation of complementary RNAs. Capturing the molecular mechanisms of miRNA interacting with its target will not only reinforce the understanding of underlying RNA interference but also fuel the design of more effective small-interfering RNA strands. To address this, in the present work the RNA-bound (Ago-miRNA, Ago-miRNA-target) and RNA-free Ago forms were analyzed by performing both molecular dynamics simulations and thermodynamic analysis. Based on the principal component analysis results of the simulation trajectories as well as the correlation analysis in fluctuations of residues, we discover that: 1) three important (PAZ, Mid and PIWI) domains exist in Argonaute which define the global dynamics of the protein; 2) the interdomain correlated movements are so crucial for the interaction of Ago-RNAs that they not only facilitate the relaxation of the interactions between residues surrounding the RNA binding channel but also induce certain conformational changes; and 3) it is just these conformational changes that expand the cavity of the active site and open putative pathways for both the substrate uptake and product release. In addition, by thermodynamic analysis we also discover that for both the guide RNA 5′-end recognition and the facilitated site-specific cleavage of the target, the presence of two metal ions (of Mg2+) plays a predominant role, and this conclusion is consistent with the observed enzyme catalytic cleavage activity in the ternary complex (Ago-miRNA-mRNA). Our results find that it is the set of arginine amino acids concentrated in the nucleotide-binding channel in Ago, instead of the conventionally-deemed seed base-paring, that makes greater contributions in stabilizing the binding of the nucleic acids to Ago

Rapid evolution of protein kinase PKR alters sensitivity to viral inhibitors.

Protein kinase PKR (also known as EIF2AK2) is activated during viral infection and phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), leading to inhibition of translation and viral replication. We report fast evolution of the PKR kinase domain in vertebrates, coupled with positive selection of specific sites. Substitution of positively selected residues in human PKR with residues found in related species altered sensitivity to PKR inhibitors from different poxviruses. Species-specific differences in sensitivity to poxviral pseudosubstrate inhibitors were identified between human and mouse PKR, and these differences were traced to positively selected residues near the eIF2alpha binding site. Our findings indicate how an antiviral protein evolved to evade viral inhibition while maintaining its primary function. Moreover, the identified species-specific differences in the susceptibility to viral inhibitors have important implications for studying human infections in nonhuman model systems

Methods for Molecular Modelling of Protein Complexes.

Biological processes are often mediated by complexes formed between proteins and various biomolecules. The 3D structures of such protein-biomolecule complexes provide insights into the molecular mechanism of their action. The structure of these complexes can be predicted by various computational methods. Choosing an appropriate method for modelling depends on the category of biomolecule that a protein interacts with and the availability of structural information about the protein and its interacting partner. We intend for the contents of this chapter to serve as a guide as to what software would be the most appropriate for the type of data at hand and the kind of 3D complex structure required. Particularly, we have dealt with protein-small molecule ligand, protein-peptide, protein-protein, and protein-nucleic acid interactions.Most, if not all, model building protocols perform some sampling and scoring. Typically, several alternate conformations and configurations of the interactors are sampled. Each such sample is then scored for optimization. To boost the confidence in these predicted models, their assessment using other independent scoring schemes besides the inbuilt/default ones would prove to be helpful. This chapter also lists such software and serves as a guide to gauge the fidelity of modelled structures of biomolecular complexes

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\u27-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

Protein-protein Interactions of Bacterial Topoisomerase I

Protein-protein interactions (PPIs) are essential features of cellular processes including DNA replication, transcription, translation, recombination, and repair. In my study, the protein interactions of bacterial DNA topoisomerase I, an essential enzyme, were investigated. The topoisomerase I in bacteria relaxes excess negative supercoiling on DNA and maintains genomic stability. Investigating the PPI network of DNA topoisomerase I can further our understanding of the various functional roles of this enzyme. My study is focused on topoisomerase I of Escherichia coli and Mycobacterium smegmatis. Firstly, we have explored the biochemical mechanisms for an interaction between RNA Polymerase, and topoisomerase I in E. coli. Molecular docking and molecular dynamic simulations have predicted that the interactions are mediated through electrostatic, and hydrogen bonding. The predicted Lysine residues (K627, K664) of topoisomerase I that are involved in the electrostatic interactions were mutated to Alanine, and its effect on the binding efficiency with RNA polymerase was reported. In a separate study, PPI partners of topoisomerase I in mycobacteria were identified. Knowledge gained from the study can provide valuable insights into the physiological functions of a validated drug target, DNA topoisomerase I, in pathogenic mycobacteria. Co-immunoprecipitation and pull-down assays were coupled to mass spectrometry for identification of the protein partners of mycobacterial topoisomerase I. The study has identified RNA polymerase, and putative helicases (DEAD/DEAH BOX helicases) as potential protein partners of mycobacterial topoisomerase I. My results indicated that the tail region of the CTD-topoisomerase I was required for direct physical interaction with the RNAP beta’ subunit. My studies have also verified the physiological relevance of the topoisomerase I - RNA polymerase interactions for survival under antibiotic, and oxidative stress. Lastly, I report a direct physical interaction between E. coli topoisomerase I and RecA by pull-down assays. Previous studies have shown that RecA, a DNA repair protein, can stimulate the relaxation activity of E. coli topoisomerase I. Our new results showed that the stimulatory effect can be attributed to the physical interaction of topoisomerase I with RecA

Structure-Guided Computational Approaches to Unravel Druggable Proteomic Landscape of Mycobacterium leprae.

Leprosy, caused by Mycobacterium leprae (M. leprae), is treated with a multidrug regimen comprising Dapsone, Rifampicin, and Clofazimine. These drugs exhibit bacteriostatic, bactericidal and anti-inflammatory properties, respectively, and control the dissemination of infection in the host. However, the current treatment is not cost-effective, does not favor patient compliance due to its long duration (12 months) and does not protect against the incumbent nerve damage, which is a severe leprosy complication. The chronic infectious peripheral neuropathy associated with the disease is primarily due to the bacterial components infiltrating the Schwann cells that protect neuronal axons, thereby inducing a demyelinating phenotype. There is a need to discover novel/repurposed drugs that can act as short duration and effective alternatives to the existing treatment regimens, preventing nerve damage and consequent disability associated with the disease. Mycobacterium leprae is an obligate pathogen resulting in experimental intractability to cultivate the bacillus in vitro and limiting drug discovery efforts to repositioning screens in mouse footpad models. The dearth of knowledge related to structural proteomics of M. leprae, coupled with emerging antimicrobial resistance to all the three drugs in the multidrug therapy, poses a need for concerted novel drug discovery efforts. A comprehensive understanding of the proteomic landscape of M. leprae is indispensable to unravel druggable targets that are essential for bacterial survival and predilection of human neuronal Schwann cells. Of the 1,614 protein-coding genes in the genome of M. leprae, only 17 protein structures are available in the Protein Data Bank. In this review, we discussed efforts made to model the proteome of M. leprae using a suite of software for protein modeling that has been developed in the Blundell laboratory. Precise template selection by employing sequence-structure homology recognition software, multi-template modeling of the monomeric models and accurate quality assessment are the hallmarks of the modeling process. Tools that map interfaces and enable building of homo-oligomers are discussed in the context of interface stability. Other software is described to determine the druggable proteome by using information related to the chokepoint analysis of the metabolic pathways, gene essentiality, homology to human proteins, functional sites, druggable pockets and fragment hotspot maps