Current Pharmaceutical Design

ISSN: 1381-6128

Current Pharmaceutical Design
Volume 12, Number 15, 2006

Contents

HIV-1 Reverse Transcriptase Inhibitors: Drug Resistance and Drug Development
Executive Editors: Nicolas Sluis-Cremer and Ted Ross


Editorial Pp. 1809-1810


Insertions and Deletions in HIV-1 Reverse Transcriptase: Consequences for Drug Resistance and Viral Fitness Pp. 1811-1825
L. Menéndez-Arias, T. Matamoros and C.E. Cases-González
[Abstract]


The Influence of Natural Substrates and Inhibitors on the Nucleotide-Dependent Excision Activity of HIV-1 Reverse Transcriptase in the Infected Cell Pp. 1827-1841
A.J. Smith and W.A. Scott
[Abstract]


Assault on Resistance: The Use of Computational Chemistry in the Development of Anti-HIV Drugs Pp. 1843-1856
M.B.K. Smith, R.H. Smith, Jr. and W.L. Jorgensen
[Abstract]


Developing Novel Nonnucleoside HIV-1 Reverse Transcriptase Inhibitors: Beyond the Butterfly Pp. 1857-1865
A. Basavapathruni and K.S. Anderson
[Abstract]


Effects of Nucleotides and Nucleotide Analogue Inhibitors of HIV 1 Reverse Transcriptase in a Ratchet Model of Polymerase Translocation Pp. 1867-1877
M. Götte
[Abstract]


Dimerization of Human Immunodeficiency Virus Type 1 Reverse Transcriptase as an Antiviral Target Pp. 1879-1894
S. Srivastava, N. Sluis-Cremer and G. Tachedjian
[Abstract]


TSAO Derivatives, Inhibitors of HIV-1 Reverse Transcriptase Dimerization: Recent Progress Pp. 1895-1907
M.J. Camarasa, S. Velázquez, A. San-Félix, M.J. Pérez-Pérez, M.C. Bonache and S. De Castro
[Abstract]


Recent Progress in the Design of Small Molecule Inhibitors of HIV RNase H Pp. 1909-1922
K. Klumpp and T. Mirzadegan
[Abstract]


Natural and Synthetic Retinoids in Prostate Cancer Pp. 1923-1921
D. Pasquali, V. Rossi, G. Bellastella, A. Bellastella and A.A. Sinisi
[Abstract]


Gq protein-Coupled Receptors as Targets for Anesthetics Pp. 1931-1937
K. Minami and Y. Uezono
[Abstract]


Abstracts
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Editorial

HIV-1 Reverse Transcriptase Inhibitors: Drug Resistance and Drug Development

HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that facilitates the conversion of the viral single-stranded (+) RNA genome into double-stranded DNA. The enzyme exhibits both a DNA polymerase activity, that can use either RNA or DNA as a template, and a ribonuclease H (RNase H) activity that specifically degrades the RNA strand of RNA:DNA duplexes. Due to its essential role in the HIV life-cycle, RT is a primary target for anti-HIV drug development. To date, the United States Food and Drug Administration has approved 11 RT inhibitors (RTIs) for clinical use. These can be classified into two distinct therapeutic groups: (i) nucleoside and nucleotide RT inhibitors (NRTI) which include zidovudine (AZT), stavudine (d4T), didanosine (ddI), zalcitabine (ddC), lamivudine (3TC), emtricitabine (FTC), abacavir (ABC) and tenofovir disoproxil fumarate (PMPA); and (ii) the nonnucleoside RT inhibitors (NNRTI) which include nevirapine, delavirdine and efavirenz.

The emergence of HIV-1 viral resistance to the available RTIs has limited their efficacy for long-term clinical use and has necessitated the development of new inhibitors that are active against both wild-type and resistant strains of the virus. In this directed issue of Current Pharmaceutical Design, we solicited review articles from leaders in the field who shed new paradigms into RTI drug resistance and strategies for developing new therapeutics against HIV-1 RT. In general the review articles can be broadly categorized into three categories: (i) those that deal with resistance; (ii) those that deal with designing better inhibitors against existing drug targets; and (iii) those that identify novel drug targets in HIV-1 RT.

The mechanism(s) of resistance of HIV-1 to RTIs has been the focus of many excellent review articles. The 3 review articles in this issue, contributed by Drs Luis Menéndez-Arias (Spain), Walter Scott (USA) and Marilyn Kroeger Smith (USA), do not re-capitulate past articles, but focus on previously under-explored areas of drug resistance. In this regard, Menéndez-Arias et al. examine the role of amino acid insertions and deletions in HIV-1 RTI multi-drug resistance and viral replication fitness [1]. Smith and Scott discuss the influence of natural substrates and inhibitors in the infected cell on the nucleotide excision mechanism of HIV-1 resistance to NRTIs [2]. Kroeger Smith et al. highlight the contribution that computational chemistry can make toward understanding HIV-1 resistance and drug development [3].

NNRTIs are allosteric inhibitors that bind to a non-active site pocket in HIV-1 RT, termed the NNRTI-binding pocket (NNRTI-BP). One major limitation of this class of inhibitors is that single mutations in the NNRTI-BP often yield cross-resistance to all NNRTI. In this issue, Dr Karen Anderson (USA) has contributed an article that provides succinct insights into the kinetic mechanisms of action of, and resistance to, NNRTI and describes the recent advances that have been made to create NNRTI which are more potent and less susceptible to existing drug resistance mutations [4].

The identification of novel drug targets and/or the development of new classes of antiviral compounds are essential in the fight against HIV/AIDS. The 11 existing FDA-approved RTIs all target the DNA polymerase activity of the enzyme, however there are other targets in HIV-1 RT that can be exploited to develop new therapeutic classes of RTIs. These include translocation, dimerization and RNase H activity.

Translocation describes the movement of polymerases along the nucleic acid template after each nucleotide addition reaction. Translocation is often viewed as a kinetically “invisible” step, but recently Dr Matthias Götte (Canada) has made significant progress into elucidating possible translocation mechanisms in HIV-1 RT. In his review, Dr Götte discusses mechanisms of translocation, the role of translocation in drug resistance, and also possible strategies for inhibiting this kinetic event [5].

HIV-1 RT is an obligate heterodimer; the enzymatic activities of RT (in particular the DNA polymerase activity) are entirely dependent on the dimeric structure of the enzyme. Dr Gilda Tachedjian (Australia) reviews the merits of HIV-1 RT dimerization as an antiviral target [6], while Dr María-José Camarasa (Spain) describes structure-activity relationships of TSAO derivatives [7], the first non-peptidic inhibitors of HIV-1 RT dimerization.

In contrast to DNA polymerase inhibitors, the discovery of potent and selective inhibitors of HIV RT RNase H has been slow, and inhibitors of this enzyme function have yet to reach the clinical development stage. Dr Klaus Klumpp (USA) reviews recent progress in a number of key areas that has provided new impetus to the discovery of HIV RNase H inhibitors [8]. In particular, he describes the design, synthesis and activity of the N-hydroxyimides and, for the first time, presents crystal structure data for the binding of these compounds in the RNase H active site.

As co-editors of this issue of Current Pharmaceutical Design, we are very grateful for the time and effort that all of the authors and co-authors have put into their review articles. Furthermore, we hope that the reader will be stimulated by this collection of insightful review articles.

References

[1] Menéndez-Arias L, Matamoros T, Cases-González, CE, Insertions and deletions in HIV-1 reverse transcriptase: Consequences for drug resistance and viral fitness. Curr Pharm Design 2006; 12(15): 1811-1825.

[2] Smith AJ, Scott WA, The influence of natural substrates and inhibitors on the nucleotide dependent excision activity of HIV-1 reverse transcriptase in the infected cell. Curr Pharm Design 2006; 12(15): 1827-1841.

[3] Kroeger Smith MB, Smith Jr RH, Jorgensen WL, Assault on resistance, the use of computacional chemistry in the development of anti-HIV drugs. Curr Pharm Design 2006; 12(15): 1843-1856.

[4] Basavapathruni A, Anderson KS, Developing novel nonnucleoside HIV-1 reverse transcriptase inhibitors: beyond the butterfly. Curr Pharm Design 2006; 12(15): 1857-1865.

[5] Götte M, Effects of Nucleotides and Nucleotide Analogue Inhibitors of HIV-1 Reverse Transcriptase in a Ratchet Model of Polymerase Translocation. Curr Pharm Design 2006; 12(15): 1867-1877.

[6] Srivastava S, Sluis-Cremer N, Tachedjian G, Dimerization of Human Immunodeficiency Virus type 1 Reverse Transcriptase as an Antiviral Target. Curr Pharm Design 2006; 12(15): 1879-1894.

[7] Camarasa MJ, Velázquez S, San-Félix A, Pérez-Pérez MJ, Bonache MC and De Castro S, TSAO Derivatives, Inhibitors of HIV-1 Reverse Transcriptase Dimerization: Recent Progress. Curr Pharm Design 2006; 12(15): 1895-1907.

[8] Klumpp K, Mirzadegan T, Recent progress in the design of small molecule inhibitors of HIV RNase H. Curr Pharm Design 2006; 12(15): 1909-1922.

Nicolas Sluis-Cremer and Ted Ross
University of Pittsburgh
Department of Medicine
Division of Infectious Diseases
Pittsburgh
Pennsylvania 15261
USA


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Insertions and Deletions in HIV-1 Reverse Transcriptase: Consequences for Drug Resistance and Viral Fitness
L. Menéndez-Arias, T. Matamoros and C.E. Cases-González

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is an important target of drugs fighting HIV infection. The introduction of potent antiretroviral therapies based on the use of RT inhibitors and/or protease inhibitors has been an important achievement towards the control of AIDS. However, the development of drug resistance constitutes a major hurdle towards long-term efficacy of those therapies. With the increasing complexity of the antiretroviral regimens, novel mutational patterns conferring high-level resistance to nucleoside and nonnucleoside RT inhibitors have been identified in viral isolates. Among them, insertions and deletions in the β3-β4 hairpin-loop-coding region of HIV-1 RT have been identified in heavily-treated patients. Insertions of one, two or several residues appear to have a significant impact on nucleoside analogue resistance. The frequently found combination of a dipeptide insertion and thymidine analogue resistance mutations (i.e. T215Y) in the viral RT confers an ATP-dependent phosphorolytic activity that facilitates the removal of the inhibitor from primers terminated with zidovudine or stavudine. Furthermore, this mechanism appears to be relevant for resistance mediated by one amino acid-deletions appearing in combination with thymidine analogue resistance mutations. However, in other sequence contexts (i.e. in the presence of Q151M), the effects of the deletion are not fully understood. Drugs targeting the excision repair mechanism could be an important aid in the fight against multinucleoside-resistant HIV isolates bearing complex mutational patterns in their RT-coding region.


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The Influence of Natural Substrates and Inhibitors on the Nucleotide-Dependent Excision Activity of HIV-1 Reverse Transcriptase in the Infected Cell
A.J. Smith and W.A. Scott

Human immunodeficiency virus type 1 resistance to nucleoside reverse transcriptase inhibitors such as 3’-azido-2’, 3’-dideoxythymidine (AZT) can arise through mutations in the coding region of reverse transcriptase (RT) that enhance the enzyme’s ability to remove the drug after it has been incorporated. This excision activity of HIV-1 RT has been well characterized in a number of in vitro systems. However, the in vitro findings do not provide a complete picture of the in vivo significance of this resistance mechanism. This review will attempt to bridge the gap between the in vitro observations and the in vivo environment by summarizing the fragmentary information that is available about the intracellular conditions that may influence drug excision in cell subpopulations that are infected by HIV-1. Topics that will be discussed include (a) intracellular compounds HIV-1 RT may use to remove chain terminators; (b) how dNTPs can affect excision activity and how these effects differ in different immune cell subpopulations; (c) the influence of HIV infection on excision activity – e.g., through immune activation of infected cells or through changes indirectly induced in cells that subsequently become infected; (d) intracellular conditions that favor selection for mutations that increase the excision-based resistance mechanism; (e) the importance of macrophages in the selection of resistance mutations. Understanding factors that control excision in the intracellular environment will greatly enhance our understanding of the process of selection for this class of drug resistance mutations and may open doors for the development of novel targets for antiviral therapy.


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Assault on Resistance: The Use of Computational Chemistry in the Development of Anti-HIV Drugs
M.B.K. Smith, R.H. Smith, Jr. and W.L. Jorgensen

While many inhibitors of the Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immunodeficiency Syndrome (AIDS), have been developed, the problem of drug resistance has continued to plague the fight against the disease. The ability of computers to aid in the drug discovery process, and by default the resistance problem, has increased dramatically as the speed of computers and sophistication of associated calculation programs has grown. In particular, the capability of predicting a compound’s ability to combat resistance prior to synthesis of drug candidates has proven particularly desirable. Since resistance can develop against a specific drug designed to inhibit only one stage of the viral cycle, combinations of drugs directed at more than one step have proven to be more effective than a single drug given alone. While the introduction of this combination therapy (termed highly active antiretroviral therapy (HAART)) has significantly decreased the death rate from HIV infections, resistance problems still arise. This paper will review previous approaches and address current and future computational strategies used in the design of second-generation and beyond drugs.


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Developing Novel Nonnucleoside HIV-1 Reverse Transcriptase Inhibitors: Beyond the Butterfly
A. Basavapathruni and K.S. Anderson

To date three nonnucleoside reverse transcriptase inhibitors (NNRTIs) have been approved by the U.S. Food and Drug Administration for the treatment of human immunodeficiency virus type 1 infection. A limiting factor in the effectiveness of these agents is the development of resistance, manifested by amino acid substitutions within the virally encoded reverse transcriptase (RT). Understanding the mechanism of action of these agents and how resistance develops have broadened the field of NNRTI research to elucidate structural and biochemical features of inhibition in hopes of creating better inhibitors. In this review, the history of NNRTIs will preface the many studies characterizing inhibition and the development of a new paradigm for understanding the molecular mechanism of drug resistance to NNRTIs. Combination therapies including nonnucleoside inhibitors will be discussed, concluding with remarks on potential new inhibitors.


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Effects of Nucleotides and Nucleotide Analogue Inhibitors of HIV 1 Reverse Transcriptase in a Ratchet Model of Polymerase Translocation
M. Götte

A single cycle of nucleotide incorporation by the reverse transcriptase of the human immunodeficiency virus type 1 (HIV-1 RT) involves the initial binding of an incoming nucleotide, a conformational change that traps the substrate, the formation of a new phosphodiester bond, the release of pyrophosphate (PPi), and ultimately polymerase translocation, which clears the nucleotide binding site. This article reviews different mechanistic models for polymerase translocation with emphasis placed on HIV-1 RT. Structure-function analyses of stalled complexes of HIV-1 RT provide strong evidence to suggest that the enzyme can oscillate between pre- and post-translocational states. Nucleotide hydrolysis is not required for the movement of the polymerase in a stalled configuration; thermal energy is sufficient to allow random bidirectional sliding. The next complementary nucleotide, following the incorporated chain-terminator, acts like a pawl of a ratchet that traps the enzyme in the post-translocation state and prevents the reverse movement. Quantitative footprinting experiments have shown that the concentration of the templated nucleotide required to shift the translocational equilibrium forward depends crucially on the structure of the 3’end of the primer. Changes in the relative population of pre- and post-translocation complexes can influence rates of excision of incorporated NRTIs, which, in turn, affects drug susceptibility. The concept of a ratchet model of HIV-1 RT translocation and its implications for drug action and resistance, and the discovery and development of novel antiviral compounds is discussed.


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Dimerization of Human Immunodeficiency Virus Type 1 Reverse Transcriptase as an Antiviral Target
S. Srivastava, N. Sluis-Cremer and G. Tachedjian

Emergence of drug resistant strains of human immunodeficiency virus type 1 (HIV-1) is a major hindrance in the long-term treatment of HIV-1 infected individuals. Alternative strategies, including those directed to structural elements of viral targets, are needed to combat the growing acquired immune deficiency syndrome (AIDS) pandemic. The HIV-1 reverse transcriptase (RT) dimer interface, critical for dimer stability and catalytic function, is a novel target for designing new anti-HIV-1 drugs. Several existing RT inhibitors are known to impair polymerase function by destabilizing RT dimer stability and can serve as useful leads in this direction. Conversely, studies have shown that potent nonnucleoside reverse transcriptase inhibitors (NNRTIs) can enhance RT subunit interaction, which may contribute in part to the inhibitory effect of these drugs. Interface peptides are reported to suppress enzyme activity by interfering with active RT heterodimer formation. This review focuses on small molecule and peptide inhibitors that interfere with the formation of the active RT heterodimer and also discusses regions in the RT that are critical for RT dimerization that can be considered as potential targets for chemotherapeutic intervention.


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TSAO Derivatives, Inhibitors of HIV-1 Reverse Transcriptase Dimerization: Recent Progress
M.J. Camarasa, S. Velázquez, A. San-Félix, M.J. Pérez-Pérez, M.C. Bonache and S. De Castro

There is an urgent need for the development of new and safer drugs for the treatment of HIV (human immunodeficiency virus) infection, active against the currently resistant viral strains or directed to novel targets in the viral replicative cycle that may be useful for multiple drug combination. TSAO derivatives are a peculiar group of highly functionalized nucleosides that belong to the so-called nonnucleoside RT inhibitors (NNRTIs). HIV-1 reverse transcriptase (RT) is a key enzyme that plays an essential and multifunctional role in the life cycle of the virus and thus represents a key target for antiviral chemotherapeutic intervention. The dimeric form of the enzyme is absolutely required for all enzymatic ac-tivities. Thus, the process of dimerization and subsequent maturation into the p66/p51 heterodimer is essential for a fully functional RT and constitutes a target for therapeutic intervention, however to date such agents have not been developed. TSAO molecules are a peculiar group of non-nucleoside RT inhibitors that exert a unique selectivity for HIV-1 through a specific interaction with the p51 subunit of HIV-1 RT. They interact at the p66/p51 heterodimer interface of the enzyme. They were the first small non peptidic molecules shown to interfere with the dimerization process of the enzyme. This re-view covers the recent work within this family of compounds aimed at enhancing their interaction with the dimer interface of HIV-1 RT.


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Recent Progress in the Design of Small Molecule Inhibitors of HIV RNase H
K. Klumpp and T. Mirzadegan

DNA polymerase and RNase H (RH) activities of HIV reverse transcriptase (RT) have been recognized as potential targets for antiretroviral therapy for more than 15 years. The development of medicines targeting the DNA polymerase activity has been highly successful, with currently 12 drugs approved for the treatment of HIV infection and more candidates in preclinical and clinical development. In contrast, the discovery of potent and selective inhibitors of HIV RH has been slow, and inhibitors of this enzyme function have yet to reach the clinical development stage. Selective HIV RH inhibitors are likely to provide significant clinical benefit in combination therapies, considering the high prevalence of HIV strains resistant to currently available antiretroviral therapies. Recent progress in a number of key areas has provided new impetus to the discovery of HIV RH inhibitors. High throughput assay systems based on fluorescence detection have been developed, which facilitate screening of inhibitor candidates. Substantial progress has been made in expression, purification, crystallisation and solution studies of HIV RT and RH, in particular with regards to aspects of structural dynamics. Crystal structures of active site binding and allosteric HIV RH inhibitors bound to HIV RT and RH have been obtained. Finally, an improved understanding of similarities and differences in enzymatic mechanisms between related nuclease enzymes has provided new concepts for achieving inhibitor selectivity. Together, these developments provide promising new starting points for the rational design of selective HIV RH inhibitors.


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Natural and Synthetic Retinoids in Prostate Cancer
D. Pasquali, V. Rossi, G. Bellastella, A. Bellastella and A.A. Sinisi

Prostate cancer (PCa) is the most common cancer for men in Europe, North America, and some parts of Africa. Initially, growth of prostate cancer is usually androgen-dependent, but often it becomes androgen-independent after androgen-deprivation therapy. Managing hormone-refractory prostate carcinoma remains a difficult challenge for clinicians. Retinoids, vitamin A and its synthetic analogs are one of the most studied class of chemopreventive drugs for PCa. Retinoids play a key role in several vital functions as vision and development, and also exert anti-proliferative actions. Anti-proliferative effects of retinoids rely on the regulation of many biological processes, including differentiation, cell proliferation, and apoptosis. Retinoid actions are mediated by two classes of nuclear proteins called retinoic acid (RARα,β and γ and retinoic α,β and γ receptors, which are ligand-regulated transcription factors. Effects of both all-trans -retinoic acid (RA), the natural active derivative of vitamin A, and its synthetic derivatives, on prostate gland or prostate cell lines implicate retinoids in the regulation of prostate growth and suppression of PCa development. Deficient retinoid availability and action at the cellular level because of either decreased content or altered metabolism in PCa cells can play a key role in abnormal cellular differentiation pathways, and the loss of anti-proliferative effects. Here we review the in vitro and in vivo effects of retinoids in PCa.

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Gq protein-Coupled Receptors as Targets for Anesthetics
K. Minami and Y. Uezono

The mechanisms of action of anesthetics are unclear. Much attention has been focused on ion channels in the central nervous system as targets for anesthetics. During the last decade, major advances have been made in our understanding of the physiology and pharmacology of G-protein-coupled receptor (GPCR) signaling. Several lines of studies have shown that GPCRs are targets for anesthetics and that some anesthetics inhibit the functions of Gq-coupled receptors, including muscarinic acetylcholine (ACh) M1, metabotropic type 5 glutamate, 5-hydroxytryptamine (5-HT) type 2A, and substance P receptors. Nearly 160 GPCRs have been identified, based on their gene sequence and ability to interact with known endogenous ligands. However, an estimated 500-800 additional GPCRs have been classified as “orphan” receptors (oGPCRs) because their endogenous ligands have not yet been identified. Given that known GPCRs are targets for anesthetics, these oGPCRs represent a rich group of receptor targets for anesthetics. This article highlights the effects of anesthetics on Gq-coupled receptors, and discusses whether GPCRs other than Gq-coupled receptors are targets for anesthetics.