Publications by authors named "Greg L Beilhartz"

21 Publications

  • Page 1 of 1

Exploiting the diphtheria toxin internalization receptor enhances delivery of proteins to lysosomes for enzyme replacement therapy.

Sci Adv 2020 12 11;6(50). Epub 2020 Dec 11.

Molecular Medicine Program, The Hospital for Sick Children, Toronto, ON, Canada.

Enzyme replacement therapy, in which a functional copy of an enzyme is injected either systemically or directly into the brain of affected individuals, has proven to be an effective strategy for treating certain lysosomal storage diseases. The inefficient uptake of recombinant enzymes via the mannose-6-phosphate receptor, however, prohibits the broad utility of replacement therapy. Here, to improve the efficiency and efficacy of lysosomal enzyme uptake, we exploited the strategy used by diphtheria toxin to enter into the endolysosomal network of cells by creating a chimera between the receptor-binding fragment of diphtheria toxin and the lysosomal hydrolase TPP1. We show that chimeric TPP1 binds with high affinity to target cells and is efficiently delivered into lysosomes. Further, we show superior uptake of chimeric TPP1 over TPP1 alone in brain tissue following intracerebroventricular injection in mice lacking TPP1, demonstrating the potential of this strategy for enhancing lysosomal storage disease therapy.
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http://dx.doi.org/10.1126/sciadv.abb0385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732195PMC
December 2020

Attenuated diphtheria toxin mediates siRNA delivery.

Sci Adv 2020 May 1;6(18). Epub 2020 May 1.

Department of Chemistry, University of Toronto, Toronto, ON, Canada.

Toxins efficiently deliver cargo to cells by binding to cell surface ligands, initiating endocytosis, and escaping the endolysosomal pathway into the cytoplasm. We took advantage of this delivery pathway by conjugating an attenuated diphtheria toxin to siRNA, thereby achieving gene downregulation in patient-derived glioblastoma cells. We delivered siRNA against integrin-β1 ()-a gene that promotes invasion and metastasis-and siRNA against eukaryotic translation initiation factor 3 subunit b ()-a survival gene. We demonstrated mRNA downregulation of both genes and the corresponding functional outcomes: knockdown of led to a significant inhibition of invasion, shown with an innovative 3D hydrogel model; and knockdown of resulted in significant cell death. This is the first example of diphtheria toxin being used to deliver siRNAs, and the first time a toxin-based siRNA delivery strategy has been shown to induce relevant genotypic and phenotypic effects in cancer cells.
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http://dx.doi.org/10.1126/sciadv.aaz4848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195190PMC
May 2020

An engineered chimeric toxin that cleaves activated mutant and wild-type RAS inhibits tumor growth.

Proc Natl Acad Sci U S A 2020 07 2;117(29):16938-16948. Epub 2020 Jul 2.

Department of Microbiology and Immunology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611;

Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small-molecule approaches has proven to be a major challenge. Here we describe the development of a pan-RAS biologic inhibitor composed of the RAS-RAP1-specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Furthermore, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutant Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising approach to developing RAS therapeutics against a broad array of cancers.
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http://dx.doi.org/10.1073/pnas.2000312117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382267PMC
July 2020

Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.

Cell 2020 07 25;182(2):345-356.e16. Epub 2020 Jun 25.

Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Molecular Architecture of Life Program, Canadian Institute for Advanced Research (CIFAR), Toronto, ON M5G 1M1, Canada. Electronic address:

Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.
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http://dx.doi.org/10.1016/j.cell.2020.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316060PMC
July 2020

bioPROTACs as versatile modulators of intracellular therapeutic targets including proliferating cell nuclear antigen (PCNA).

Proc Natl Acad Sci U S A 2020 03 2;117(11):5791-5800. Epub 2020 Mar 2.

Quantitative Biosciences, MSD International, Singapore 138665;

Targeted degradation approaches such as proteolysis targeting chimeras (PROTACs) offer new ways to address disease through tackling challenging targets and with greater potency, efficacy, and specificity over traditional approaches. However, identification of high-affinity ligands to serve as PROTAC starting points remains challenging. As a complementary approach, we describe a class of molecules termed biological PROTACs (bioPROTACs)-engineered intracellular proteins consisting of a target-binding domain directly fused to an E3 ubiquitin ligase. Using GFP-tagged proteins as model substrates, we show that there is considerable flexibility in both the choice of substrate binders (binding positions, scaffold-class) and the E3 ligases. We then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA) to elicit rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Overall, bioPROTACs are powerful tools for interrogating degradation approaches, target biology, and potentially for making therapeutic impacts.
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http://dx.doi.org/10.1073/pnas.1920251117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084165PMC
March 2020

Host-targeted niclosamide inhibits C. difficile virulence and prevents disease in mice without disrupting the gut microbiota.

Nat Commun 2018 12 7;9(1):5233. Epub 2018 Dec 7.

Molecular Medicine, Hospital for Sick Children, 686 Bay St., Toronto, ON, M5G 0A4, Canada.

Clostridium difficile is the leading cause of nosocomial diarrhea and colitis in the industrialized world. Disruption of the protective gut microbiota by antibiotics enables colonization by multidrug-resistant C. difficile, which secrete up to three different protein toxins that are responsible for the gastrointestinal sequelae. Oral agents that inhibit the damage induced by toxins, without altering the gut microbiota, are urgently needed to prevent primary disease and break the cycle of antibiotic-induced disease recurrence. Here, we show that the anthelmintic drug, niclosamide, inhibits the pathogenesis of all three toxins by targeting a host process required for entry into colonocytes by each toxin. In mice infected with an epidemic strain of C. difficile, expressing all three toxins, niclosamide reduced both primary disease and recurrence, without disrupting the diversity or composition of the gut microbiota. Given its excellent safety profile, niclosamide may address an important unmet need in preventing C. difficile primary and recurrent diseases.
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http://dx.doi.org/10.1038/s41467-018-07705-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286312PMC
December 2018

Intracellular Delivery of Human Purine Nucleoside Phosphorylase by Engineered Diphtheria Toxin Rescues Function in Target Cells.

Mol Pharm 2018 11 26;15(11):5217-5226. Epub 2018 Sep 26.

Division of Immunology and Allergy , The Hospital for Sick Children , Toronto , ON M5G 0A4 , Canada.

Despite a wealth of potential applications inside target cells, protein-based therapeutics are largely limited to extracellular targets due to the inability of proteins to readily cross biological membranes and enter the cytosol. Bacterial toxins, which deliver a cytotoxic enzyme into cells as part of their intoxication mechanism, hold great potential as platforms for delivering therapeutic protein cargo into cells. Diphtheria toxin (DT) has been shown to be capable of delivering an array of model proteins of varying sizes, structures, and stabilities into mammalian cells as amino-terminal fusions. Here, seeking to expand the utility of DT as a delivery vector, we asked whether an active human enzyme, purine nucleoside phosphorylase (PNP), could be delivered by DT into cells to rescue PNP deficiency. Using a series of biochemical and cellular readouts, we demonstrate that PNP is efficiently delivered into target cells in a receptor- and translocation-dependent manner. In patient-derived PNP-deficient lymphocytes and pluripotent stem cell-differentiated neurons, we show that human PNP is efficiently translocated into target cells by DT, where it is able to restore intracellular hypoxanthine levels. Further, through replacement of the native receptor-binding moiety of DT with single-chain variable fragments that were selected to bind mouse HBEGF, we show that PNP can be retargeted into mouse splenocytes from PNP-deficient mice, resulting in restoration of the proliferative capacity of T-cells. These findings highlight the versatility of the DT delivery platform and provide an attractive approach for the delivery of PNP as well as other cytosolic enzymes implicated in disease.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b00735DOI Listing
November 2018

Repurposing bacterial toxins for intracellular delivery of therapeutic proteins.

Biochem Pharmacol 2017 10 10;142:13-20. Epub 2017 Apr 10.

Molecular Medicine Program, The Hospital for Sick Children Research Institute, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada. Electronic address:

Despite enormous efforts, achieving efficacious levels of proteins inside mammalian cells remains one of the greatest challenges in biologics-based drug discovery and development. The inability of proteins to readily cross biological membranes precludes access to the wealth of intracellular targets and applications that lie within mammalian cells. Existing methods of delivery commonly suffer from an inability to target specific cells and tissues, poor endosomal escape, and limited in vivo efficacy. The aim of the present commentary is to highlight the potential of certain classes of bacterial toxins, which naturally deliver a large protein into the cytosolic compartment of target cells after binding a host cell-surface receptor with high affinity, as robust protein delivery platforms. We review the progress made in recent years toward demonstrating the utility of these systems at delivering a wide variety of protein cargo, with special attention paid to three distinct toxin-based platforms. We contend that with recent advances in protein deimmunization strategies, bacterial toxins are poised to introduce biologics into the inner sanctum of cells and treat a wealth of heretofore untreatable diseases with a new generation of therapeutics.
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http://dx.doi.org/10.1016/j.bcp.2017.04.009DOI Listing
October 2017

Comment on "A small-molecule antivirulence agent for treating Clostridium difficile infection".

Sci Transl Med 2016 12;8(370):370tc2

Molecular Structure & Function, The Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada.

New insights into the mechanism of action of ebselen, a small-molecule antivirulence agent that reduces disease pathology in a mouse model of Clostridium difficile infection, suggest a different molecular target may be responsible for its efficacy.
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http://dx.doi.org/10.1126/scitranslmed.aad8926DOI Listing
December 2016

Small Molecules Take A Big Step Against Clostridium difficile.

Trends Microbiol 2015 Dec 5;23(12):746-748. Epub 2015 Nov 5.

Molecular Structure & Function, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

Effective treatment of Clostridium difficile infections demands a shift away from antibiotics towards toxin-neutralizing agents. Work by Bender et al., using a drug that attenuates toxin action in vivo without affecting bacterial survival, demonstrates the exciting potential of small molecules as a new modality in the fight against C. difficile.
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http://dx.doi.org/10.1016/j.tim.2015.10.009DOI Listing
December 2015

Efficient Delivery of Structurally Diverse Protein Cargo into Mammalian Cells by a Bacterial Toxin.

Mol Pharm 2015 Aug 2;12(8):2962-71. Epub 2015 Jul 2.

‡Department of Biochemistry, University of Toronto, Toronto, ON, Canada.

Platforms enabling targeted delivery of proteins into cells are needed to fully realize the potential of protein-based therapeutics with intracellular sites-of-action. Bacterial toxins are attractive systems to consider as templates for designing protein transduction systems as they naturally bind and enter specific cells with high efficiency. Here we investigated the capacity of diphtheria toxin to function as an intracellular protein delivery vector. We report that diphtheria toxin delivers an impressive array of passenger proteins spanning a range of sizes, structures, and stabilities into cells in a manner that indicates that they are "invisible" to the translocation machinery. Further, we show that α-amylase delivered into cells by a detoxified diphtheria toxin chimera digests intracellular glycogen in live cells, providing evidence that delivered cargo is folded, active, and abundant. The efficiency and versatility of diphtheria toxin over existing systems open numerous possibilities for intracellular delivery of bioactive proteins.
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http://dx.doi.org/10.1021/acs.molpharmaceut.5b00233DOI Listing
August 2015

Small molecule inhibitors of Clostridium difficile toxin B-induced cellular damage.

Chem Biol 2015 Feb 22;22(2):175-85. Epub 2015 Jan 22.

Molecular Structure & Function, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

Clostridium difficile causes life-threatening diarrhea through the actions of its homologous toxins TcdA and TcdB on human colonocytes. Therapeutic agents that block toxin-induced damage are urgently needed to prevent the harmful consequences of toxin action that are not addressed with current antibiotic-based treatments. Here, we developed an imaging-based phenotypic screen to identify small molecules that protected human cells from TcdB-induced cell rounding. A series of structurally diverse compounds with antitoxin activity were identified and found to act through one of a small subset of mechanisms, including direct binding and sequestration of TcdB, inhibition of endosomal maturation, and noncompetitive inhibition of the toxin glucosyltransferase activity. Distinct classes of inhibitors were used further to dissect the determinants of the toxin-mediated necrosis phenotype occurring at higher doses of toxin. These findings validate and inform novel targeting strategies for discovering small molecule agents to treat C. difficile infection.
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http://dx.doi.org/10.1016/j.chembiol.2014.12.010DOI Listing
February 2015

Derivatives of mesoxalic acid block translocation of HIV-1 reverse transcriptase.

J Biol Chem 2015 Jan 29;290(3):1474-84. Epub 2014 Oct 29.

From the Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada, the Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada, the Department of Medicine, Division of Experimental Medicine, McGill University, Quebec H3A 1A3, Canada

The pyrophosphate mimic and broad spectrum antiviral phosphonoformic acid (PFA, foscarnet) was shown to freeze the pre-translocational state of the reverse transcriptase (RT) complex of the human immunodeficiency virus type 1 (HIV-1). However, PFA lacks a specificity domain, which is seen as a major reason for toxic side effects associated with the clinical use of this drug. Here, we studied the mechanism of inhibition of HIV-1 RT by the 4-chlorophenylhydrazone of mesoxalic acid (CPHM) and demonstrate that this compound also blocks RT translocation. Hot spots for inhibition with PFA or CPHM occur at template positions with a bias toward pre-translocation. Mutations at active site residue Asp-185 compromise binding of both compounds. Moreover, divalent metal ions are required for the formation of ternary complexes with either of the two compounds. However, CPHM contains both an anchor domain that likely interacts with the catalytic metal ions and a specificity domain. Thus, although the inhibitor binding sites may partly overlap, they are not identical. The K65R mutation in HIV-1 RT, which reduces affinity to PFA, increases affinity to CPHM. Details with respect to the binding sites of the two inhibitors are provided on the basis of mutagenesis studies, structure-activity relationship analyses with newly designed CPHM derivatives, and in silico docking experiments. Together, these findings validate the pre-translocated complex of HIV-1 RT as a specific target for the development of novel classes of RT inhibitors.
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http://dx.doi.org/10.1074/jbc.M114.614305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340395PMC
January 2015

Inhibition of the ribonuclease H activity of HIV-1 reverse transcriptase by GSK5750 correlates with slow enzyme-inhibitor dissociation.

J Biol Chem 2014 Jun 9;289(23):16270-7. Epub 2014 Apr 9.

From the Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada, the Department of Biochemistry, McGill University, Montreal, Quebec H3G1Y6, Canada, and the Department of Medicine, Division of Experimental Medicine, McGill University, Montreal, Quebec H3A 1A3, Canada

Compounds that efficiently inhibit the ribonuclease (RNase) H activity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have yet to be developed. Here, we demonstrate that GSK5750, a 1-hydroxy-pyridopyrimidinone analog, binds to the enzyme with an equilibrium dissociation constant (K(d)) of ~400 nM. Inhibition of HIV-1 RNase H is specific, as DNA synthesis is not affected. Moreover, GSK5750 does not inhibit the activity of Escherichia coli RNase H. Order-of-addition experiments show that GSK5750 binds to the free enzyme in an Mg(2+)-dependent fashion. However, as reported for other active site inhibitors, binding of GSK5750 to a preformed enzyme-substrate complex is severely compromised. The bound nucleic acid prevents access to the RNase H active site, which represents a possible biochemical hurdle in the development of potent RNase H inhibitors. Previous studies suggested that formation of a complex with the prototypic RNase H inhibitor β-thujaplicinol is slow, and, once formed, it dissociates rapidly. This unfavorable kinetic behavior can limit the potency of RNase H active site inhibitors. Although the association kinetics of GSK5750 remains slow, our data show that this compound forms a long lasting complex with HIV-1 RT. We conclude that slow dissociation of the inhibitor and HIV-1 RT improves RNase H active site inhibitors and may circumvent the obstacle posed by the inability of these compounds to bind to a preformed enzyme-substrate complex.
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http://dx.doi.org/10.1074/jbc.M114.569707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047396PMC
June 2014

Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore.

Proc Natl Acad Sci U S A 2014 Mar 24;111(10):3721-6. Epub 2014 Feb 24.

Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S1A8.

Disease associated with Clostridium difficile infection is caused by the actions of the homologous toxins TcdA and TcdB on colonic epithelial cells. Binding to target cells triggers toxin internalization into acidified vesicles, whereupon cryptic segments from within the 1,050-aa translocation domain unfurl and insert into the bounding membrane, creating a transmembrane passageway to the cytosol. Our current understanding of the mechanisms underlying pore formation and the subsequent translocation of the upstream cytotoxic domain to the cytosol is limited by the lack of information available regarding the identity and architecture of the transmembrane pore. Here, through systematic perturbation of conserved sites within predicted membrane-insertion elements of the translocation domain, we uncovered highly sensitive residues--clustered between amino acids 1,035 and 1,107--that when individually mutated, reduced cellular toxicity by as much as >1,000-fold. We demonstrate that defective variants are defined by impaired pore formation in planar lipid bilayers and biological membranes, resulting in an inability to intoxicate cells through either apoptotic or necrotic pathways. These findings along with the unexpected similarities uncovered between the pore-forming "hotspots" of TcdB and the well-characterized α-helical diphtheria toxin translocation domain provide insights into the structure and mechanism of formation of the translocation pore for this important class of pathogenic toxins.
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http://dx.doi.org/10.1073/pnas.1400680111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3956163PMC
March 2014

Telbivudine exerts no antiviral activity against HIV-1 in vitro and in humans.

Antivir Ther 2011 ;16(7):1123-30

Department of Medical Microbiology, Virology, University Medical Center Utrecht, Utrecht, The Netherlands.

Background: HIV-HBV-coinfected individuals who need to be treated only for their HBV infection have limited therapeutic options, since most approved anti-HBV agents have a risk of selecting for drug-resistant HIV mutants. In vivo data are inconclusive as to whether telbivudine (LdT) may exert antiviral effects against HIV. Thus, we investigated in further detail the antiviral activity and the biochemical properties of LdT against HIV-1.

Methods: To investigate the activity of LdT against HIV-1 in humans we analysed viral dynamics and genotypic and phenotypic resistance development in two HIV-HBV-coinfected individuals with no prior antiviral exposure. To investigate the activity of LdT against HIV-1 in vitro, LdT susceptibility for HIV-1 wild-type strains as well as drug-resistant strains was determined. Furthermore, we studied whether the 5'-triphosphate form of LdT (LdT-TP) can act as a substrate for wild-type HIV-1 RT.

Results: In the two patients studied, LdT treatment did not result in a significant decline of HIV-1 RNA load nor in selection of genotypic or phenotypic resistance in HIV-1 RT. In vitro virological analyses demonstrated that LdT had no activity (50% effective concentration >100 μM) against wild type HIV and drug-resistant variants. Biochemical analyses demonstrated that LdT-TP is not incorporated by wild-type HIV-1 RT.

Conclusions: Based on the in vivo and in vitro evidence obtained in this study, we conclude that LdT has no anti-HIV-1 activity and is currently the only selective anti-HBV agent among the five FDA-approved nucleoside/nucleotide analogues for treatment of HBV infections in HIV-infected individuals.
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http://dx.doi.org/10.3851/IMP1912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732022PMC
April 2012

Impact of primer-induced conformational dynamics of HIV-1 reverse transcriptase on polymerase translocation and inhibition.

J Biol Chem 2011 Aug 7;286(34):29575-83. Epub 2011 Jul 7.

Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec, Canada.

The rapid emergence and the prevalence of resistance mutations in HIV-1 reverse transcriptase (RT) underscore the need to identify RT inhibitors with novel binding modes and mechanisms of inhibition. Recently, two structurally distinct inhibitors, phosphonoformic acid (foscarnet) and INDOPY-1 were shown to disrupt the translocational equilibrium of RT during polymerization through trapping of the enzyme in the pre- and the post-translocation states, respectively. Here, we show that foscarnet and INDOPY-1 additionally display a shared novel inhibitory preference with respect to substrate primer identity. In RT-catalyzed reactions using RNA-primed substrates, translocation inhibitors were markedly less potent at blocking DNA polymerization than in equivalent DNA-primed assays; i.e. the inverse pattern observed with marketed non-nucleoside inhibitors that bind the allosteric pocket of RT. This potency profile was shown to correspond with reduced binding on RNA·DNA primer/template substrates versus DNA·DNA substrates. Furthermore, using site-specific footprinting with chimeric RNA·DNA primers, we demonstrate that the negative impact of the RNA primer on translocation inhibitor potency is overcome after 18 deoxyribonucleotide incorporations, where RT transitions primarily into polymerization-competent binding mode. In addition to providing a simple means to identify similarly acting translocation inhibitors, these findings suggest a broader role for the primer-influenced binding mode on RT translocation equilibrium and inhibitor sensitivity.
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http://dx.doi.org/10.1074/jbc.M111.268235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3190998PMC
August 2011

N348I in HIV-1 reverse transcriptase can counteract the nevirapine-mediated bias toward RNase H cleavage during plus-strand initiation.

J Biol Chem 2010 Aug 8;285(35):26966-26975. Epub 2010 Jun 8.

Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada. Electronic address:

Drug resistance-associated mutations in HIV-1 reverse transcriptase (RT) can affect the balance between polymerase and ribonuclease H (RNase H) activities of the enzyme. We have recently demonstrated that the N348I mutation in the connection domain causes selective dissociation from RNase H-competent complexes, whereas the functional integrity of the polymerase-competent complex remains largely unaffected. N348I has been associated with resistance to the non-nucleoside RT inhibitor (NNRTI), nevirapine; however, a possible mechanism that links changes in RNase H activity to changes in NNRTI susceptibility remains to be established. To address this problem, we consider recent findings suggesting that NNRTIs may affect the orientation of RT on its nucleic acid substrate and increase RNase H activity. Here we demonstrate that RNase H-mediated primer removal is indeed more efficient in the presence of NNRTIs; however, the N348I mutant enzyme is able to counteract this effect. Efavirenz, a tight binding inhibitor, restricts the influence of the mutation. These findings provide strong evidence to suggest that N348I can thwart the inhibitory effects of nevirapine during initiation of (+)-strand DNA synthesis, which provides a novel mechanism for resistance. The data are in agreement with clinical data, which demonstrate a stronger effect of N348I on susceptibility to nevirapine as compared with efavirenz.
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http://dx.doi.org/10.1074/jbc.M110.105775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2930696PMC
August 2010

HIV-1 Ribonuclease H: Structure, Catalytic Mechanism and Inhibitors.

Viruses 2010 Apr 30;2(4):900-26. Epub 2010 Mar 30.

Department of Microbiology and Immunology, McGill University, Lyman Duff Medical Building (D6), 3775 University St., Montreal, QC, H3A 2B4, Canada.

Since the human immunodeficiency virus (HIV) was discovered as the etiological agent of acquired immunodeficiency syndrome (AIDS), it has encouraged much research into antiviral compounds. The reverse transcriptase (RT) of HIV has been a main target for antiviral drugs. However, all drugs developed so far inhibit the polymerase function of the enzyme, while none of the approved antiviral agents inhibit specifically the necessary ribonuclease H (RNase H) function of RT. This review provides a background on structure-function relationships of HIV-1 RNase H, as well as an outline of current attempts to develop novel, potent chemotherapeutics against a difficult drug target.
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http://dx.doi.org/10.3390/v2040900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185654PMC
April 2010

HIV-1 reverse transcriptase can simultaneously engage its DNA/RNA substrate at both DNA polymerase and RNase H active sites: implications for RNase H inhibition.

J Mol Biol 2009 May 13;388(3):462-74. Epub 2009 Mar 13.

Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec, Canada.

Reverse transcriptase of the human immunodeficiency virus possesses DNA polymerase and ribonuclease (RNase) H activities. Although the nucleic acid binding cleft separating these domains can accommodate structurally diverse duplexes, it is currently unknown whether regular DNA/RNA hybrids can simultaneously contact both active sites. In this study, we demonstrate that ligands capable of trapping the 3'-end of the primer at the polymerase active site affect the specificity of RNase H cleavage without altering the efficiency of the reaction. Experiments under single-turnover conditions reveal that complexes with a bound nucleotide substrate show specific RNase H cleavage at template position -18, while complexes with the pyrophosphate analogue foscarnet show a specific cut at position -19. This pattern is indicative of post-translocated and pre-translocated conformations. The data are inconsistent with models postulating that the substrate toggles between both active sites, such that the primer 3'-terminus is disengaged from the polymerase active site when the template is in contact with the RNase H active site. In contrast, our findings provide strong evidence to suggest that the nucleic acid substrate can engage both active sites at the same time. As a consequence, the bound and intact DNA/RNA hybrid can restrict access of RNase H active site inhibitors. We have mapped the binding site of the recently discovered inhibitor beta-thujaplicinol between the RNase H active site and Y501 of the RNase H primer grip, and have shown that the inhibitor is unable to bind to a preformed reverse transcriptase-DNA/RNA complex. In conclusion, the bound nucleic acid substrate and in turn, active DNA synthesis can represent an obstacle to RNase H inhibition with compounds that bind to the RNase H active site.
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http://dx.doi.org/10.1016/j.jmb.2009.03.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4285699PMC
May 2009

Connection domain mutations N348I and A360V in HIV-1 reverse transcriptase enhance resistance to 3'-azido-3'-deoxythymidine through both RNase H-dependent and -independent mechanisms.

J Biol Chem 2008 Aug 10;283(32):22222-32. Epub 2008 Jun 10.

Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada.

Thymidine analogue-associated mutations (TAMs) in reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) cause resistance to 3'-azido-3'-deoxythymidine (AZT) through excision of the incorporated monophosphate. Mutations in the connection domain of HIV-1 RT can augment AZT resistance. It has been suggested that these mutations compromise RNase H cleavage, providing more time for AZT excision to occur. However, the underlying mechanism remains elusive. Here, we focused on connection mutations N348I and A360V that are frequently observed in clinical samples of treatment-experienced patients. We show that both N348I and A360V, in combination with TAMs, decrease the efficiency of RNase H cleavage and increase excision of AZT in the presence of the pyrophosphate donor ATP. The TAMs/N348I/A360V mutant accumulates transiently formed, shorter hybrids that can rebind to RT before the template is irreversibly degraded. These hybrids dissociate selectively from the RNase H-competent complex, whereas binding in the polymerase-competent mode is either not affected with N348I or modestly improved with A360V. Both connection domain mutations can compensate for TAM-mediated deficits in processive DNA synthesis, and experiments with RNase H negative mutant enzymes confirm an RNase H-independent contribution to increased levels of resistance to AZT. Moreover, the combination of diminished RNase H cleavage and increased processivity renders the use of both PP(i) and ATP advantageous, whereas classic TAMs solely enhance the ATP-dependent reaction. Taken together, our findings demonstrate that distinct, complementary mechanisms can contribute to higher levels of excision of AZT, which in turn can amplify resistance to this drug.
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http://dx.doi.org/10.1074/jbc.M803521200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2494928PMC
August 2008
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