Publications by authors named "Amaravadhi Harikishore"

21 Publications

  • Page 1 of 1

Targeting C-terminal Helical bundle of NCOVID19 Envelope (E) protein.

Int J Biol Macromol 2021 Apr 4;175:131-139. Epub 2021 Feb 4.

Department of Biophysics, Bose Institute, Kolkata 700 054, India. Electronic address:

One of the most crucial characteristic traits of Envelope (E) proteins in the severe acute respiratory syndrome SARS-CoV-1 and NCOVID19 viruses is their membrane-associated oligomerization led ion channel activity, virion assembly, and replication. NMR spectroscopic structural studies of envelope proteins from both the SARS CoV-1/2 reveal that this protein assembles into a homopentamer. Proof of concept studies via truncation mutants on either transmembrane (VFLLV), glycosylation motif (CACCN), hydrophobic helical bundle (PVYVY) as well as replacing C-terminal "DLLV" segments or point mutants such as S68, E69 residues with cysteine have significantly reduced viral titers of SARS-CoV-1. In this present study, we have first developed SARS-2 E protein homology model based on the pentamer coordinates of SARS-CoV-1 E protein (86.4% structural identity) with good stereochemical quality. Next, we focused on the glycosylation motif and hydrophobic helical bundle regions of E protein shown to be important for viral replication. A four feature (4F) model comprising of an acceptor targeting S60 hydroxyl group, a donor feature anchoring the C40 residue, and two hydrophobic features anchoring the V47 L28, L31, Y55, and P51 residues formed the protein based pharmacophore model targeting the glycosylation motif and helical bundle of E protein. Database screening with this 4F protein pharmacophore, ADMET property filtering on enamine small molecule discovery collection yielded a focused library of ~7000 hits. Further molecular docking and visual inspection of docked pose interactions at the above mention V47 L28, L31, Y55, P51, S60, C40 residues led to the identification of 10 best hits. Our STD NMR binding assay results demonstrate that the ligand 3, 2-(2-amino-2-oxo-ethoxy)-N-benzyl-benzamide, binds to NCOVID19 E protein with a binding affinity (K) of 141.7 ± 13.6 μM. Furthermore, the ligand 3 also showed binding to C-terminal peptide (NR25) as evidenced with the STD spectrums of wild type E protein would serve to confirm the involvement of C-terminal helical bundle as envisaged in this study.
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http://dx.doi.org/10.1016/j.ijbiomac.2021.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7859708PMC
April 2021

Antiviral activity against Middle East Respiratory Syndrome coronavirus by Montelukast, an anti-asthma drug.

Antiviral Res 2021 01 10;185:104996. Epub 2020 Dec 10.

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore. Electronic address:

Middle East Respiratory Syndrome (MERS) is a respiratory disease caused by a coronavirus (MERS-CoV). Since its emergence in 2012, nosocomial amplifications have led to its high epidemic potential and mortality rate of 34.5%. To date, there is an unmet need for vaccines and specific therapeutics for this disease. Available treatments are either supportive medications in use for other diseases or those lacking specificity requiring higher doses. The viral infection mode is initiated by the attachment of the viral spike glycoprotein to the human Dipeptidyl Peptidase IV (DPP4). Our attempts to screen antivirals against MERS led us to identify montelukast sodium hydrate (MSH), an FDA-approved anti-asthma drug, as an agent attenuating MERS-CoV infection. We showed that MSH directly binds to MERS-CoV-Receptor-Binding Domain (RBD) and inhibits its molecular interaction with DPP4 in a dose-dependent manner. Our cell-based inhibition assays using MERS pseudovirions demonstrated that viral infection was significantly inhibited by MSH and was further validated using infectious MERS-CoV culture. Thus, we propose MSH as a potential candidate for therapeutic developments against MERS-CoV infections.
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http://dx.doi.org/10.1016/j.antiviral.2020.104996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726485PMC
January 2021

A systematic assessment of mycobacterial F -ATPase subunit ε's role in latent ATPase hydrolysis.

FEBS J 2021 02 4;288(3):818-836. Epub 2020 Jul 4.

School of Biological Sciences, Nanyang Technological University, Singapore City, Singapore.

In contrast to most bacteria, the mycobacterial F F -ATP synthase (α :β :γ:δ:ε:a:b:b':c ) does not perform ATP hydrolysis-driven proton translocation. Although subunits α, γ and ε of the catalytic F -ATPase component α :β :γ:ε have all been implicated in the suppression of the enzyme's ATPase activity, the mechanism remains poorly defined. Here, we brought the central stalk subunit ε into focus by generating the recombinant Mycobacterium smegmatis F -ATPase (MsF -ATPase), whose 3D low-resolution structure is presented, and its ε-free form MsF αβγ, which showed an eightfold ATP hydrolysis increase and provided a defined system to systematically study the segments of mycobacterial ε's suppression of ATPase activity. Deletion of four amino acids at ε's N terminus, mutant MsF αβγε , revealed similar ATP hydrolysis as MsF αβγ. Together with biochemical and NMR solution studies of a single, double, triple and quadruple N-terminal ε-mutants, the importance of the first N-terminal residues of mycobacterial ε in structure stability and latency is described. Engineering ε's C-terminal mutant MsF αβγε and MsF αβγε with deletion of the C-terminal residue D121 and the two C-terminal ɑ-helices, respectively, revealed the requirement of the very C terminus for communication with the catalytic α β -headpiece and its function in ATP hydrolysis inhibition. Finally, we applied the tools developed during the study for an in silico screen to identify a novel subunit ε-targeting F-ATP synthase inhibitor.
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http://dx.doi.org/10.1111/febs.15440DOI Listing
February 2021

Discovery of a Novel Mycobacterial F-ATP Synthase Inhibitor and its Potency in Combination with Diarylquinolines.

Angew Chem Int Ed Engl 2020 08 26;59(32):13295-13304. Epub 2020 May 26.

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Republic of Singapore.

The F F -ATP synthase is required for growth and viability of Mycobacterium tuberculosis and is a validated clinical target. A mycobacterium-specific loop of the enzyme's rotary γ subunit plays a role in the coupling of ATP synthesis within the enzyme complex. We report the discovery of a novel antimycobacterial, termed GaMF1, that targets this γ subunit loop. Biochemical and NMR studies show that GaMF1 inhibits ATP synthase activity by binding to the loop. GaMF1 is bactericidal and is active against multidrug- as well as bedaquiline-resistant strains. Chemistry efforts on the scaffold revealed a dynamic structure activity relationship and delivered analogues with nanomolar potencies. Combining GaMF1 with bedaquiline or novel diarylquinoline analogues showed potentiation without inducing genotoxicity or phenotypic changes in a human embryonic stem cell reporter assay. These results suggest that GaMF1 presents an attractive lead for the discovery of a novel class of anti-tuberculosis F-ATP synthase inhibitors.
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http://dx.doi.org/10.1002/anie.202002546DOI Listing
August 2020

Targeting the menaquinol binding loop of mycobacterial cytochrome bd oxidase.

Mol Divers 2021 Feb 14;25(1):517-524. Epub 2020 Jan 14.

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Republic of Singapore.

Mycobacteria have shown enormous resilience to survive and persist by remodeling and altering metabolic requirements. Under stringent conditions or exposure to drugs, mycobacteria have adapted to rescue themselves by shutting down their major metabolic activity and elevate certain survival factor levels and efflux pathways to survive and evade the effects of drug treatments. A fundamental feature in this adaptation is the ability of mycobacteria to vary the enzyme composition of the electron transport chain (ETC), which generates the proton motive force for the synthesis of adenosine triphosphate via oxidative phosphorylation. Mycobacteria harbor dehydrogenases to fuel the ETC, and two terminal respiratory oxidases, an aa3-type cytochrome c oxidase (cyt-bcc-aa3) and a bacterial specific cytochrome bd-type menaquinol oxidase (cyt-bd). In this study, we employed homology modeling and structure-based virtual screening studies to target mycobacteria-specific residues anchoring the b558 menaquinol binding region of Mycobacterium tuberculosis cyt-bd oxidase to obtain a focused library. Furthermore, ATP synthesis inhibition assays were carried out. One of the ligands MQL-H inhibited both NADH- and succinate-driven ATP synthesis inhibition of Mycobacterium smegmatis inside-out vesicles in micromolar potency. Similarly, MQL-H also inhibited NADH-driven ATP synthesis in inside-out vesicles of the cytochrome-bcc oxidase deficient M. smegmatis strain. Since neither varying the electron donor substrates nor deletion of the cyt-bcc oxidase, a major source of protons, hindered the inhibitory effects of the MQL-H, reflecting that MQL-H targets the terminal oxidase cytochrome bd oxidase, which was consistent with molecular docking studies. Characterization of novel cytochrome bd oxidase Menaquinol binding domain inhibitor (MQL-H2) using virtual screening and ATP synthesis inhibition assays.
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http://dx.doi.org/10.1007/s11030-020-10034-0DOI Listing
February 2021

Disrupting coupling within mycobacterial F-ATP synthases subunit ε causes dysregulated energy production and cell wall biosynthesis.

Sci Rep 2019 11 14;9(1):16759. Epub 2019 Nov 14.

Nanyang Technological University, School of Biological Sciences, 60 Nanyang Drive, Singapore, 637551, Republic of Singapore.

The dynamic interaction of the N- and C-terminal domains of mycobacterial F-ATP synthase subunit ε is proposed to contribute to efficient coupling of H-translocation and ATP synthesis. Here, we investigate crosstalk between both subunit ε domains by introducing chromosomal atpC missense mutations in the C-terminal helix 2 of ε predicted to disrupt inter domain and subunit ε-α crosstalk and therefore coupling. The ε mutant εR105A,R111A,R113A,R115A (ε) showed decreased intracellular ATP, slower growth rates and lower molar growth yields on non-fermentable carbon sources. Cellular respiration and metabolism were all accelerated in the mutant strain indicative of dysregulated oxidative phosphorylation. The ε mutant exhibited an altered colony morphology and was hypersusceptible to cell wall-acting antimicrobials suggesting defective cell wall biosynthesis. In silico screening identified a novel mycobacterial F-ATP synthase inhibitor disrupting ε's coupling activity demonstrating the potential to advance this regulation as a new area for mycobacterial F-ATP synthase inhibitor development.
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http://dx.doi.org/10.1038/s41598-019-53107-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856130PMC
November 2019

Ursolic acid exerts anti-cancer activity by suppressing vaccinia-related kinase 1-mediated damage repair in lung cancer cells.

Sci Rep 2015 Sep 28;5:14570. Epub 2015 Sep 28.

Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea.

Many mitotic kinases have been targeted for the development of anti-cancer drugs, and inhibitors of these kinases have been expected to perform well for cancer therapy. Efforts focused on selecting good targets and finding specific drugs to target are especially needed, largely due to the increased frequency of anti-cancer drugs used in the treatment of lung cancer. Vaccinia-related kinase 1 (VRK1) is a master regulator in lung adenocarcinoma and is considered a key molecule in the adaptive pathway, which mainly controls cell survival. We found that ursolic acid (UA) inhibits the catalytic activity of VRK1 via direct binding to the catalytic domain of VRK1. UA weakens surveillance mechanisms by blocking 53BP1 foci formation induced by VRK1 in lung cancer cells, and possesses synergistic anti-cancer effects with DNA damaging drugs. Taken together, UA can be a good anti-cancer agent for targeted therapy or combination therapy with DNA damaging drugs for lung cancer patients.
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http://dx.doi.org/10.1038/srep14570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585938PMC
September 2015

Immunophilins: Structures, Mechanisms and Ligands.

Curr Mol Pharmacol 2015 ;9(1):37-47

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551.

Immunophilins consist of a family of highly conserved proteins which possess binding abilities to immunosuppressive drugs. Cyclophilins (Cyps) and FK506-binding proteins (FKBP) are family proteins collectively referred as immunophilins. Most Cyps and FKBP family members catalyse peptidyl-prolyl cis/trans isomerase (PPIase) mediated reactions and form binary complexes with their ligands cyclosporine A and FK506. Immunophilins are also involved in key biochemical processes including protein folding, receptor signalling, protein trafficking, and transcription and exhibit versatile biological functions, when complexed with their ligands. Therapeutic implications of immunophilins and effects of their ligands in neurodegenerative disorders, cancer, and infectious diseases have been accumulating in recent years. This review focuses on molecular characteristics of the canonical and non-canonical immunophilin family members from human and Plasmodium falciparum and P. vivax, recent progress on immunophilin inhibitor development, and future perspectives of structure-based design of non-immunosuppressive immunophilin ligands with potential pharmacological activities against infectious diseases.
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http://dx.doi.org/10.2174/1874467208666150519113427DOI Listing
October 2016

Combination of pharmacophore hypothesis and molecular docking to identify novel inhibitors of HCV NS5B polymerase.

Mol Divers 2015 Aug 11;19(3):529-39. Epub 2015 Apr 11.

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore,

Hepatitis C virus (HCV) infection or HCV-related liver diseases are now shown to cause more than 350,000 deaths every year. Adaptability of HCV genome to vary its composition and the existence of multiple strains makes it more difficult to combat the emergence of drug-resistant HCV infections. Among the HCV polyprotein which has both the structural and non-structural regions, the non-structural protein NS5B RNA-dependent RNA polymerase (RdRP) mainly mediates the catalytic role of RNA replication in conjunction with its viral protein machinery as well as host chaperone proteins. Lack of such RNA-dependent RNA polymerase enzyme in host had made it an attractive and hotly pursued target for drug discovery efforts. Recent drug discovery efforts targeting HCV RdRP have seen success with FDA approval for sofosbuvir as a direct-acting antiviral against HCV infection. However, variations in drug-binding sites induce drug resistance, and therefore targeting allosteric sites could delay the emergence of drug resistance. In this study, we focussed on allosteric thumb site II of the non-structural protein NS5B RNA-dependent RNA polymerase and developed a five-feature pharmacophore hypothesis/model which estimated the experimental activity with a strong correlation of 0.971 & 0.944 for training and test sets, respectively. Further, the Güner-Henry score of 0.6 suggests that the model was able to discern the active and inactive compounds and enrich the true positives during a database search. In this study, database search and molecular docking results supported by experimental HCV viral replication inhibition assays suggested ligands with best fitness to the pharmacophore model dock to the key residues involved in thumbs site II, which inhibited the HCV 1b viral replication in sub-micro-molar range.
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http://dx.doi.org/10.1007/s11030-015-9591-5DOI Listing
August 2015

Suprafenacine, an indazole-hydrazide agent, targets cancer cells through microtubule destabilization.

PLoS One 2014 29;9(10):e110955. Epub 2014 Oct 29.

Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, Singapore, Singapore; Department of Genetic Engineering, College of Life Sciences, Kyung Hee University, Yongin-si, Gyeonggi-do, Republic of Korea.

Microtubules are a highly validated target in cancer therapy. However, the clinical development of tubulin binding agents (TBA) has been hampered by toxicity and chemoresistance issues and has necessitated the search for new TBAs. Here, we report the identification of a novel cell permeable, tubulin-destabilizing molecule--4,5,6,7-tetrahydro-1H-indazole-3-carboxylic acid [1p-tolyl-meth-(E)-ylidene]-hydrazide (termed as Suprafenacine, SRF). SRF, identified by in silico screening of annotated chemical libraries, was shown to bind microtubules at the colchicine-binding site and inhibit polymerization. This led to G2/M cell cycle arrest and cell death via a mitochondria-mediated apoptotic pathway. Cell death was preceded by loss of mitochondrial membrane potential, JNK-mediated phosphorylation of Bcl-2 and Bad, and activation of caspase-3. Intriguingly, SRF was found to selectively inhibit cancer cell proliferation and was effective against drug-resistant cancer cells by virtue of its ability to bypass the multidrug resistance transporter P-glycoprotein. Taken together, our results suggest that SRF has potential as a chemotherapeutic agent for cancer treatment and provides an alternate scaffold for the development of improved anti-cancer agents.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0110955PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4212991PMC
June 2015

Luteolin suppresses cancer cell proliferation by targeting vaccinia-related kinase 1.

PLoS One 2014 13;9(10):e109655. Epub 2014 Oct 13.

Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea; Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Republic of Korea.

Uncontrolled proliferation, a major feature of cancer cells, is often triggered by the malfunction of cell cycle regulators such as protein kinases. Recently, cell cycle-related protein kinases have become attractive targets for anti-cancer therapy, because they play fundamental roles in cellular proliferation. However, the protein kinase-targeted drugs that have been developed so far do not show impressive clinical results and also display severe side effects; therefore, there is undoubtedly a need to investigate new drugs targeting other protein kinases that are critical in cell cycle progression. Vaccinia-related kinase 1 (VRK1) is a mitotic kinase that functions in cell cycle regulation by phosphorylating cell cycle-related substrates such as barrier-to-autointegration factor (BAF), histone H3, and the cAMP response element (CRE)-binding protein (CREB). In our study, we identified luteolin as the inhibitor of VRK1 by screening a small-molecule natural compound library. Here, we evaluated the efficacy of luteolin as a VRK1-targeted inhibitor for developing an effective anti-cancer strategy. We confirmed that luteolin significantly reduces VRK1-mediated phosphorylation of the cell cycle-related substrates BAF and histone H3, and directly interacts with the catalytic domain of VRK1. In addition, luteolin regulates cell cycle progression by modulating VRK1 activity, leading to the suppression of cancer cell proliferation and the induction of apoptosis. Therefore, our study suggests that luteolin-induced VRK1 inhibition may contribute to establish a novel cell cycle-targeted strategy for anti-cancer therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0109655PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4195671PMC
September 2015

Revisiting de novo drug design: receptor based pharmacophore screening.

Curr Top Med Chem 2014 ;14(16):1890-8

School of Biological Science, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551.

De novo drug design methods such as receptor or protein based pharmacophore modeling present a unique opportunity to generate novel ligands by employing the potential binding sites even when no explicit ligand information is known for a particular target. Recent developments in molecular modeling programs have enhanced the ability of early programs such as LUDI or Pocket that not only identify the key interactions or hot spots at the suspected binding site, but also and convert these hot spots into three-dimensional search queries and virtual screening of the property filtered synthetic libraries. Together with molecular docking studies and consensus scoring schemes they would enrich the lead identification processes. In this review, we discuss the ligand and receptor based de novo drug design approaches with selected examples.
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http://dx.doi.org/10.2174/1568026614666140929115506DOI Listing
June 2015

Adamantyl derivative as a potent inhibitor of Plasmodium FK506 binding protein 35.

ACS Med Chem Lett 2013 Nov 16;4(11):1097-101. Epub 2013 Sep 16.

School of Biological Sciences, Nanyang Technological University , 60 Nanyang Drive, 637665 Singapore.

FKBP35, FK506 binding protein family member, in Plasmodium species displays a canonical peptidyl-prolyl isomerase (PPIase) activity and is intricately involved in the protein folding process. Inhibition of PfFKBP35 by FK506 or its analogues were shown to interfere with the in vitro growth of Plasmodium falciparum. In this study, we have synthesized adamantyl derivatives, Supradamal (SRA/4a) and its analogues SRA1/4b and SRA2/4c, which demonstrate submicromolar inhibition of Plasmodium falciparum FK506 binding domain 35 (FKBD35) PPIase activity. SRA and its analogues not only inhibit the in vitro growth of Plasmodium falciparum 3D7 strain but also show stage specific activity by inhibiting the trophozoite stage of the parasite. SRA/4a also inhibits the Plasmodium vivax FKBD35 PPIase activity and our crystal structure of PvFKBD35 in complex with the SRA provides structural insights in achieving selective inhibition against Plasmodium FKBPs.
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http://dx.doi.org/10.1021/ml400306rDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027365PMC
November 2013

Crystal structure of Plasmodium vivax FK506-binding protein 25 reveals conformational changes responsible for its noncanonical activity.

Proteins 2014 Jul 26;82(7):1235-44. Epub 2013 Dec 26.

Division of Structural Biology and Biochemistry, School of Biological Science, Nanyang Technological University, Singapore, 637665, Singapore.

The malarial parasites currently remain one of the most dreadful parasites, which show increasing trend of drug resistance to the currently available antimalarial drugs. Thus, the need to identify and characterize new protein targets in these parasites can aid to design novel therapeutic strategies to combat malaria. Recently, the conserved FK506-binding protein family members with molecular weight of 35 kDa from Plasmodium falciparum and Plasmodium vivax (referred to as PfFKBP35 and PvFKBP35, respectively) were identified for drug targeting. Further data mining revealed a 25-kDa FKBP (FKBP25) family member present in the parasites. FKBP25 belongs to a unique class of FKBP, because it is a nuclear FKBP with multiple protein-binding partners. Apart from immune regulation, it is also known for its chaperoning role in various cellular processes such as transcription regulation and trafficking. Here, we present the biochemical characterization and 1.9-Å crystal structure of an N-terminal truncated FKBP25 from P. vivax (PvFKBP25(72-209)). The protein reveals the noncanonical nature with unique structural changes observed in the loops flanking the active site, concealing the binding pocket. Further, a potential calmodulin-binding domain, which is absent in human FKBP25, is observed in this protein. Although the functional implication of Plasmodium FKBP25 in malaria still remains elusive, we speculate that the notable conformational changes in its structure might serve as an overture in understanding its molecular mechanism.
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http://dx.doi.org/10.1002/prot.24487DOI Listing
July 2014

The flavonoid myricetin reduces nocturnal melatonin levels in the blood through the inhibition of serotonin N-acetyltransferase.

Biochem Biophys Res Commun 2013 Oct 25;440(2):312-6. Epub 2013 Sep 25.

Pohang Center for Evaluation of Biomaterials, Pohang Technopark, Jigok-dong, Pohang, Gyeongbuk, South Korea; The College of Veterinary Medicine, Kyungpook National University, Sangyeok-dong, Daegu, South Korea.

Melatonin is secreted during the hours of darkness and is thought to influence the circadian and seasonal timing of a variety of physiological processes. AANAT, which is expressed in the pineal gland, retina, and various other tissues, catalyzes the conversion of serotonin to N-acetylserotonin and is the rate-limiting enzyme in the biosynthetic pathway of melatonin. The compounds that modulate the activity of AANAT can be used to treat patients with circadian rhythm disorders that are associated with specific circadian rhythm alterations, such as shift work disorder. In the present study, we screened modulators of AANAT activity from the water extracts of medicinal plants. Among the 267 tested medicinal plant extracts, Myricae Cortex (Myrica rubra), Perillae Herba (Perilla sikokiana), and Eriobotryae Folium (Eriobotrya japonica) showed potent inhibition of AANAT activity. Myricetin (5,7,3',4',5'-pentahydroxyflavonol), a main component of the Myricae Cortex, strongly inhibited the activity of AANAT and probably block the access to the substrate by docking to the catalytic residues that are important for AANAT activity. Myricetin significantly decreased the nocturnal serum melatonin levels in rats. In addition, the locomotor activity of rats treated with myricetin decreased during the nighttime and slightly increased throughout the day. These results suggest that myricetin could be used as a therapy to increase nighttime alertness by changing the circadian rhythm of serum melatonin and locomotor activity.
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http://dx.doi.org/10.1016/j.bbrc.2013.09.076DOI Listing
October 2013

Small molecule Plasmodium FKBP35 inhibitor as a potential antimalaria agent.

Sci Rep 2013 ;3:2501

1] School of Biological Science, Nanyang Technological University, 60 Nanyang Drive, Singapore 637665 [2].

Malaria parasite strains have emerged to tolerate the therapeutic effects of the prophylactics and drugs presently available. This resistance now poses a serious challenge to researchers in the bid to overcome malaria parasitic infection. Recent studies have shown that FK520 and its analogs inhibit malaria parasites growth by binding to FK506 binding proteins (FKBPs) of the parasites. Structure based drug screening efforts based on three-dimensional structural information of FKBPs from Plasmodium falciparum led us to identify new chemical entities that bind to the parasite FKBP35 and inhibit its growth. Our experimental results verify that this novel compound (D44) modulate the PPIase activity of Plasmodium FKBP35 and demonstrate the stage-specific growth inhibition of Plasmodium falciparum strains. Here, we present the X-ray crystallographic structures of FK506 binding domains (FKBDs) of PfFKBP35 and PvFKBP35 in complex with the newly identified inhibitor providing molecular insights into its mode of action.
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http://dx.doi.org/10.1038/srep02501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3752609PMC
February 2014

NMR solution structure of C2 domain of MFG-E8 and insights into its molecular recognition with phosphatidylserine.

Biochim Biophys Acta 2013 Mar 21;1828(3):1083-93. Epub 2012 Dec 21.

School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

MFG-E8 (also known as lactadherin), which is a secreted glycoprotein from a variety of cell types, possesses two EGF domains and tandem C domains with sequence homology to that of blood coagulation proteins factor V and factor VIII. MFG-E8 binds to phosphatidylserine (PS) in membranes with high affinity. We have recently shown that the C2 domain of MFG-E8 bears more specificity toward PS when compared with phosphatidylcholine (PC), another phospholipid thought to be involved in the immune function of phagocytes. In our current study, we have determined the solution structure of the C2 domain by nuclear magnetic resonance (NMR) spectroscopy, and characterized the molecular basis of binding between the C2 domain and PS by (31)P-NMR spectroscopy. Furthermore, we also verified that that positively charged and aromatic residues clustered in loops 1-3 of the C2 domain play key roles in recognizing PS in apoptotic cells.
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http://dx.doi.org/10.1016/j.bbamem.2012.12.009DOI Listing
March 2013

Solution structure of FK506-binding protein 12 from Aedes aegypti.

Proteins 2012 Oct 31;80(10):2476-81. Epub 2012 Jul 31.

Division of Structural Biology and Biochemistry, School of Biological Science, Nanyang Technological University, Singapore.

Dengue remains one of the major public concerns as the virus eludes the immune response. Currently, no vaccines or antiviral therapeutics are available for dengue prevention or treatment. Immunosuppressive drug FK506 shows an antimalarial activity, and its molecular target, FK506-binding protein (FKBP), was identified in human Plasmodium parasites. Likewise, a conserved FKBP family protein has also been identified in Aedes aegypti (AaFKBP12), which is expected to play a similar role in the life cycle of Aedes aegypti, the primary vector of dengue virus infection. As FKBPs belong to a highly conserved class of immunophilin family and are involved in key biological regulations, they are considered as attractive pharmacological targets. In this study, we have determined the nuclear magnetic resonance solution structure of AaFKBP12, a novel FKBP member from Aedes aegypti, and presented its structural features, which may facilitate the design of potential inhibitory ligands against the dengue-transmitting mosquitoes.
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http://dx.doi.org/10.1002/prot.24146DOI Listing
October 2012

Crystallographic structure of the tetratricopeptide repeat domain of Plasmodium falciparum FKBP35 and its molecular interaction with Hsp90 C-terminal pentapeptide.

Protein Sci 2009 Oct;18(10):2115-24

Nanyang Technological University, Singapore.

Plasmodium falciparum FK506-binding protein 35 (PfFKBP35) that binds to FK506 contains a conserved tetratricopeptide repeat (TPR) domain. Several known TPR domains such as Hop, PPP5, CHIP, and FKBP52 are structurally conserved and are able to interact with molecular chaperones such as Hsp70/Hsp90. Here, we present the crystal structure of PfFKBP35-TPR and demonstrate its interaction with Hsp90 C-terminal pentapeptide (MEEVD) by surface plasmon resonance and nuclear magnetic resonance spectroscopy-based binding studies. Our sequence and structural analyses reveal that PfFKBP35 is similar to Hop and PPP5 in possessing all the conserved residues which are important for carboxylate clamping with Hsp90. Mutational studies were carried out on positively charged clamp residues that are crucial for binding to carboxylate groups of aspartate, showing that all the mutated residues are important for Hsp90 binding. Molecular docking and electrostatic calculations demonstrated that the MEEVD peptide of Hsp90 can form aspartate clamp unlike FKBP52. Our results provide insightful information and structural basis about the molecular interaction between PfFKBP35-TPR and Hsp90.
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http://dx.doi.org/10.1002/pro.226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2786975PMC
October 2009
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