Kenneth A. Johnson - US grants

Previous work in this laboratory by scanning transmission electron microscopy (STEM) has shown that Tetrahymena 21S dynein consist of a bouquet of three globular heads connected by three separate strands to a common base. Mass analysis by integration of electron scattering intensities gave a molecular weight of 2x10 to the 6 g/mole and indicated that two of the globular heads were identical with a mass of approximately 400 kdaltons while the third head exhibited a mass of approximately 550 kdaltons. Moreover, the present data suggests that the rootlike base of the bouquet forms the structural attachment site and the globular heads interact with the B-subfiber in an ATP-dependent reaction coupled to force production. Specifically, the current proposal seeks to follow up on these studies to (1) establish the absolute orientation of the molecule with respect to the A-subfiber of the outer doublet. (2) Determine whether two heads are identical and one head is different. (3) Localize the ATP and microtubule binding sites. (4) Localize the major polypeptides of the dynein molecule. (5) Begin to address the question of the roles of the three dynein heads in motility. These problems will be addressed by STEM and conventional TEM analysis of isolated dynein, intact ciliary outer doublets and dynein subfragments produced by proteolytic digestion. Monoclonal antibodies will be used to localize functional domains of the dynein molecule. This structural analysis is absolutely essential to the longer term objectives of establishing the mechanism of force pro-production and control of the dynein in ciliary motility and for subsequent analysis of dynein-like ATPases in other microtubule-dependent movements.

The overall goals of the proposed research are to establish the mechanism of action and control of the dynein ATPase in generating ciliary and flagellar movement. The specific goals of these studies can be divided into four parts: (a) we will work to complete our description of the kinetics and thermodynamics of the ATPase cycle by transient and steady state kinetic analysis of the microtubule activation of the dynein ATPase and by equilibrium binding measurements; (b) we will determine the functions and potential interactions of the three dynein heads by examining the ATPase kinetics of dynein subfragments and attempt to relate those studies to the more complex kinetics observed with the three-headed dynein by computer modeling; (c) we will test possible mechanisms of regulation by exploring the effects of calcium, calmodulin and phosphorylation on each step of the ATPase cycle, especially the reactions involved in microtubule activation of the ATPase; (d) finally, we will work to extend these results to the intact axoneme, by examining the kinetics of the intact axoneme directly, and by determining the effects of well characterized monoclonal antibodies on wave propagation in reactivated flagella. The current work builds upon our previous results and well established methods in examining the structure and ATPase pathway of dynein islated from Tetrahymena cilia. We will use stopped-flow and chemical-quench flow methods for rapid kinetic analysis, 18O-isotope exchange studies to examine the lifetime of intermediates in the reaction pathway, and light and electron microscopy to examine the intact axoneme. These studies are expected to establish the complete pathway and mechanism of control by which the hydrolysis of ATP is coupled to dynein crossbridge interaction with microtubules to produce a force for ciliary movement. The work also provides a basis for analysis of dynein-like ATPases in other microtubule systems such as chromosome movement or the transport of membrane bound particles.

The long term goals of this proposal are to establish the kinetic, thermodynamic and structural basis for force production in microtubule- dependent motility. Kinesin, the putative anterograde motor in axoplasmic transport, will be examined because it is the most simple of the ATPases involved in force production for sliding filament motility. Kinesin will be purified from cloned and overexpressed gene of Drosophila kinesin heavy chains or as native protein, form bovine brain. Conditions will be optimized for expression and purification of the kinesin head fragments, consisting of amino acids 10447 of Drosophila kinesin heavy chain. The microtubule-kinesin complex will be characterized to determine the stoichiometry of kinesin binding to tubulin and to establish conditions for kinetic and equilibrium measurements on the formation of the complex. The kinetics and thermodynamics of the microtubule-kinesin ATPase pathway will be established by transient state kinetic methods. Stopped-flow light scattering methods will be used to measure the rate of ATP-induced dissociation and reformation of the microtubule-kinesin complex. Chemical- quench-flow methods will be used to determine the rates of ATP binding and hydrolysis and ATP synthesis. These kinetic measurements will be extended to examine the potential regulation of kinesin by Ca+2 or by phosphorylation. Site-directed mutagenesis will be used to relate structural domains of the kinesin head to enzymatic functions responsible for energy transduction. Mutation of residues thought to be involved in ATP- or microtubule-binding or in regulation will allow a direct quantitative assessment of the roles of individual or groups of amino acids in each step in the ATPase cycle. Attempts will be made to grow crystals of the cloned kinesin head fragment, in order solve the structure of the kinesin head in the presence and absence of ATP and thereby define in molecular detail the nature of the conformational changes responsible for movement. Microtubule-dependent ATPases have been implicated in a large number of fundamental life processes including anterograde and retrograde axoplasmic transport in neurons and in chromosome movement during cell division. Thus, these studies into the fundamental molecular events leading to generation and control of force production have a wide applicability to health related research.

The goal of this proposal is to establish the structural, kinetic and thermodynamic basis for the fidelity and efficiency of DNA replication. Analysis of HIV reverse transcriptase will allow definition of the structural constraints that ultimately may limit the ability of the virus to avoid an appropriate combination of nucleoside analogs in the treatment of AIDS and other viral infections. Analysis of the mitochondrial DNA polymerase will define the origins of the toxicity of nucleoside analogs thought to be due to incorporation of the analogs into mitochondrial DNA. Studies on T7 DNA polymerase and HIV RT have shown that the selectivity of the polymerase is a function of a two-step nucleotide binding reaction involving a nucleotide-induced change in conformation from an "open" to a "cloned" state of the ternary E.DNA.dNTP complex. Based upon the crystal structure of HIV RT complexed with DNA, a working model has been formulated proposing the movement of the "fingers" domain of the polymerase into the major groove of DNA in response to the binding of a correct base pair. The specific aims of this proposal are to: (1.) Identify the protein structural domains involved in the conformational change leading to tight nucleotide binding and rapid polymerization; specific photo-affinity labeling will be used to localize amino acids which are brought into close contact with the DNA major groove following the conformational change. (2.) Transient state kinetic analysis of site- directed mutants will be employed to further define the contacts made in the "closed" conformational state and to quantify the contributions of individual amino acids. The kinetics of the conformational change will be examined by stopped-flow methods using fluorescently labeled nucleotides, DNA and protein. (3.) The effects of DNA structure on the conformational change and the kinetics of polymerization will be examined. In particular, the role of DNA bends will be investigated, and the mechanisms of frameshift mutagenesis will be examined by measuring the kinetics of extension over premutational frameshift intermediates. (4.) The effect of RNA secondary structure on the kinetics of polymerization catalyzed by RT will be investigated and the effect of the nucleocapsid protein on the ability of the polymerase to read through hairpins will be assessed. (5.) Genes for the mitochondrial polymerase will be cloned and conditions will be optimized for the overexpression and purification of active protein. The mechanism and fidelity of the mitochondrial polymerase will be established by kinetic measurements.

The long term goals of this proposal are to establish the kinetic, thermodynamic and structural basis for force production in microtubule- dependent motility. Kinesin, the putative anterograde motor in axoplasmic transport, will be examined because it is the most simple of the ATPases involved in force production for sliding filament motility. Kinesin will be purified from cloned and overexpressed gene of Drosophila kinesin heavy chains or as native protein, form bovine brain. Conditions will be optimized for expression and purification of the kinesin head fragments, consisting of amino acids 10447 of Drosophila kinesin heavy chain. The microtubule-kinesin complex will be characterized to determine the stoichiometry of kinesin binding to tubulin and to establish conditions for kinetic and equilibrium measurements on the formation of the complex. The kinetics and thermodynamics of the microtubule-kinesin ATPase pathway will be established by transient state kinetic methods. Stopped-flow light scattering methods will be used to measure the rate of ATP-induced dissociation and reformation of the microtubule-kinesin complex. Chemical- quench-flow methods will be used to determine the rates of ATP binding and hydrolysis and ATP synthesis. These kinetic measurements will be extended to examine the potential regulation of kinesin by Ca+2 or by phosphorylation. Site-directed mutagenesis will be used to relate structural domains of the kinesin head to enzymatic functions responsible for energy transduction. Mutation of residues thought to be involved in ATP- or microtubule-binding or in regulation will allow a direct quantitative assessment of the roles of individual or groups of amino acids in each step in the ATPase cycle. Attempts will be made to grow crystals of the cloned kinesin head fragment, in order solve the structure of the kinesin head in the presence and absence of ATP and thereby define in molecular detail the nature of the conformational changes responsible for movement. Microtubule-dependent ATPases have been implicated in a large number of fundamental life processes including anterograde and retrograde axoplasmic transport in neurons and in chromosome movement during cell division. Thus, these studies into the fundamental molecular events leading to generation and control of force production have a wide applicability to health related research.

The long-term aims of this proposal are to establish the structural and mechanistic basis for force production by biological motors in general and the microtubule-kinesin system specifically. Kinesin is a microtubule- dependent motor responsible for the intracellular movements of membrane- bound organelles. It is the father of a class of kinesin-like motor proteins implicated in a wide range of biological processes including axoplasmic transport, chromosome and nuclear movements, pigment granule translocation, and endoplasmic reticulum and Golgi membrane dynamics, and axonemal assembly and motility. Of the three known classes of eukaryotic motors, kinesin is the smallest and most simple in terms of its structural complexity. Moreover, recent work has established conditions for obtaining biochemical quantities of the kinesin motor domain by expression of a truncated Drosophila gene in E. coli. The truncated kinesin, containing 401 N-terminal amino acids and referred to as K401, retains the key features expected for a native kinesin molecule. This form of kinesin binds to microtubules with an 8 nm repeat with one kinesin head per tubulin heterodimer. Previous studies establish the essential features of the reaction pathway of the microtubule-activated ATPase. Further studies outlined in this proposal have three broad specific aims: (1) to complete the kinetic and thermodynamic description of the ATPase pathway; (2) to examine the kinetic and mechanistic basis for apparent processivity in kinesin motility and possible cooperativity between kinesin heads; and (3) to establish the structural basis for force production. These goals will be addressed by a comprehensive kinetic analysis of the ATPase pathway using transient kinetic methods, measurements of the free energy change occurring in each step in the pathway, and structural studies by electron microscopy and crystallography to define the molecular basis for force production. A comprehensive kinetic and mechanistic analysis of mutant and wild-type proteins will serve to define the relationship of the structure to energy transduction. The combination of approaches outlined here will provide rigorous and direct information to define the structural and mechanistic basis for force production by kinesin. The work will thereby provide an understanding of the biological phenomena pertaining to the role of kinesin in intracellular movement.

The goal of this application is to acquire a new Beckman analytical ultracentrifuge and accessories to be used to establish a facility for the analysis of macromolecules and their interactions between and within macromolecular assemblages are frequently the key to understanding many complex cellular processes of medical importance. A group of 8 investigators with 25 graduate students and 10 post-doctoral fellows will form the initial user group in studies of multi-subunit protein assembly, ligand-dependent self-association processes, other protein-protein interactions, protein-nucleic acid interactions, and micelle-micelle interactions. The facility will become part of an established Protein Microanalysis Core Facility of the Institute for Cellular and Molecular Biology of the University. The ultracentrifuge will be an important tool for research and training of graduate students and postdoctoral fellows in biophysical chemistry.

DESCRIPTION (provided by applicant): The long term goals of this proposal are to define the structural, kinetic, thermodynamic and mechanistic basis for nucleotide selectivity during DNA replication by a high fidelity DNA polymerase. Elementary steps in the reactions governing nucleotide selectivity will be defined by comprehensive and rigorous kinetic analysis using transient-state kinetic methods (stopped-flow and chemical-quench-flow), combined with site-directed mutagenesis and site-specific labeling. The T7 DNA polymerase will be used as a model system, because it represents the best available high fidelity polymerase with a high resolution crystal structure of the E-DNA-nucleotide ternary complex, poised to carry out catalysis. The kinetics of nucleotide-induced changes in protein structure, and the role of this important rearrangement of the active site residues in the specificity and efficiency of DNA replication, will be established. The kinetic partitioning between the DNA polymerase active site and the proofreading exonuclease active site will be examined, including analysis of the role of structural elements in the protein that sense mismatches at the polymerase site and those that stabilize the binding and catalysis of single-stranded DNA at the exonuclease site. This work will provide a comprehensive picture of the mechanisms by which DNA polymerases achieve such extraordinary fidelity in replicating DNA. The work is important, in that it will provide the standard for critical events responsible for nucleotide recognition and discrimination against mismatches or nucleotide analogs. The results will be applied to improve our understanding of processes underlying the origins of some cancers, aging and hereditary disorders related to mitochondria! DNA replication errors, the selective incorporation of nucleoside analogs by viral polymerases, and the toxic side effects of nucleoside analogs used to treat viral infections. The fundamental molecular details provided by this work will provide the basis for understanding nucleotide selectivity in systems that may not be amenable to such detailed analysis and are causally related to these significant human health issues.

DESCRIPTION (provided by applicant): The effectiveness of nucleoside analogs used to treat HIV infections is limited by the evolution of resistance by HIV reverse transcriptase (RT), and by toxicity due to incorporation into mitochondrial DNA by the human mitochondrial DNA polymerase. The clinically observable effectiveness versus toxicity of nucleoside analogs can be understood at the molecular level in terms of the discrimination against these analogs during DNA polymerization catalyzed by HIV RT relative to that by the human mitochondrial DNA polymerase. Understanding polymerase specificity is at the heart of the problems inherent in developing less toxic and more effective nucleoside analogs. This application is a continuation of our studies to understand nucleotide selectivity by the human mitochondrial DNA polymerase and its role in the toxicity of nucleoside analogs. In collaboration with Whitney Yin, we will solve the structure of the mitochondrial DNA polymerase and use the new structural information to examine the efficiency and specificity of nucleotide incorporation. We will use site-directed mutagenesis and comprehensive kinetic analysis to evaluate the roles of individual amino acids. We will examine mutants in the mitochondrial polymerase gene that are linked to heritable diseases and attempt to correlate changes in structure and function of the polymerase to the physiological effects of mutations. These studies will provide additional data to understand the physiological basis for the toxicity of nucleoside analogs, and the role of mutations and oxidative damage in ageing. Initiation of DNA polymerization at the mitochondrial replication origin will be studied using synthetic RNA/DNA duplex. We will also reconstitute the replisome using the mitochondrial helicase with the polymerase to examine leading strand synthesis. The roles of the accessory protein and the possible involvement of p53, a tumor suppressor, in regulating polymerase activity will be assessed. We will use pre-steady state and single turnover rapid kinetic studies to directly examine reactions occurring at the active. These methods enable reaction pathways to be established and reaction rates to be quantified by direct measurement and these kinetic parameters can be related directly to structure. This research will provide a better understanding of the role of the mitochondrial polymerase in diseases related to mitochondrial function, and will provide new information to define the molecular basis for nucleotide discrimination by the human mitochondrial DNA polymerase, and it will facilitate the continued development of more effective, less toxic nucleoside analog to combat HIV infections. PUBLIC HEALTH RELEVANCE The effectiveness of nucleoside analogs used to treat HIV infections is limited by their toxicity due to incorporation into mitochondrial DNA by the human mitochondrial DNA polymerase. This research will provide a better understanding of the role of the mitochondrial polymerase in diseases related to mitochondrial function, will provide new information to define the molecular basis for nucleotide discrimination, and will facilitate the development of more effective, less toxic nucleoside analog to combat HIV infections.

DESCRIPTION (provided by applicant): Nucleotide selectivity is at the heart of the problem of understanding the effectiveness of nucleoside analogs used to treat HIV infections, and the evolution of resistance. In order to continue to develop new drugs and most efficiently use the ones available, it will be critical to understand the mechanisms by which HIV reverse transcriptase (RT) achieves nucleotide selectivity during DNA polymerization and how the enzyme changes in evolving to increase selectivity against nucleoside analogs while retaining sufficiently efficient incorporation of normal nucleotides. There has been considerable debate over the role of conformational changes in contributing to the selectivity of DNA polymerases as well as for other enzymes. New data suggest that a conformational switch dictates whether a dNTP will be incorporated or rejected. In this proposal we will investigate whether HIV RT follows this new paradigm for DNA polymerase selectivity. In preliminary data, we show that we can label HIV RT with a fluorophore on the fingers domain in a position that provides a signal to monitor the conformational changes upon nucleotide binding, and we present data to define the rates of nucleotide-induced changes in structure that precede incorporation. We will exploit this new signal to establish the pathway of reactions governing selectivity by HIV RT and define the role played by enzyme conformational changes in discrimination against nucleoside analogs. In addition, we will use this signal to examine changes in the dynamics of nucleotide binding and incorporation that underlie resistance to nucleoside analogs. We will also explore the mechanism by which nonnucleoside inhibitors alter dynamics of nucleotide binding and attenuate chemistry at the active site. These studies will be achieved using a combination of kinetic and structural methods, including stopped-flow fluorescence, rapid chemical quench-flow and single molecule fluorescence kinetic studies. This work will define better the reactions governing nucleotide selectivity by HIV RT, allow us to more rigorously interpret observed changes in enzyme structure thought to be responsible for resistance to nucleoside analogs, and provide insights to into the design and evaluation of new drugs needed to manage HIV infections. PUBLIC HEALTH RELEVANCE: Nucleotide selectivity is at the heart of the problem of understanding the effectiveness of nucleoside analogs used to treat HIV infections, and the evolution of resistance. This work will define the elementary steps governing nucleotide selectivity by HIV RT, allow us to more rigorously interpret observed changes in enzyme structure responsible for resistance to nucleoside analogs, and provide insights to into the design and evaluation of new drugs needed to manage HIV infections.

DESCRIPTION (provided by applicant): The hepatitis C virus infects approximately 3% of the world's population, including 4-5 million in the USA. Chronic infection leads to liver cirrhosis an cancer, and in 2007, HCV surpassed HIV in mortality rates. It is expected that successful treatment of HCV infections will require a combination therapy, analogous to current treatments for HIV infections, and that inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) will be a cornerstone of that treatment. The FDA has recently approved a new treatment based upon the first direct antiviral nucleoside analog and new potential nonnucleoside inhibitors (NNI's) are currently in the pipeline. These pharmaceuticals have been developed by using screens based on subgenomic replicons that self- replicates in human hepatoma cell lines. However, biochemical screens for enzyme activity have been limited because of the inefficient de novo initiation of RNA synthesis in vitro and the inability of the viral polymerase to bind duplex RNA (primer/template) from solution, and it is commonly accepted that all crystal structures of NS5B are of an inactive state. Current enzyme assays present an unresolved mixture of slow initiation kinetics and fast elongation and, therefore, it is not possible to know whether a given drug inhibits initiation or elongation. Quantitative data on binding affinity and mechanism of action of each class of drugs are lacking. There is a need for a quantitative assay for enzyme function to establish the kinetic parameters governing nucleotide incorporation, extension and nucleotide-dependent excision, a reaction that we recently showed can effectively remove nucleoside analogs. Non-nucleoside inhibitors (NNI's) have been discovered that bind to at least four distinct sites on the polymerase. These data on various inhibitors raise important questions regarding the mechanisms of action of the different NNI's binding to distinct enzyme sites. We have established conditions for efficient formation and purification of an active, highly processive elongation complex, overcoming the major obstacle to detailed biochemical analysis of NS5B-catalyzed replication. In this proposal, we will use state of the art single turnover kinetic methods to: (1) Establish the fidelity and baseline kinetic parameters governing cognate and noncognate base pair incorporation; (2) Examine the kinetics of incorporation, extension and excision of nucleotide analogs; (3) Establish modes of action for each class of nonnucleoside inhibitors; and (4) Quantify the effects of drug resistance mutations. In addition, hydrogen/deuterium exchange studies will reveal changes in enzyme flexibility in the transition from inactive to active enzyme, and we will attempt to determine the crystal structure of the elongation complex. This work lays the foundation for understanding structure/function relationships governing RNA-dependent RNA polymerization, the mechanisms of action of various drugs currently being investigated, and the evolution of drug resistance.

? DESCRIPTION (provided by applicant): An important goal of biomedical research is to establish the molecular basis for disease so that more effective therapies can be devised. Relating the biochemical effects of heritable point mutations to their physiological and clinical consequences is a challenging but important step toward reaching this goal. Mutations in the human mitochondrial DNA (mtDNA) polymerase have been correlated with various mitochondrial disorders, including mtDNA depletion syndrome, Alpers Syndrome, and progressive external opthalmoplegia (PEO). Symptoms of Alpers Syndrome include liver disease and refractory seizures, while patients with PEO present with progressive weakness of the external ocular muscles and skeletal myopathy. Many of the nucleoside analogs used to treat viral infections have toxic side effects due to inhibition of mtDNA replication, which are seen first as peripheral neuropathy. Mitochondrial DNA replication is performed by a replisome comprised of a nuclearly-encoded DNA polymerase, processivity factor, single-stranded DNA binding protein (mtSSB), and DNA helicase. The major challenge in interpreting the clinical effects of mutations in the mtDNA polymerase lies in understanding the molecular basis for the slow onset of the symptoms. Like other heritable disorders of the mitochondrial genes and the toxic side effects of nucleoside analogs used to treat HIV infection, mutations in the mtDNA polymerase lead to diseases often characterized by slow onset due to the accumulation of mtDNA defects and oxidative damage, although certain mutations lead to more severe symptoms resulting in death within one to two years of birth. Understanding the clinical consequences of point mutations in the mtDNA polymerase requires precise and accurate measurements and rigorous data analysis. We will use site-directed mutagenesis and comprehensive kinetic analysis to evaluate the effects of mutations on the mtDNA polymerase in vitro. In addition, we will work to correlate changes in structure and function of the polymeras to the physiological consequences of these mutations observable in a humanized yeast model system expressing the human mtDNA polymerase, which appears to be a good model system to predict the long term consequences of mutations in humans. We will use single turnover rapid kinetic studies to directly examine reactions occurring at the active site in order to quantify key kinetic parameters governing DNA replication. We will also work to examine the role of the mtDNA helicase and mtSSB in the coordinated DNA unwinding and leading strand synthesis. This research will provide a better understanding of the role of the mtDNA polymerase in diseases related to mitochondrial function, and will provide new information to define the molecular basis for nucleotide discrimination by the human mtDNA polymerase, the physiological basis for the toxicity of nucleoside analogs used to treat HIV infections, and the role of mtDNA polymerase and helicase mutations in heritable disorders.

Project Summary/Abstract Although there is much hope for an effective vaccine to combat COVID-19, a pressing need remains to develop direct acting antivirals in the event that vaccines fail to provide protective immunity, for the treatment of acute infections, and for future coronavirus strains that might evade existing vaccines. The SARS coronavirus (CoV- 2) RNA-dependent RNA polymerase (RdRp) is an attractive target because inhibitors of viral RNA-dependent polymerases form the cornerstone of antiviral drug combination therapy for successful treatment of HIV and hepatitis C virus infections. Remdesivir, a nucleotide analog developed by Gilead, is already showing promise in clinical trials. The long-term goal of this research is to facilitate the development of more effective, less toxic drugs directed against the SARS CoV-2 RdRp. The rationale for this research is based on prior experience demonstrating that accurate measurements of the kinetics of nucleotide incorporation and excision by the viral polymerase/exonuclease translates directly to understanding viral RNA replication and can guide the design of robust assays to find effective inhibitors. Kinetic analysis will be based on single turnover rapid-kinetic measurements of polymerization to provide definitive results to define the mechanistic basis for nucleotide selectivity. Our working hypothesis is that an effective nucleotide analog can be identified and its therapeutic potential quantified based on analysis of the kinetics of incorporation relative to the kinetics of excision by the proofreading exonuclease. Specifically, the aims of this research are to quantify the kinetics of nucleotide incorporation using single turnover kinetic analysis in order to establish the mechanism and overall fidelity of the RNA replication. Parallel studies will establish the kinetic and mechanistic basis for inhibition for nucleotide analogs. We will also include extensive characterization of the kinetics of the proofreading exonuclease to define the rules governing removal of mismatched base pairs and nucleotide analogs. We will also us cryoEM with samples based on our biochemical knowledge to obtain structures of the polymerase with Remdesivir incorporated and of the RdRp with the exonuclease. These studies are innovative in that they take advantage of the most advanced methods of single turnover kinetic analysis and global data fitting developed by the PI to establish the kinetic and thermodynamic basis for polymerase specificity to reveal the basis for discrimination against nucleotide analogs. No other lab is applying such standards to this important problem. Moreover, this quantitative analysis provides an accurate vector pointing toward more effective inhibitors in structure/activity relationship studies. The work is soundly based the the PI's prior work and on preliminary data explaining the kinetic basis for the effectiveness of Remdesivir in competing with ATP. The proposed research will significantly advance our understanding the mechanism and kinetics of CoV RNA replication and provide a sound quantitative basis to find inhibitors acting directly against viral replication. This research has a strong potential to play a key role in the developing direct acting antiviral drugs to combat SARS CoV-2 and future coronaviruses.