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Studies of Retroviral Reverse Transcriptase and Flaviviral Protease Enzymes as Antiviral Drug Targets: Applications in Antiviral Drug Discovery & Therapy
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Viruses are a major threat to humans due to their unique adaptability, evolvability and  capability to control their hosts as parasites and genetic elements. HIV/AIDS is the third largest cause of death by infectious diseases in the world, and drug resistance due to the viral mutations is still the leading cause of treatment failure. The flaviviruses, such as Dengue virus (DEN) and Japanese encephalitis virus (JEV), represent other major cause of morbidity and mortality, and the areas where these viruses are endemic are spreading rapidly. No curative therapy for any flavivirus could be made available as yet.

The first part of this thesis focuses on the HIV-1 drug resistance caused by mutations in a major HIV drug target, the HIV-1 reverse transcriptase (RT) as a response to the largest class of clinically used anti-retrovirals, the NRTIs. A robust proteochemometric model was created to analyse the complex mutation patterns in RT drug resistance. The model identified more than ten frequently-occurring mutations, each conferring at least two-fold decrease in susceptibility for one or several NRTIs. Using our prediction server (hivdrc.org), the model can be applied to propose optimum combination therapy for patients harbouring mutated HIV variants.

The second part of the thesis encompasses studies on a promising drug target, the NS2B(H)-NS3pro, in two flaviviruses, namely the dengue virus (DEN) and Japanese encephalitis virus (JEV). Functional determinants of DEN NS2B(H)-NS3pro were identified by site-directed mutagenesis. Further, peptide inhibitors were designed using proteochemometrics (PCM) and statistical molecular design (SMD), synthesized and assayed on DEN proteases, which resulted in some novel peptides with low micromolar or sub-micromolar inhibitor activity. The very poorly characterised JEV NS2B(H)-NS3pro  was cloned, purified and the kinetic parameters of this attractive drug target were determined for a series of model substrates and inhibitor. The results identified the role in target-ligand interaction of different residues on specific positions in the target (NS2B(H)-NS3pro) and ligands (substrates/inhibitors).

Overall, the findings in this thesis contribute to rational antiviral drug discovery and therapy.

Place, publisher, year, edition, pages
Acta Universitatis Upsaliensis: Uppsala universitet, 2012. , 68 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 163
Keyword [en]
Virus, enzymes, HIV/AIDS, retroviral reverse transcriptase, flaviviral protease, NRTIs, proteochemometrics, drug resistance, DEN, JEV, NS2B(H)-NS3pro, antiviral, drug targets, drug discovery, drug therapy.
National Category
Pharmaceutical Sciences
Research subject
Pharmacology
Identifiers
URN: urn:nbn:se:uu:diva-173504ISBN: 978-91-554-8388-3 (print)OAI: oai:DiVA.org:uu-173504DiVA: diva2:523717
Public defence
2012-06-14, C4:305, BMC, Husargatan 3, Uppsala, 09:00 (English)
Supervisors
Available from: 2012-05-23 Created: 2012-04-25 Last updated: 2012-08-01Bibliographically approved
List of papers
1. Proteochemometric Modeling of the Susceptibility of Mutated Variants of the HIV-1 Virus to Reverse Transcriptase Inhibitors
Open this publication in new window or tab >>Proteochemometric Modeling of the Susceptibility of Mutated Variants of the HIV-1 Virus to Reverse Transcriptase Inhibitors
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2010 (English)In: PLoS ONE, ISSN eISSN-1932-6203, Vol. 5, no 12, e14353- p.Article in journal (Refereed) Published
Abstract [en]

Background

Reverse transcriptase is a major drug target in highly active antiretroviral therapy (HAART) against HIV, which typically comprises two nucleoside/nucleotide analog reverse transcriptase (RT) inhibitors (NRTIs) in combination with a non-nucleoside RT inhibitor or a protease inhibitor. Unfortunately, HIV is capable of escaping the therapy by mutating into drug-resistant variants. Computational models that correlate HIV drug susceptibilities to the virus genotype and to drug molecular properties might facilitate selection of improved combination treatment regimens.

Methodology/Principal Findings

We applied our earlier developed proteochemometric modeling technology to analyze HIV mutant susceptibility to the eight clinically approved NRTIs. The data set used covered 728 virus variants genotyped for 240 sequence residues of the DNA polymerase domain of the RT; 165 of these residues contained mutations; totally the data-set covered susceptibility data for 4,495 inhibitor-RT combinations. Inhibitors and RT sequences were represented numerically by 3D-structural and physicochemical property descriptors, respectively. The two sets of descriptors and their derived cross-terms were correlated to the susceptibility data by partial least-squares projections to latent structures. The model identified more than ten frequently occurring mutations, each conferring more than two-fold loss of susceptibility for one or several NRTIs. The most deleterious mutations were K65R, Q151M, M184V/I, and T215Y/F, each of them decreasing susceptibility to most of the NRTIs. The predictive ability of the model was estimated by cross-validation and by external predictions for new HIV variants; both procedures showed very high correlation between the predicted and actual susceptibility values (Q2 = 0.89 and Q2ext = 0.86). The model is available at www.hivdrc.org as a free web service for the prediction of the susceptibility to any of the clinically used NRTIs for any HIV-1 mutant variant.

Conclusions/Significance

Our results give directions how to develop approaches for selection of genome-based optimum combination therapy for patients harboring mutated HIV variants.

National Category
Pharmacology and Toxicology
Research subject
Bioinformatics; Pharmacology
Identifiers
urn:nbn:se:uu:diva-141096 (URN)10.1371/journal.pone.0014353 (DOI)000285340000015 ()21179544 (PubMedID)
Available from: 2011-01-10 Created: 2011-01-10 Last updated: 2015-05-04Bibliographically approved
2. Structure-guided mutagenesis of active site residues in the dengue virus two-component protease NS2B-NS3
Open this publication in new window or tab >>Structure-guided mutagenesis of active site residues in the dengue virus two-component protease NS2B-NS3
2010 (English)In: Journal of Biomedical Science, ISSN 1021-7770, E-ISSN 1423-0127, Vol. 17, 68- p.Article in journal (Refereed) Published
Abstract [en]

Background

 The dengue virus two-component protease NS2B/NS3 mediates processing of the viral polyprotein precursor and is therefore an important determinant of virus replication. The enzyme is not intensively studied with a view to the structure-based development of antiviral inhibitors. Although 3-dimensional structures have now been elucidated for a number of flaviviral proteases, enzyme substrate interactions are characterized only to a limited extend. The high selectivity of the dengue virus protease for the polyprotein precursor offers the distinct advantage of disigning inhibitors with exquisite specificity for the viral enzyme. To identify important determinants of substrate binding and catalysis in the active site of the dengue virus NS3 protease, nine residues, L115, D129, G133, T134, Y150, G151, N152, S163 and I165, located within the S1 and S2 pockets of the enzyme were targeted by alanine substitution mutagenesis and effects on enzyme activity were fluorometrically assayed.

Methods

 Alanine substitutions were introduced by site directed mutagenesis at residues L115, D129, G133, T134, T150, G151, N152, S163 and I165 and recombinant proteins were purified from overexpressing E. coli. Effects of these substitutions on enzymatic activity of the NS3 protease were assayed by fluorescence release from the synthetic model substrate GRR-amc and kinetic parameters K-m, K-cat and K-cat/K-m were determined.

Results

 Kinetic data for mutant derivatives in the active site of the dengue virus NS3 protease were essentially in agreement with a functional role of the selected residues for substrate binding and/or catalysis. Only the L115A mutant displayed activity comparable to the wild-type enzyme, whereas mutation of residues Y150 and G151 to alanine completely abrogated enzyme activiey. A G133A mutant had an approximately 10-fold reduced catalytic efficiency thus suggesting a critical role for this residue seemingly as part of the oxyanion binding hole.

Conclusions

 Kinetic data obtained for mutants in the NS3 protease have confirmed predictions for the conformation of the active site S1 and S2 pockets based on earlier observations. The data presented herein will be useful to further explore structure-activity relationships of the flaviviral proteases important for the structure-guided design of novel antiviral therapeutics.

National Category
Pharmaceutical Sciences Medical and Health Sciences Biological Sciences
Identifiers
urn:nbn:se:uu:diva-134695 (URN)10.1186/1423-0127-17-68 (DOI)000282337300001 ()
Available from: 2010-11-30 Created: 2010-11-30 Last updated: 2017-12-12Bibliographically approved
3. ­­­­­­­­Enzymatic Analysis of Recombinant Japanese Encephalitis Virus NS2B(H)-NS3pro Protease with Fluorogenic Model Peptide Substrates
Open this publication in new window or tab >>­­­­­­­­Enzymatic Analysis of Recombinant Japanese Encephalitis Virus NS2B(H)-NS3pro Protease with Fluorogenic Model Peptide Substrates
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 5, e36872- p.Article in journal (Refereed) Published
Abstract [en]

Background

Japanese encephalitis virus (JEV), a member of the Flaviviridae family, causes around 68,000 encephalitis cases annually, of which 20–30% are fatal, while 30–50% of the recovered cases develop severe neurological sequelae. Specific antivirals for JEV would be of great importance, particularly in those cases where the infection has become persistent. Being indispensable for flaviviral replication, the NS2B-NS3 protease is a promising target for design of anti-flaviviral inhibitors. Contrary to related flaviviral proteases, the JEV NS2B-NS3 protease is structurally and mechanistically much less characterized. Here we aimed at establishing a straightforward procedure for cloning, expression, purification and biochemical characterization of JEV NS2B(H)-NS3pro protease.

Methodology/Principal Findings

The full-length sequence of JEV NS2B-NS3 genotype III strain JaOArS 982 was obtained as a synthetic gene. The sequence of NS2B(H)-NS3pro was generated by splicing by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblotting of the purified enzyme demonstrated NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 36, 21, and 10 kDa bands, respectively. Kinetic parameters, Km and kcat, for fluorogenic protease model substrates, Boc-GRR-amc, Boc-LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)4SAG-nY-amide, were obtained using inner filter effect correction. The highest catalytic efficiency kcat/Km was found for Pyr-RTKR-amc (kcat/Km: 1962.96±85.0 M−1 s−1) and the lowest for Boc-LRR-amc (kcat/Km: 3.74±0.3 M−1 s−1). JEV NS3pro is inhibited by aprotinin but to a lesser extent than DEN and WNV NS3pro.

Conclusions/Significance

A simplified procedure for the cloning, overexpression and purification of the NS2B(H)-NS3pro was established which is generally applicable to other flaviviral proteases. Kinetic parameters obtained for a number of model substrates and inhibitors, are useful for the characterization of substrate specificity and eventually for the design of high-throughput assays aimed at antiviral inhibitor discovery.

National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-173500 (URN)10.1371/journal.pone.0036872 (DOI)000305336300044 ()
Available from: 2012-04-25 Created: 2012-04-25 Last updated: 2017-12-07Bibliographically approved
4. Design and Evaluation of Substrate-based Octapeptide and Nonsubstrate-based Tetrapeptide Inhibitors of Dengue Virus NS2B/NS3 Proteases
Open this publication in new window or tab >>Design and Evaluation of Substrate-based Octapeptide and Nonsubstrate-based Tetrapeptide Inhibitors of Dengue Virus NS2B/NS3 Proteases
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(English)Manuscript (preprint) (Other academic)
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Pharmacology
Identifiers
urn:nbn:se:uu:diva-173234 (URN)
Available from: 2012-04-25 Created: 2012-04-21 Last updated: 2013-04-11
5. Design and Evaluation of Dengue NS3 Protease Peptide Inhibitors
Open this publication in new window or tab >>Design and Evaluation of Dengue NS3 Protease Peptide Inhibitors
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(English)Manuscript (preprint) (Other academic)
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-173501 (URN)
Available from: 2012-04-25 Created: 2012-04-25 Last updated: 2013-04-11

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