Current Drug Targets - Infectious Disorders - Volume 3, Issue 4, 2003

HIV DRUG RESISTANCE AND APPROACHES TO NEW THERAPIES In the absence of preventative or therapeutic vaccines for the foreseeable future, antiretroviral therapy is the most effective weapon in the fight against HIV / AIDS. Currently, there are 19 drugs that are commercially available for the treatment of HIV-1 infection, including eight nucleoside analog inhibitors of reverse transcriptase (NRTI), three allosteric (non-nucleoside) RT inhibitors (NNRTI), seven protease inhibitors (PI) and one recently approved entry inhibitor. In developed countries, treatments with combinations of these agents have proven highly effective at suppressing viral replication resulting in dramatic reductions in HIV morbidity and mortality. However, treatment failure is almost inevitable and is most frequently associated with the emergence of drug resistant virus. Efforts to reduce the occurrence and long-term clinical impact of treatment failures include the development of new and improved antiretroviral therapeutics and treatment strategies. The HIV / AIDS pandemic has emerged as the defining public health crisis of today's developing countries. The delivery of effective drug treatment to patient populations most in need is long overdue, but will require careful planning and implementation. These efforts can benefit from the many lessons learned since the approval of the first antiretroviral drug (AZT) in 1987. The collection of articles in this special issue address recent advancements in HIV-1 drug resistance research and the development of effective antiretroviral drug treatment strategies. A Review of Resistance to the Nucleoside and Nucleotide Reverse Transcriptase Inhibitors N. Shulman* and M. Winters Although members of the NRTI class were among the first antiretroviral drugs available for the treatment of HIV-1 infection, the field has only recently gained a firm understanding of the molecular mechanisms that confer reduced NRTI susceptibility. In this article, the authors describe an improved appreciation for the incidence and patterns of NRTI cross resistance, including the newer members of this drug class. With this new information, the field is better equipped to develop new candidates with more favorable / less extensive cross-resistance profiles and to optimize gene sequencing strategies for an important drug class that continues to serve as the cornerstone for most of today's HAART regimens. Resistance to HIV-1 Entry Inhibitors W.C. Olson* and P.J. Maddon Broad cross-resistance within the NRTI, NNRTI, and PI classes significantly limits the treatment options for a growing number of patients that have experienced multiple treatment regimen failures. This has prompted the search for drugs that target other steps in the HIV-1 replication cycle. The process of virus attachment / entry represents a promising target for antiretroviral drug development and the first member (enfuvirtide) of this new drug class is now available for the treatment of HIV / AIDS in the US and EU. In this review, the authors describe the preclinical and clinical characterization of each of the leading entry inhibitor candidates, and also discuss mechanisms of drug resistance. Members of this new drug family can be sub-classified as inhibitors of attachment, co-receptor binding, or fusion based on their ability to disrupt distinct steps in the entry process. The use of multiple entry inhibitors may offer advantages that include synergistic antiviral activity and non-overlapping resistance profiles. Is Resistance Futile? Victoria D. Kutilek, Dennis A. Sheeter, John H. Elder, and Bruce E. Torbett* In this article, the authors detail the structure and function of HIV-1 protease and discuss the limitations in the transition state design of existing protease inhibitors that have led to the widespread selection of viral variants exhibiting broad cross-resistance. These limitations include the structural rigidity of inhibitors and proteaseinhibitor binding interactions that are less reliant on backbone atoms and more sensitive to changes in side chain atoms than the normal protease-substrate complex. The authors discuss several new considerations for the design of second-generation competitive inhibitors, including inhibitor flexibility and inhibitor binding on rates that are more competitive with normal substrate binding. The authors also suggest the means for targeting other non-active site features, including the dimer interface, and entertain the attractive concept that viral evolution can be constrained in favor of drug resistant variants that are incapable of high rates of replication. Structural and Thermodynamic Basis of Resistance to HIV-1 Protease Inhibition: Implications for Inhibitor Design Adrian Velazquez-Campoy, Salman Muzammil, Hiroyasu Ohtaka, Arne Schön, Sonia Vega and Ernesto Freire* The available protease inhibitors were developed to inhibit the protease activity of wild-type (i.e. protease inhibitor naïve) HIV-1. Because the design of these first generation inhibitors was based on a constrained target, they are all vulnerable to resistance mutations that are capable of conferring broad cross-resistance to other members of this class. In this review, the authors discuss a paradigm shift in drug development strategy that includes the design of inhibitors that can structurally adapt to the binding site changes conferred by resistance mutations or polymorphisms without sacrificing binding specificity and affinity. Predicting the Impact of Antiretrovirals in Resource-Poor Settings: Preventing HIV Infections whilst Controlling Drug Resistance Sally Blower*, Li Ma, Paul Farmer, and Serena Koenig The introduction of antiretroviral drug treatment into resource limited settings that are in desperate need of HIV treatment intervention has been the subject of much debate. However, there are limited data to substantiate the beneficial and detrimental consequences of such a course of action, or how best to proceed. This will likely be the single most important decision that the world health community will make in the foreseeable future addressing this devastating global epidemic. In this article, the authors develop and apply newly developed mathematical models to estimate the impact of low to moderate levels of antiretroviral drug treatment in developing countries. The outcome of this endeavor stresses the importance and value of developing community specific AIDS prevention and care policies that carefully balance the prevention of new infections against the emergence and transmission of drug resistant strains. Mathematical Approaches in the Study of Viral Kinetics and Drug Resistance in HIV-1 Infection V. Muller* and S. Bonhoeffer The authors provide an in depth and comprehensive discussion of the application of mathematical modeling to the study of HIV dynamics. This work builds on earlier dynamic models in attempts to explain common treatment observations, including persistent low-level viremia and the emergence of drug resistant variants in the face of fully suppressive therapy. The authors also discuss the roles of viral “fitness”, recombination and intermittent drug pressure on the selection and outgrowth of resistant variants. The refined model further highlights the critical importance of treatment adherence to the durability of fully suppressive antiretroviral drug treatments. Fitness Variations and their Impact on the Evolution of Antiretroviral Drug Resistance Luis Menendez-Arias*, Miguel A. Martinez , Miguel E. Quinones-Mateu , and Javier Martinez-Picado Over the past several years, it has become increasingly apparent that many antiretroviral drug resistance mutations impair the normal function of the viral target protein, which in turn leads to attenuated viral replication. In this review, the authors describe the various methods that have been developed to measure the degree of impairment and also provide a comprehensive description of the contribution of specific NRTI, NNRTI and PI mutations to decreased viral “fitness”. The authors review several recent studies that have revealed associations between viruses with impaired replication and immune system preservation. More studies are needed to determine whether / how the impaired replication of drug resistant strains directly impacts pathogenesis, disease progression, and transmission. Variety of Interpretation Systems for Human Immunodeficiency Virus Type 1 Genotyping: Confirmatory Information of Additional Confusion M. Sturmer*, H. W. Doerr, and W. Preiser Genotyping assays are the most widely used form of antiretroviral resistance testing. The increasing complexities of mutational patterns that have emerged in response to the successive use of multi-drug treatment regimens have prompted the development of a wide variety of assay interpretation systems. In this article, the authors provide an overview of the most commonly used systems, followed by a drug by drug comparison of available interpretation algorithms. The authors also discuss the likely causes for common discordant interpretations and stress the importance of linking genotypic interpretations to clinical outcome data. Anti-HIV Inhibitors Based on Nucleic Acids: Emergence of Aptamers as Potent Antivirals P. J. Joshi, T. S. Fisher and V. R. Prasad* Cross resistance has not only prompted the search for new drug targets, but also new inhibitors that are structurally and mechanistically distinct from existing members of the NRTI, NNRTI, and PI classes. The authors provide an extensive review of nucleic acid-based inhibitors of HIV-1 replication. These versatile approaches can be applied to a wide variety of new (e.g. regulatory and structural proteins) and existing (enzymes) viral targets. Progress on the identification and development of inhibitory RNAs, referred to as “aptamers”, which exhibit impressive binding specificities and affinities is discussed in detail. The authors close with a discussion of several important technical obstacles, including delivery and intracellular expression, which will need to be addressed before this new therapeutic strategy can be fully exploited. I would like to close by thanking all of the authors for their excellent and thoughtful contributions, as well as Dr. Robert Goldman for his assistance and cooperation in the assembly of this special topics issue of CDT-ID.

The nucleoside reverse transcriptase inhibitors (NRTIs) were the first class of agents used for the treatment of HIV and remain an important component of combination antiretroviral therapy. Resistance to the NRTIs occurs by the acquisition of mutations in the reverse transcriptase gene that result in a structural change that either decreases the NRTI incorporation into the extending nucleotide chain or enhances removal of the NRTI from the terminated chain, also known as primer unblocking or pyrophosphorylysis. There are several major genetic mutational patterns of resistance and crossresistance that evolve with the NRTIs including the thymidine analog mutations M41L, D67N, K70R, L210W, T215Y, and K219Q / E / W, the non-thymidine mutations M184V, L74V, and K65R, and the mutidrug resistant Q151M complex, as well as others. Increasing knowledge of resistance and cross-resistance patterns that evolve on the NRTIs as well as the other antiretroviral classes will help optimize antiretroviral treatment strategies. Advancing knowledge of the biochemical and structural basis of resistance will aid in the design of newer compounds that are active against HIV resistant to the currently available drugs, ultimately prolonging virologic suppression and life in the millions of people who are infected with HIV.

Resistance-testing technology has been incorporated into the standard of care for human immunodeficiency virus type 1 (HIV-1) infection and therapy with protease and reverse transcriptase inhibitors. Inhibitors of HIV-1 entry represent an emerging mode of antiretroviral therapy, and HIV-1 entry inhibitors encompass three mechanistically distinct classes of agents known as attachment inhibitors, coreceptor inhibitors, and fusion inhibitors. Each class of agent has demonstrated promise in controlled clinical trials, and understanding the determinants and evolution of viral resistance will be critical for the optimal development and deployment of these new treatment classes. Advances in resistance-testing technologies have paralleled the development of HIV-1 entry inhibitor therapies, and the available data support the notion that attachment, coreceptor and fusion inhibitors offer complementary modes of therapy and distinct resistance profiles.

A global effort has been undertaken to control human immunodeficiency virus (HIV) though the development of vaccines and pharmacologics. Current FDA approved pharmacological inhibitors target two of the three viral enzymes critical to replication and maturation of infectious viral particles: reverse transcriptase (RT) and protease (Pr). Although combination therapies targeting RT and Pr have significantly reduced AIDS related morbidity and mortality, resistance to individual inhibitors is a growing concern. Currently, there are six protease inhibitors in clinical use. These inhibitors target the active site of protease using peptidomimetic transition state analogs based on natural substrates. However, treatment failures arise as a lack of compliance due to HIV-inhibitor pharmacokinetics, toxicity, and tolerance. This allows reduced HIV-inhibitor pressure, increased viral replication, and the emergence of drug resistant mutations. Continued use of protease inhibitors in the face of incomplete viral suppression may result in HIV-1 escape mutants not only being resistant to the protease inhibitor used, but to all clinically available protease inhibitors. Thus, new broad-based protease inhibitors are needed to control the emerging multi-drug, cross-resistant HIV-1. Moreover, given the emergence of cross-resistant HIV-1, there is a need to target novel protease structural sites to reduce the risk of multi-drug cross-resistance. In this review, we discuss the resistance to protease inhibitors and the rationale for new strategies towards drug design for suppressing protease activity. We focus on the structure and function relationship and the influence that drug resistance mutants exert on the evolution of HIV-1 protease.

One of the most serious side effects associated with the therapy of HIV-1 infection is the appearance of viral strains that exhibit resistance to protease inhibitors. At the molecular level, resistance to protease inhibition predominantly takes the form of mutations within the protease molecule that preferentially lower the affinity of protease inhibitors with respect to protease substrates, while still maintaining a viable catalytic activity. Mutations associated with drug resistance occur within the active site cavity as well as distal sites. Active site mutations affect directly inhibitor / protease interactions while non-active site mutations affect inhibitor binding through long range cooperative perturbations. The effects of mutations associated with drug resistance are compounded by the presence of naturally occurring polymorphisms, especially those observed in non-B subtypes of HIV-1. The binding thermodynamics of all clinical inhibitors against the wild type protease, drug resistant mutations and non-B subtype HIV-1 proteases has been determined by high sensitivity isothermal titration calorimetry. In conjunction with structural information, these data have provided a precise characterization of the binding mechanism of different inhibitors and their response to mutations. Inhibitors that exhibit extremely high affinity and low susceptibility to the effects of mutations share common features and binding determinants even if they belong to different chemical scaffolds. These binding determinants define a set of rules and constraints for the design of better HIV-1 protease inhibitors.

We review some crucial aspects of drug therapy and viral resistance that have been investigated within the framework developed for the modelling of virus kinetics. First, we give a general overview on the use of mathematical models in the field of HIV research. We seek to identify the factors that determine the steady state virus load and show that stable reductions during antiviral therapy are difficult to explain within the standard model of virus dynamics. We discuss possible extensions that enable the models to account for the moderately reduced virus loads during non-suppressive treatment and argue that the residual viremia under suppressive treatment can probably be attributed to the survival of long-lived infected cells, rather than to new rounds of replication. Next, we address the emergence of resistance during suppressive therapy and demonstrate that the resistant virus is more likely to be present already at the start of treatment than to be generated during therapy. The appearance of resistance after a prolonged period of initial suppression indicates that drug efficacy is not continuously maintained over time. We investigate the potential risks and benefits of therapy interruptions. Considering the effect of recombination, we argue that it probably decelerates, rather than accelerates the evolution of multidrug-resistant virus. We also review state-of-the-art methods for the estimation of fitness, which is crucial to the understanding of the emergence of resistance during therapy or the re-emergence of wild type upon the cessation of therapy.

There is currently an opportunity to carefully plan the implementation of antiretroviral (ARV) therapy in the developing world. Here, we use mathematical models to predict the potential impact that low to moderate usage rates of ARVs might have in developing countries. We use our models to predict the relationship between the specific usage rate of ARVs (in terms of the percentage of those infected with HIV who receive such treatment) and: (i) the prevalence of drug-resistant HIV that will arise, (ii) the future transmission rate of drug-resistant strains of HIV, and (iii) the cumulative number of HIV infections that will be prevented through more widespread use of ARVs. We also review the current state of HIV / AIDS treatment programs in resource-poor settings and identify the essential elements of a successful treatment project, noting that one key element is integration with a strong prevention program. We apply both program experience from Haiti and Brazil and the insights gleaned from our modeling to address the emerging debate regarding the increased availability of ARVs in developing countries. Finally, we show how mathematical models can be used as tools for designing robust health policies for implementing ARVs in developing countries. Our results demonstrate that designing optimal ARV-based strategies to control HIV epidemics is extremely complex, as increasing ARV usage has both beneficial and detrimental epidemic-level effects. Control strategies should be based upon the overall impact on the epidemic and not simply upon the impact ARVs will have on the transmission and / or prevalence of ARV-resistant strains.

The human immunodeficiency virus (HIV) exhibits extensive heterogeneity due to its rapid turnover, high mutation rate, and high frequency of recombination. Its remarkable genetic diversity plays a key role in virus adaptation, including development of drug resistance. The increasing complexity of antiretroviral regimens has favored selection of HIV variants harboring multiple drug resistance mutations. Evolution of drug resistance is characterized by severe fitness losses, which can be partially overcome by compensatory mutations or other adaptive changes that restore virus replication capacity. Recent reports have addressed the impact of drugresistance mutations on viral fitness. Methods include in vitro estimates based on the determination of viral replication kinetics, viral infectivity in single-cycle assays and growth competition experiments; as well as estimates of the relative fitness of viral populations in vivo calculated from standard population genetics theory. This review focuses on the effects in viral fitness of mutations arising during treatment with reverse transcriptase and protease inhibitors, and the molecular mechanisms (including compensatory mutations) that improve the viral fitness of drug-resistant variants.

The emergence of drug resistance remains a major problem during antiretroviral treatment of patients infected with human immunodeficiency virus type 1 (HIV-1). As phenotypic drug resistance is laborious and expensive to determine, and because numerous specific mutations are known to be correlated with different resistance patterns, genotyping of the reverse transcriptase and protease genes of HIV is fast becoming an integral part of HIV management in industrialized countries. A number of software-based interpretation systems have been developed for the interpretation of the resulting complex nucleotide sequences. These programs either employ rule-based algorithms or are based on a genotype-phenotype database. This paper reviews recent publications that compare different such systems, trying to identify the degree of discordance between different systems and the reasons underlying such discrepant interpretations. The highest discordance rate was observed for nucleoside reverse transcriptase inhibitors (NRTIs) followed by protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). For the NRTIs, it is the role of nucleoside analogue associated mutations, for the PIs and for the NNRTIs, that of secondary mutations that causes most discrepancies. As the complexity of the mutation pattern is likely to increase further with new drugs becoming available, rule-based genotype interpretation algorithms need to be updated frequently. Whilst not recommending one particular system, the authors believe that the correlation of genotypic with clinical data is probably the best way to develop an optimal algorithm.

The development of resistance and the inability of currently approved antiretroviral drugs to completely eradicate HIV-1 have led to increased focus on therapies other than small molecules. Although nucleic acid-based intervention requires complex tasks involving intracellular delivery and / or stable expression in target cells, recent advances in gene therapy methods combined with continued progress in stem cell approaches have made nucleic acidbased compounds excellent candidates for effectively inhibiting intracellular targets. Consequently, multiple nucleic acidbased therapies are being developed. These include antisense nucleic acids, peptide nucleic acids and RNA decoys, which can interfere with HIV-1 replication. More recently, RNA interference, which exploits a novel cellular pathway, has been shown to effectively reduce viral titers in cell culture and promises to be a potential candidate for suppressing HIV replication in vivo. A promising candidate in the midst of these emerging approaches is the aptamer approach, which involves the use of a class of small nucleic acid molecules isolated from combinatorial libraries by an in vitro evolution protocol termed SELEX. Aptamers exhibit exquisite specificity, high affinity and the virtual lack of immunogenicity, features that make them exceptionally well-suited to combat HIV without affecting the host. The powerful nature of these specific antagonists of protein function could lead to the development of an effective anti-HIV therapy. Several highly specific, nucleic acid aptamers targeting select HIV proteins have been described. Investigations with anti- HIV RNA aptamers have shown an effective block to viral replication. This review summarizes the existing nucleic-acid based approaches to block HIV replication and attempts to chart the current progress in the development of aptamers against HIV, their use in inhibiting the virus replication, prospects for their use in the clinic and potential drawbacks.