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Prakasha Kempaiah

Bio: Prakasha Kempaiah is an academic researcher from Mayo Clinic. The author has contributed to research in topics: Virtual screening & Docking (molecular). The author has an hindex of 4, co-authored 11 publications receiving 32 citations. Previous affiliations of Prakasha Kempaiah include Loyola University Medical Center.

Papers
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Journal ArticleDOI
14 Jan 2021-Methods
TL;DR: In this article, a high-throughput virtual screen of drugs and investigational molecules was performed against viral targets using three sequential docking modes (i.e., HTVS, SP, and XP).
Abstract: SARS-CoV-2, the virus that causes COVID-19 consists of several enzymes with essential functions within its proteome. Here, we focused on repurposing approved and investigational drugs/compounds. We targeted seven proteins with enzymatic activities known to be essential at different stages of the viral cycle including PLpro, 3CLpro, RdRP, Helicase, ExoN, NendoU, and 2′-O-MT. For virtual screening, energy minimization of a crystal structure of the modeled protein was carried out using the Protein Preparation Wizard (Schrodinger LLC 2020-1). Following active site selection based on data mining and COACH predictions, we performed a high-throughput virtual screen of drugs and investigational molecules (n = 5903). The screening was performed against viral targets using three sequential docking modes (i.e., HTVS, SP, and XP). Virtual screening identified ∼290 potential inhibitors based on the criteria of energy, docking parameters, ligand, and binding site strain and score. Drugs specific to each target protein were further analyzed for binding free energy perturbation by molecular mechanics (prime MM-GBSA) and pruning the hits to the top 32 candidates. The top lead from each target pool was further subjected to molecular dynamics simulation using the Desmond module. The resulting top eight hits were tested for their SARS-CoV-2 anti-viral activity in-vitro. Among these, a known inhibitor of protein kinase C isoforms, Bisindolylmaleimide IX (BIM IX), was found to be a potent inhibitor of SARS-CoV-2. Further, target validation through enzymatic assays confirmed 3CLpro to be the target. This is the first study that has showcased BIM IX as a COVID-19 inhibitor thereby validating our pipeline.

22 citations

Journal ArticleDOI
TL;DR: In this article, the authors reported results of highly efficacious viral entry blocking properties of heparin (IC50 = 12.3 nM) in the complete virus assay, and further, propose ways to use it as a potential transmission blocker.
Abstract: The world is currently facing a novel coronavirus (SARS-CoV-2) pandemic. The greatest threat that is disrupting the normal functioning of society is the exceptionally high species independent transmission. Drug repurposing is understood to be the best strategy to immediately deploy well-characterized agents against new pathogens. Several repurposable drugs are already in evaluation for determining suitability to treat COVID-19. One such promising compound includes heparin, which is widely used in reducing thrombotic events associated with COVID-19 induced pathology. As part of identifying target-specific antiviral compounds among FDA and world-approved libraries using high-throughput virtual screening (HTVS), we previously evaluated top hits for anti-SARS-CoV-2 activity. Here, we report results of highly efficacious viral entry blocking properties of heparin (IC50 = 12.3 nM) in the complete virus assay, and further, propose ways to use it as a potential transmission blocker. Exploring further, our in-silico analysis indicated that the heparin interacts with post-translational glycoconjugates present on spike proteins. The patterns of accessible spike-glycoconjugates in open and closed states are completely contrasted by one another. Heparin-binding to the open conformation of spike structurally supports the state and may aid ACE2 binding as reported with cell surface-bound heparan sulfate. We also studied spike protein mutant variants' heparin interactions for possible resistance. Based on available data and optimal absorption properties by the skin, heparin could potentially be used to block SARS-CoV-2 transmission. Studies should be designed to exploit its nanomolar antiviral activity to formulate heparin as topical or inhalation-based formulations, particularly on exposed areas and sites of primary viremia e.g. ACE2 rich epithelia of the eye (conjunctiva/lids), nasal cavity, and mouth.

21 citations

Journal ArticleDOI
18 Aug 2021
TL;DR: A derivative of piperazine based '(2S,3S)-3-amino-1-(4-(tert-butyl)benzyl)piperazin-1-yl)-4-phenylbutan-2-ol' (IV) from the authors' in-house libraries having potential efficacy against SARS-CoV-2 in in vitro assays is reported.
Abstract: NendoU (NSP15) is an Mn(2+)-dependent, uridylate-specific enzyme, which leaves 2'-3'-cyclic phosphates 5' to the cleaved bond. Our in-house library was subjected to high throughput virtual screening (HTVS) to identify compounds with potential to inhibit NendoU enzyme, high-rank compounds (those that bound to multiple target structures) were further subjected to 100 nanoseconds MD simulations. Among these, one was found to be bound highly stable within the active site of the NendoU protein structure. Here, we are reporting a derivative of piperazine based '(2S,3S)-3-amino-1-(4-(4-(tert-butyl)benzyl)piperazin-1-yl)-4-phenylbutan-2-ol' (IV) from our in-house libraries having potential efficacy against SARS-CoV-2 in in vitro assays. This compound demonstrated inhibition of viral replication at the same level as Ivermectin, a known SARS-CoV-2 inhibitor, which is not used due to its toxicity at a higher than the currently approved dosage. Compound IV was not toxic to the cell lines up to a 50 μM concentration and exhibited IC50s of 4.97 μM and 8.46 μM in viral entry and spread assay, respectively. Therefore, this novel class of NendoU inhibitor could provide new insights for the development of treatment options for COVID-19.

14 citations

Journal ArticleDOI
TL;DR: This review complements existing ones by discussing current state of treatment options, therapeutic bottlenecks including chemoresistance and toxicity, as well as drug targets, and highlights innovative applications of nanotherapeutics-based formulations, inhibitory potential of leishmanicides, anti-microbial peptides and organometallic compounds on leishmania species.
Abstract: The global prevalence of leishmaniasis has increased with skyrocketed mortality in the past decade. The causative agent of leishmaniasis is Leishmania species, which infects populations in almost all the continents. Prevailing treatment regimens are consistently inefficient with reported side effects, toxicity and drug resistance. This review complements existing ones by discussing the current state of treatment options, therapeutic bottlenecks including chemoresistance and toxicity, as well as drug targets. It further highlights innovative applications of nanotherapeutics-based formulations, inhibitory potential of leishmanicides, anti-microbial peptides and organometallic compounds on leishmanial species. Moreover, it provides essential insights into recent machine learning-based models that have been used to predict novel leishmanicides and also discusses other new models that could be adopted to develop fast, efficient, robust and novel algorithms to aid in unraveling the next generation of anti-leishmanial drugs. A plethora of enriched functional genomic, proteomic, structural biology, high throughput bioassay and drug-related datasets are currently warehoused in both general and leishmania-specific databases. The warehoused datasets are essential inputs for training and testing algorithms to augment the prediction of biotherapeutic entities. In addition, we demonstrate how pharmacoinformatics techniques including ligand-, structure- and pharmacophore-based virtual screening approaches have been utilized to screen ligand libraries against both modeled and experimentally solved 3D structures of essential drug targets. In the era of data-driven decision-making, we believe that highlighting intricately linked topical issues relevant to leishmanial drug discovery offers a one-stop-shop opportunity to decipher critical literature with the potential to unlock implicit breakthroughs.

9 citations

Journal ArticleDOI
TL;DR: In this article, a hydroxyethylamine (HEA) library was constructed from harvested chemical structures from all the series being used in our laboratories for screening against malaria and Leishmania parasites.
Abstract: The continued toll of COVID-19 has halted the smooth functioning of civilization on a global scale. With a limited understanding of all the essential components of viral machinery and the lack of structural information of this new virus, initial drug discovery efforts had limited success. The availability of high-resolution crystal structures of functionally essential SARS-CoV-2 proteins, including 3CLpro, supports the development of target-specific therapeutics. 3CLpro, the main protease responsible for the processing of viral polypeptide, plays a vital role in SARS-CoV-2 viral replication and translation and is an important target in other coronaviruses. Additionally, 3CLpro is the target of repurposed drugs, such as lopinavir and ritonavir. In this study, target proteins were retrieved from the protein data bank (PDB IDs: 6 M03, 6LU7, 2GZ7, 6 W63, 6SQS, 6YB7, and 6YVF) representing different open states of the main protease to accommodate macromolecular substrate. A hydroxyethylamine (HEA) library was constructed from harvested chemical structures from all the series being used in our laboratories for screening against malaria and Leishmania parasites. The database consisted of ∼1000 structure entries, of which 70% were new to ChemSpider at the time of screening. This in-house library was subjected to high throughput virtual screening (HTVS), followed by standard precision (SP) and then extra precision (XP) docking (Schrodinger LLC 2021). The ligand strain and complex energy of top hits were calculated by Molecular Mechanics Generalized Born Surface Area (MM/GBSA) method. Promising hit compounds (n = 40) specifically binding to 3CLpro with high energy and average MM/GBSA scores were then subjected to (100-ns) MD simulations. Using this sequential selection followed by an in-silico validation approach, we found a promising HEA-based compound (N,N'-((3S,3′S)-piperazine-1,4-diylbis(3-hydroxy-1-phenylbutane-4,2-diyl))bis(2-(5-methyl-1,3-dioxoisoindolin-2-yl)-3-phenylpropanamide)), which showed high in vitro antiviral activity against SARS-CoV-2. Further to reduce the size of the otherwise larger ligand, a pharmacophore-based predicted library of ∼42 derivatives was constructed, which were added to the previous compound library and rescreened virtually. Out of several hits from the predicted library, two compounds were synthesized, tested against SARS-CoV-2 culture, and found to have markedly improved antiviral activity.

7 citations


Cited by
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Journal ArticleDOI
TL;DR: SARS-CoV-2 is an extremely contagious respiratory virus causing adult atypical pneumonia COVID-19 with severe acute respiratory syndrome (SARS) as discussed by the authors, which has a single-stranded, positive-sense RNA (+RNA) genome of 29.9 kb and exhibits significant genetic shift from different isolates.
Abstract: SARS-CoV-2 is an extremely contagious respiratory virus causing adult atypical pneumonia COVID-19 with severe acute respiratory syndrome (SARS). SARS-CoV-2 has a single-stranded, positive-sense RNA (+RNA) genome of ~ 29.9 kb and exhibits significant genetic shift from different isolates. After entering the susceptible cells expressing both ACE2 and TMPRSS2, the SARS-CoV-2 genome directly functions as an mRNA to translate two polyproteins from the ORF1a and ORF1b region, which are cleaved by two viral proteases into sixteen non-structural proteins (nsp1-16) to initiate viral genome replication and transcription. The SARS-CoV-2 genome also encodes four structural (S, E, M and N) and up to six accessory (3a, 6, 7a, 7b, 8, and 9b) proteins, but their translation requires newly synthesized individual subgenomic RNAs (sgRNA) in the infected cells. Synthesis of the full-length viral genomic RNA (gRNA) and sgRNAs are conducted inside double-membrane vesicles (DMVs) by the viral replication and transcription complex (RTC), which comprises nsp7, nsp8, nsp9, nsp12, nsp13 and a short RNA primer. To produce sgRNAs, RTC starts RNA synthesis from the highly structured gRNA 3' end and switches template at various transcription regulatory sequence (TRSB) sites along the gRNA body probably mediated by a long-distance RNA-RNA interaction. The TRS motif in the gRNA 5' leader (TRSL) is responsible for the RNA-RNA interaction with the TRSB upstream of each ORF and skipping of the viral genome in between them to produce individual sgRNAs. Abundance of individual sgRNAs and viral gRNA synthesized in the infected cells depend on the location and read-through efficiency of each TRSB. Although more studies are needed, the unprecedented COVID-19 pandemic has taught the world a painful lesson that is to invest and proactively prepare future emergence of other types of coronaviruses and any other possible biological horrors.

90 citations

Journal ArticleDOI
TL;DR: In this paper, a new fucosylated chondroitin sulfate (FucCS) from the sea cucumber Pentacta pygmaea was isolated and structurally characterized by NMR.
Abstract: Certain sulfated glycans, including those from marine sources, can show potential effects against SARS-CoV-2. Here, a new fucosylated chondroitin sulfate (FucCS) from the sea cucumber Pentacta pygmaea (PpFucCS) (MW ∼10–60 kDa) was isolated and structurally characterized by NMR. PpFucCS is composed of {→3)-β-GalNAcX-(1→4)-β-GlcA-[(3→1)Y]-(1→}, where X = 4S (80%), 6S (10%) or nonsulfated (10%), Y = α-Fuc2,4S (40%), α-Fuc2,4S-(1→4)-α-Fuc (30%), or α-Fuc4S (30%), and S = SO3−. The anti-SARS-CoV-2 activity of PpFucCS and those of the FucCS and sulfated fucan isolated from Isostichopus badionotus (IbFucCS and IbSF) were compared with that of heparin. IC50 values demonstrated the activity of the three holothurian sulfated glycans to be ∼12 times more efficient than heparin, with no cytotoxic effects. The dissociation constant (KD) values obtained by surface plasmon resonance of the wildtype SARS-CoV-2 spike (S)-protein receptor-binding domain (RBD) and N501Y mutant RBD in interactions with the heparin-immobilized sensor chip were 94 and 1.8 × 103 nM, respectively. Competitive surface plasmon resonance inhibition analysis of PpFucCS, IbFucCS, and IbSF against heparin binding to wildtype S-protein showed IC50 values (in the nanomolar range) 6, 25, and 6 times more efficient than heparin, respectively. Data from computational simulations suggest an influence of the sulfation patterns of the Fuc units on hydrogen bonding with GlcA and that conformational change of some of the oligosaccharide structures occurs upon S-protein RBD binding. Compared with heparin, negligible anticoagulant action was observed for IbSF. Our results suggest that IbSF may represent a promising molecule for future investigations against SARS-CoV-2.

26 citations

Journal ArticleDOI
07 Jun 2021-Cells
TL;DR: In this paper, the effects of various heparins on ACE2-independent spike protein interaction with cells were investigated, and it was shown that the furin cleavage site might also be a heparin-binding site and potentially important for interactions with host cells.
Abstract: Coronaviruses such as SARS-CoV-2, which is responsible for COVID-19, depend on virus spike protein binding to host cell receptors to cause infection. The SARS-CoV-2 spike protein binds primarily to ACE2 on target cells and is then processed by membrane proteases, including TMPRSS2, leading to viral internalisation or fusion with the plasma membrane. It has been suggested, however, that receptors other than ACE2 may be involved in virus binding. We have investigated the interactions of recombinant versions of the spike protein with human epithelial cell lines that express low/very low levels of ACE2 and TMPRSS2 in a proxy assay for interaction with host cells. A tagged form of the spike protein containing the S1 and S2 regions bound in a temperature-dependent manner to all cell lines, whereas the S1 region alone and the receptor-binding domain (RBD) interacted only weakly. Spike protein associated with cells independently of ACE2 and TMPRSS2, while RBD required the presence of high levels of ACE2 for interaction. As the spike protein has previously been shown to bind heparin, a soluble glycosaminoglycan, we tested the effects of various heparins on ACE2-independent spike protein interaction with cells. Unfractionated heparin inhibited spike protein interaction with an IC50 value of <0.05 U/mL, whereas two low-molecular-weight heparins were less effective. A mutant form of the spike protein, lacking the arginine-rich putative furin cleavage site, interacted only weakly with cells and had a lower affinity for unfractionated and low-molecular-weight heparin than the wild-type spike protein. This suggests that the furin cleavage site might also be a heparin-binding site and potentially important for interactions with host cells. The glycosaminoglycans heparan sulphate and dermatan sulphate, but not chondroitin sulphate, also inhibited the binding of spike protein, indicating that it might bind to one or both of these glycosaminoglycans on the surface of target cells.

25 citations

Journal ArticleDOI
TL;DR: Future antimalarial drug discovery efforts seeking to identify plasmepin inhibitors should focus on incorporating non-haemoglobinase plasmepsins such as V, IX and X in their screening in order to maximise chances of success.
Abstract: Plasmepsins represent novel antimalarial drug targets. However, plasmepsin-based antimalarial drug discovery efforts in the past 2 decades have generally suffered some drawbacks including lack of translatability of target inhibition to potent parasite inhibition in vitro and in vivo as well as poor selectivity over the related human aspartic proteases. Most studies reported in this period have over-relied on the use of hemoglobinase plasmepsins I-IV (particularly I and II) as targets for the new inhibitors even though these are known to be nonessential at the asexual stage of parasite development. Therefore, future antimalarial drug discovery efforts seeking to identify plasmepsin inhibitors should focus on incorporating non-hemoglobinase plasmepsins such as V, IX, and X in their screening in order to maximize chances of success. Additionally, there is need to go beyond just target enzymatic activity profiling to establishing cellular activity, physicochemical as well as drug metabolism and pharmacokinetics properties and finally in vivo proof-of-concept while ensuring selectivity over related human host proteases.

21 citations

Journal ArticleDOI
TL;DR: In this article, the authors reported results of highly efficacious viral entry blocking properties of heparin (IC50 = 12.3 nM) in the complete virus assay, and further, propose ways to use it as a potential transmission blocker.
Abstract: The world is currently facing a novel coronavirus (SARS-CoV-2) pandemic. The greatest threat that is disrupting the normal functioning of society is the exceptionally high species independent transmission. Drug repurposing is understood to be the best strategy to immediately deploy well-characterized agents against new pathogens. Several repurposable drugs are already in evaluation for determining suitability to treat COVID-19. One such promising compound includes heparin, which is widely used in reducing thrombotic events associated with COVID-19 induced pathology. As part of identifying target-specific antiviral compounds among FDA and world-approved libraries using high-throughput virtual screening (HTVS), we previously evaluated top hits for anti-SARS-CoV-2 activity. Here, we report results of highly efficacious viral entry blocking properties of heparin (IC50 = 12.3 nM) in the complete virus assay, and further, propose ways to use it as a potential transmission blocker. Exploring further, our in-silico analysis indicated that the heparin interacts with post-translational glycoconjugates present on spike proteins. The patterns of accessible spike-glycoconjugates in open and closed states are completely contrasted by one another. Heparin-binding to the open conformation of spike structurally supports the state and may aid ACE2 binding as reported with cell surface-bound heparan sulfate. We also studied spike protein mutant variants' heparin interactions for possible resistance. Based on available data and optimal absorption properties by the skin, heparin could potentially be used to block SARS-CoV-2 transmission. Studies should be designed to exploit its nanomolar antiviral activity to formulate heparin as topical or inhalation-based formulations, particularly on exposed areas and sites of primary viremia e.g. ACE2 rich epithelia of the eye (conjunctiva/lids), nasal cavity, and mouth.

21 citations