Computational study targeting anti-fungal Tavaborole analogs and anti-cancer BRACO19

This thesis comprises of three computer aided drug design studies utilizing molecular docking and molecular dynamic simulations: (i) a lead optimization study virtually screening an initial library of ~120000 lead compounds targeting fungal leucyl tRNA synthetase, (ii) an exploratory study to understand the binding pathway of BRACO19 to a parallel telomeric DNA G-quadruplex by MD simulations and compare with experimentally solved X-ray crystal structure (iii) a comparative study to understand the lack of selectivity of BRACO19 to various topologies of human telomeric DNA G-quadruplex over DNA duplex. The first chapter provides the background information required to understand the molecular docking studies and molecular dynamics simulation (MD) studies conducted and discussed in this thesis. This introductory chapter is organized as follows: the first section is an introduction to molecular recognition in protein-ligand interactions, the second section introduces computer-aided drug design, the third section introduces homology modelling, the fourth section discusses molecular docking and virtual screening, the fifth section introduces methods for binding affinity prediction and the sixth section explains MD simulations. The second chapter of this thesis proposes a library of compounds with enhanced activity compared to the parent molecule it had been modified from. Tavaborole, the recently approved topological anti-fungal drug, inhibits leucyl tRNA synthetase by irreversible covalent bonding and hinders protein synthesis. The benzo-boroxole pharmacophore of tavaborole is responsible for its unique activity. This study theoretically proposes molecules with improved anti-fungal affinity. The third chapter of this thesis explores the binding pathway of anti-cancer drug, BRACO19 and human telomeric DNA G-quadruplex. G-quadruplex specific ligands that stabilizes the G-quadruplex, have great potential to be developed as anticancer agents. A free human telomeric DNA G-quadruplex and an unbound BRACO19 are simulated and the resulting structure is then compared with an experimentally solved X-ray structure of human telomeric G-quadruplex with a bound BRACO19 intercalated within the G-quadruplex. Three binding modes have been identified: top end stacking, bottom intercalation and groove binding. Bottom intercalation mode (51% of the population) is identical to the binding pose in the X-ray solved crystal structure. The fourth chapter of this thesis compares different topological folds of human telomeric DNA G-quadruplexes (parallel, antiparallel and hybrid) that have been experimentally solved using molecular dynamic simulation to understand the 62-fold preferential selectivity of BRACO19 towards human telomeric DNA G-quadruplex over DNA duplex. Groove binding mode was found to be the most stable binding mode for the duplex and top stacking mode for the G-quadruplexes. The non-existential binding selectivity of BRACO19 can be accounted to the similar groove binding to both the duplex and the G-quadruplex. For that reason, a modification should be induced such that this prospective ligand destabilizes binding to the duplex but stabilizes the G-quadruplex binding

A mechanical study of cancer drug-receptor interactions, specifically in G-Quadruplex DNA and Topoisomerase I enzymes

Computational methods are becoming essential in drug discovery as they provide information that traditional drug development methods lack. Using these methods to understand drug-receptor interactions in detail, researchers are able to efficiently design promising drug candidates. In this study, extra precision Glide docking, molecular dynamics simulations and MMGBSA binding energy calculations provided information about the binding behavior of small molecules to two specific targets for current cancer therapeutics: G-quadruplex DNA and Topoisomerase I enzyme. The first study focuses on the compound Telomestatin, which induces apoptosis of various cancer cells with a relatively low effect on somatic cells due to its high selectivity toward G-quadruplex over duplex DNA. Three major binding poses were discovered: top end stacking, bottom end stacking and a groove binding. A high resolution structure of this complex does not yet exist, so this is the first time Telomestatin binding modes have been reported. The second study focuses on 8 Camptothecin class Topoisomerase I inhibitors, which have been reported to effectively treat multiple types of cancer, however are limited by their drug resistance. Recent computational studies have indicated that the mutations near the active binding site of the drug can significantly weaken the drug binding and may be a major cause of the drug resistance. Here, a complete study of each Camptothecin analog in each mutated complex in the active binding site is presented. On this set of mutant complexes, Topotecan and Camptothecin have much smaller binding energy decrease than a set of new Camptopthcin derivatives (Lurtotecan, LESN-38, Gimatecan, Exatecan and Belotecan) currently under clinical trials. Lucanthone, a non-Camptothecin, shows comparable results to Topotecan and Camptothecin, indicating that it may exhibit the least drug resistance and is therefore a promising candidate for future studies as a Topoisomerase I inhibitor. In addition, a trend is observed from our binding energy data that the shorter the distance of a mutant to a ligand, the greater the decrease in binding energy (with one exception). The results found in each of these binding studies will be utilized to further advance effective cancer therapeutics in the future

Ligand-induced unfolding mechanism of an RNA G-quadruplex

The cationic porphyrin, TMPyP4, is a well-established DNA G-quadruplex (G4) binding ligand that can stabilize different topologies via multiple binding modes. However, TMPyP4 has completely opposite destabilizing and unwinding effect on RNA G4 structures. The structural mechanisms that mediate RNA G4 unfolding remains unknown. Here, we report on the TMPyP4-induced RNA G4 unfolding mechanism studied by well-tempered metadynamics (WT-MetaD) with supporting biophysical experiments. The simulations predict a two-state mechanism of TMPyP4 interaction via a groove-bound and a top-face bound conformation. The dynamics of TMPyP4 stacking on the top tetrad disrupts Hoogsteen H-bonds between guanine bases resulting in the consecutive TMPyP4 intercalation from top-to-bottom G-tetrads. The results reveal a striking correlation between computational and experimental approaches and validate WT-MetaD simulations as a powerful tool for studying RNA G4-ligand interactions

Exploring the Role of Molecular Dynamics Simulations in Most Recent Cancer Research: Insights into Treatment Strategies

Cancer is a complex disease that is characterized by uncontrolled growth and division of cells. It involves a complex interplay between genetic and environmental factors that lead to the initiation and progression of tumors. Recent advances in molecular dynamics simulations have revolutionized our understanding of the molecular mechanisms underlying cancer initiation and progression. Molecular dynamics simulations enable researchers to study the behavior of biomolecules at an atomic level, providing insights into the dynamics and interactions of proteins, nucleic acids, and other molecules involved in cancer development. In this review paper, we provide an overview of the latest advances in molecular dynamics simulations of cancer cells. We will discuss the principles of molecular dynamics simulations and their applications in cancer research. We also explore the role of molecular dynamics simulations in understanding the interactions between cancer cells and their microenvironment, including signaling pathways, proteinprotein interactions, and other molecular processes involved in tumor initiation and progression. In addition, we highlight the current challenges and opportunities in this field and discuss the potential for developing more accurate and personalized simulations. Overall, this review paper aims to provide a comprehensive overview of the current state of molecular dynamics simulations in cancer research, with a focus on the molecular mechanisms underlying cancer initiation and progression.Comment: 49 pages, 2 figure

Exosite Binding in Thrombin: A Global Structural/Dynamic Overview of Complexes with Aptamers and Other Ligands

: Thrombin is the key enzyme of the entire hemostatic process since it is able to exert both procoagulant and anticoagulant functions; therefore, it represents an attractive target for the developments of biomolecules with therapeutic potential. Thrombin can perform its many functional activities because of its ability to recognize a wide variety of substrates, inhibitors, and cofactors. These molecules frequently are bound to positively charged regions on the surface of protein called exosites. In this review, we carried out extensive analyses of the structural determinants of thrombin partnerships by surveying literature data as well as the structural content of the Protein Data Bank (PDB). In particular, we used the information collected on functional, natural, and synthetic molecular ligands to define the anatomy of the exosites and to quantify the interface area between thrombin and exosite ligands. In this framework, we reviewed in detail the specificity of thrombin binding to aptamers, a class of compounds with intriguing pharmaceutical properties. Although these compounds anchor to protein using conservative patterns on its surface, the present analysis highlights some interesting peculiarities. Moreover, the impact of thrombin binding aptamers in the elucidation of the cross-talk between the two distant exosites is illustrated. Collectively, the data and the work here reviewed may provide insights into the design of novel thrombin inhibitors

Biophysical and computational investigations into G-quadruplex structural polymorphism and interaction with small molecules.

In the cell, guanine-rich nucleic acids can self-assemble into unique four stranded tertiary structures known as G-quadruplexes. G-quadruplex formation in the telomere leads inhibits telomerase, an enzyme activated in cancer cells to maintain the telomere and allowing for cancer cells to achieve immortality. G-quadruplex formation in the promoters and 5’-untranslated regions regulates the expression of many oncogenes. Furthermore, G-quadruplex formation during cellular replication promotes genomic instability, a characteristic which enables tumor development. Because of their implication in cancer, G-quadruplex structures have emerged as attractive drug targets for anti-tumor therapeutics. In the current dissertation work, we present three experimental approaches to investigate G-quadruplex structures, biophysical properties, small molecule interaction, and the thermodynamics of G-quadruplex formation. Current approaches to study G-quadruplex structures often employ sequence modifications or changes to the experimental condition, as a way of resolving the structural polymorphism associated with many G-quadruplex-forming sequences, to select for a single conformation for high-resolution structural studies. Our strategy for resolving G-quadruplex structural polymorphism is superior in that the experimental approaches do not result in drastic perturbation of the system. In the first approach, we employed size exclusion chromatography to separate a mixture of G-quadruplex structures formed from a G-quadruplex-forming sequence. We demonstrated that it is possible to isolate distinct species of G-quadruplex structures for further biophysical studies. In the second approach, we employed hydrodynamic bead modeling to study the structural polymorphism of a G-quadruplex-forming sequence. We showed that properties calculated from models agreed with experimentally determined values and could be used to predict the folding of G-quadruplex-forming oligonucleotides whose high-resolution structures are ambiguous or not available. In our third approach, we presented a virtual screening platform that was successful in identifying a new Gquadruplex-interacting small molecule. The results of the virtual screen were validated with extensive biophysical testing. Our target for the virtual screen was a G-quadruplex structure generated in silico, which represents one approach to receptor-based drug discovery when high-resolution structures of the binding site are not available. Taken together, our three approaches represent a new paradigm for drug discovery from guaninerich sequence to anti-cancer drugs

Disentangling the Structure-Activity Relationships of Naphthalene Diimides as Anticancer G-Quadruplex-Targeting Drugs

In the context of developing efficient anticancer therapies aimed at eradicating any sort of tumors, G-quadruplexes represent excellent targets. Small molecules able to interact with G-quadruplexes can interfere with cell pathways specific of tumors and common to all cancers. Naphthalene diimides (NDIs) are among the most promising, putative anticancer G-quadruplextargeting drugs, due to their ability to simultaneously target multiple G-quadruplexes and their strong, selective in vitro and in vivo anticancer activity. Here, all the available biophysical, biological, and structural data concerning NDIs targeting Gquadruplexes were systematically analyzed. Structure−activity correlations were obtained by analyzing biophysical data of their interactions with G-quadruplex targets and control duplex structures, in parallel to biological data concerning the antiproliferative activity of NDIs on cancer and normal cells. In addition, NDI binding modes to G-quadruplexes were discussed in consideration of the structures and properties of NDIs by in-depth analysis of the available structural models of G-quadruplex/NDI complexes

The Renaissance of Isothermal Titration Calorimetry

This dissertation is a composite of some of the research that I have conducted during the course of my PhD study. The larger goal of this dissertation is to renew the interests among the scientific community for an otherwise under-appreciated technique called Isothermal Titration Calorimetry. The resurgence of calorimetry in the biophysical community and the shift to investigations of more complex biological systems signal a real need for more sophisticated analysis techniques. This dissertation expounds on new ITC analysis methods that we have developed as well as results from the study of thermodynamic properties of higher order DNA structures. In 1978, Peter Privalov described the first use of microcalorimetry to obtain the thermodynamic properties for removing calcium from parvalbumin III protein. Fast forward 36 years: modern day electronics, highly efficient thermally conductive and chemically inert materials, in conjunction with sensitive thermal detectors, has transformed the original calorimeter into a device capable of measuring heat changes as small as 0.05 nanowatts, which is equivalent to capturing heat from an incandescent light bulb a kilometer away. However, analytical methods have not kept pace with this technology. Commercial ITC instruments are typically supplied with software that only includes a number of simple interaction models. As a result, the lack of analysis tools for more complex models has become a limiting factor for many researchers. We have recently developed new ITC fitting algorithms that we have incorporated into a userriendly program (CHASM©) for the analysis of complex ITC equilibria. In a little over a year, CHASM© has been downloaded by over 370 unique users. Several chapters in this dissertation demonstrate this software’s power and versatility in the thermodynamic investigations of two model systems in both aqueous and non-aqueous media. In chapter VI, we assembled a model NHE-III1 : a novel structure of Gquadruplex in a double stranded form and studied its structural complexity and binding interactions with a classical G-quaduplex interactive ligand known as TMPyP4. In chapter VII, we reported the thermodynamic properties of a novel PAH system in which weak dispersion forces are solely responsible for formation of the supramolecular complexes

Binding of Gemini Bisbenzimidazole Drugs with Human Telomeric G-Quadruplex Dimers: Effect of the Spacer in the Design of Potent Telomerase Inhibitors

The study of anticancer agents that act via stabilization of telomeric G-quadruplex DNA (G4DNA) is important because such agents often inhibit telomerase activity. Several types of G4DNA binding ligands are known. In these studies, the target structures often involve a single G4 DNA unit formed by short DNA telomeric sequences. However, the 3′-terminal single-stranded human telomeric DNA can form higher-order structures by clustering consecutive quadruplex units (dimers or n-mers). Herein, we present new synthetic gemini (twin) bisbenzimidazole ligands, in which the oligo-oxyethylene spacers join the two bisbenzimidazole units for the recognition of both monomeric and dimeric G4DNA, derived from d(T2AG3)4 and d(T2AG3)8 human telomeric DNA, respectively. The spacer between the two bisbenzimidazoles in the geminis plays a critical role in the G4DNA stability. We report here (i) synthesis of new effective gemini anticancer agents that are selectively more toxic towards the cancer cells than the corresponding normal cells; (ii) formation and characterization of G4DNA dimers in solution as well as computational construction of the dimeric G4DNA structures. The gemini ligands direct the folding of the single-stranded DNA into an unusually stable parallel-stranded G4DNA when it was formed in presence of the ligands in KCl solution and the gemini ligands show spacer length dependent potent telomerase inhibition properties

The Use of Ruthenium Complexes as Molecular Probes for Non-Canonical DNA

This study considered the preparation of a new DNA binding Ruthenium polypyridyl complex possessing an infrared active nitrile group. The binding abilities of a novel Ruthenium complex, [Ru(TMP)2DPPZ-10-CN], to various forms of DNA—both canonical and non-canonical—were examined by performing multiple DNA titrations. DNA is of great interest as it is the carrier of genetic information for all living things. Damage to DNA can have drastically detrimental effects, so the study of its structure and replication is of great importance. Two non-canonical structures that are important are the G-quadruplex and i-motif which form at the telomeric and regulatory regions of genes, respectively, and have the ability to block telomerase activity and influence transcription. The complex was synthesized by microwave irradiation and purified using a silica column and an ion exchange with Amberlite 402. Six titrations were, then, performed with salmon sperm dsDNA, guanine monophosphate (GMP), G4T4G4, human telomere G-quadruplex, i-motif C5T3, and i-motif C30. The complex was found to favor non-canonical structures, particularly the G-quadruplex structure, because of its high [bp]/[Ru] concentrations. The higher concentration of base pairs or structures per Ruthenium molecule indicated that the complex had a high binding affinity for that particular DNA structure. These results support the notion that Ruthenium metal complexes can be used for theragnostic purposes and can be used to target the telomeric region of genes where G-quadruplex structures can be found and influence transcription initiation and inhibit telomerase activity