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Inhibitors of HIV-1 Entry and Integration: Recent Developments and Impact on Treatment Anil K. Sharma1, Varghese George2, Ranjini Valiathan3, Sudheesh Pilakka-Kanthikeel4 and Suresh Pallikkuth2,* 1
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA; 2Department of Microbiology & Immunology, University of Miami Miller School of Medicine, Miami, FL 33136, USA; 3Department of Pathology, Laboratory of Clinical and Biological Studies- University of Miami, Miami, Fl 33136, US; 4Dept. of Immunology, Herbert Wertheim College of Medicine, Institute of NeuroImmune Pharmacology, Florida International University, Miami, FL -33199,USA Received: March 25, 2013; Accepted: April 2, 2013; Revised: April 8, 2013
Abstract: Advances in the drug development against HIV-1 have lead to the identification of new compounds which could be used to target cellular entry and nuclear integration of virus in addition to drugs that commonly target reverse transcriptase and protease. These additional targets have added a new dimension to fight against HIV. Cellular entry of HIV is a multistep procedure involving a range of cellular and molecular interactions between virus envelope protein and receptors expressed on the surface of the target cells, thus providing many opportunities to block infection. Some of these entry inhibitors are currently being used in the clinic and more compounds are under various stages of development. Integration of the HIV-1 DNA is required and essential to maintain the viral DNA in the infected cell. The design and discovery of integrase inhibitors were first focused at targeting the catalytic site of integrase that selectively acting on strand transfer and thus inhibits integration of virus DNA with host cell genome. Thus, entry and integrase inhibitors present a real added value in combined treatment against HIV infection. This review discusses the recent development in the discovery of inhibitors of HIV entry and integration along with some of recent patents in the field.
Keywords: HIV-1, entry inhibitors, integrase inhibitors, Raltegravir, HIV patents, ART and HIV, HIV treatment. 1. INTRODUCTION The entry of HIV-1 into susceptible target cells and its integration to host cell genome is a multistep process that leads to the fusion of viral and cell membranes and ultimately productive infection and latency development. Antiretroviral drugs that interact with each step in the HIV-1 entry process have been developed, but only two compounds (maraviroc and enfuvirtide) are currently approved for the usage in clinics. The integrase enzyme facilitates the incorporation of HIV-1 proviral DNA into the host cell genome and catalyzes a function vital to replication of the virus. Inhibitors of integrase are the newest class of antiretroviral drugs developed in an effort to treat HIV-1infection. Raltegravir, an integrase strand transfer inhibitor, was the first drug of this class approved by the US FDA; it is a potent and well tolerated antiviral agent. However, numerous limitations exist in the usage of these newly developed compounds; their daily dosing, adverse events and genetic barrier to the development of resistance. Given the potential for these agents to block viral entry and integration, there has been increased interest in using them to prevent acquisition *Address correspondence to this author at the Microbiology and Immunology, University of Miami Miller School of Medicine, 1580 NW 10th Avenue, BCRI 708, Miami, FL 33136; Tel: 305-243-7733; Fax: 305-243-7211; E-mail:
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and establishment of HIV-1 infection. Here, we summarize progress in the development of HIV-1 entry and integrase inhibitors, with an emphasis on molecules in different stages of clinical development, advanced pre-clinical studies in humans along with information about their recent patents. Each of them contributes to their respective class and carries potential as treatment options for patients with HIV-1 infection. 2. INTERACTIONS BETWEEN HIV-1 AND HOST CELL DURING VIRAL ENTRY The entry of HIV into a cell is mediated by the binding of its surface subunit; the hetero-trimeric envelops glycoprotein gp120 to its primary receptor on the host cell surface. CD4 is the primary surface receptor for lymphocytes and macrophages [1, 2]. Binding of gp120 with CD4 leads to conformational changes within gp120 and the interaction is further stabilized by engaging one of the two chemokine receptors, either CCR5 or CXCR4. Coreceptor binding further induces conformational changes within the fusion peptide of the envelope gp41 that leads to insertion of gp41 ectodomain into the cell membrane of target cells. This process allows the virus to fuse with host cell membrane and allows the viral nucleocapsid to enter the cytoplasm of host cells. Viral protein Vpr mediated signals allow the nucleocapsid to enter into host cell nucleus to be transcribed later into cDNA [1, 2].
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3. INHIBITORS OF VIRAL ENTRY Initial attempts made to block the entry of HIV were based on manipulating the host cell surface expression of primary HIV receptor, CD4. This approach was mainly based on usage of recombinant soluble CD4 molecules. Without having a transmembrane and cytoplasmic domain, this form of CD4 acted as a decoy that efficiently bound to gp120 and demonstrated a moderate in vitro activity against HIV-1. However, the clinical trials using these molecules were not encouraging [3-7]. Another attempt was the usage of small molecule inhibitors to target the virus by binding to a specific region within the CD4 binding site of gp120 and thereby preventing the gp120–CD4 interaction [8, 9]. Therapeutic usage of this approach was tested in chronic treatment-naive HIV infected subjects using a compound known as BMS-488043. In this study, a log10 reduction in plasma HIV-1 RNA was observed [10]. However, suboptimal pharmacokinetics led to discontinuation of this compound in further clinical trials. Another compound, BMS-663068 had demonstrated a better pharmacokinetics and enhanced potency against a wide range of HIV-1 subtypes [11]. In a randomized, open-label, phase-2a clinical trial in treatment naïve chronic HIV-infected patients, BMS-663068 with or without ritonavir boosting showed a well-tolerated profile with upto1.7 log10 reduction in plasma HIV-1 RNA levels after eight days of treatment [12]. 4. INHIBITION AFTER VIRAL ATTACHMENT TO CD4 T CELLS These inhibitors are a novel class of anti-CD4 monoclonal antibody (mAb) that do not prevent interaction between CD4 and gp120, but through blocking the virus and cell fusion by targeting a second (C2) domain of CD4, inhibiting viral attachment. This mAb known as ibalizumab (formerly TNX-355) is one such humanized IgG4 mAb that performs this function [13]. Unlike attachment inhibitors, this mAb decreases the plasticity of CD4 by interfering with the access of CD4-bound gp120, leading to distortion of the CD4–gp120 complex, thereby preventing binding to the chemokine receptors. In vitro experiments demonstrated higher potency of ibalizumab and its effect when synergized with gp120 antibodies or the fusion inhibitor enfuvirtide. In addition, in vitro effect of this molecule was associated with limited interference with immunological functions including antigen presentation [14-17]. This compound entered clinical trials and phase-1 studies of intravenous ibalizumab administration in the treatment of naïve chronic HIV infected patients showed up to a 1.5 log10 reduction in plasma HIV-1 RNA levels within 14–21 days of a single dose administration [18]. However, repeated dosing over nine weeks induced the emergence of viral resistance [19]. Studies in treatment-experienced patients showed that ibalizumab along with an optimized background regimen resulted in significant reductions in plasma HIV-1 RNA compared to the background regimen alone in a phase-2 study [20]. Higher infectivity was noted for viruses isolated from participants of phase-1b trials with reduced susceptibility to ibalizumab in comparison with baseline viruses obtained from paired samples. However, susceptibility to CCR5 antagonist maraviroc
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and fusion inhibitor enfuviritide was still intact for these low ibalizumab susceptible viruses [21]. The safety, tolerability, and pharmacokinetics of ibalizumab with three dosing schedules among at-risk, HIV-negative volunteers are currently ongoing as a randomized, double-blinded, placebocontrolled, phase-1 pilot study [22]. A recent study characterized the in vitro breadth and potency of ibalizumab against a diverse panel of 116 HIV-1 envs and showed that ibalizumabs in vitro breadth and potency were comparable to the broadly neutralizing antibodies PG9 and VRC01 suggesting its potential as an ideal candidate for long-acting preexposure prophylaxis (PrEP) strategies [23]. 5. INHIBITORS OF HIV CO-RECEPTOR USAGE After the CD4-gp120 binding, interaction with the appropriate chemokine receptor CCR5 or CXCR4 triggers the final conformational changes in env, resulting in fusion between the viral and cellular membrane [24, 25]. Different HIV-1 variants use either CCR5 or CXCR4 or both. CCR5 using R5 virus predominates during early HIV-1 infection and is a key to transmission of infection. During early stages of disease, CXCR4 usage (X4) or dual usage (both CCR5 and CXCR4) are rare, but emerge over time [26-28]. 5.1. CCR5 Antagonists CCR5 antogonists are the attractive options for early inhibition of HIV-1 entry. Several small molecule antagonists, mAbs and CCR5 ligands such as AOP-RANTES have been used to prevent HIV-1 entry through blocking/inhibiting viral interaction with CCR5. However, only small-molecule CCR5 antagonists are currently approved for use such as orally available compounds like INCB009471, aplaviroc, vicriviroc, maraviroc and cenicriviroc among others that have advanced to phase-2 or 3 clinical trials. In vitro experiments showed potent inhibition of HIV-1 replication by these compounds against laboratory-adapted and primary isolates across all clades of group M HIV-1. Treatment with Aplaviroc resulted in moderate reduction in plasma HIV-1 RNA levels during treatment, but trials were abandoned after nonfatal, drug-induced hepatitis developed in some of the treated subjects during phase-2b and 3 trials [29-31].In addition, virologic failures were also noted with aplaviroc resistance in treatment naïve subjects [32]. Another molecule, vicriviroc along with an optimized background regimen showed potent suppression of HIV-1 in a placebo-controlled phase-2b studies in cART treated individuals, but was later discontinued due to increased rates of virologic failure in treatment-naive patients [33-35]. Another CCR5 antagonist maraviroc showed potent in vitro and in vivo anti-HIV-1 activity and remains as the only clinically approved chemokine receptor antagonist [36]. The efficacy of maraviroc was established in a pair of phase 3 randomized, placebocontrolled trials known as MOTIVATE 1 and MOTIVATE 2 [37, 38]. In both of these studies, more than two-fold greater reduction in plasma virus levels than those in the control arms receiving optimized background regimens alone was noticed [37, 38]. Reduction in plasma VL was also associated with an increase in CD4 cell counts in the maraviroc arms with similar frequency of adverse events in all groups.
Targeting HIV Entry and Integration
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Virologic response to maraviroc was long lasting and remained through 96 weeks of therapy. However, the possibility of emergence of X4 [39] viruses during treatment with CCR5 antagonists cannot be ruled out and studies have shown that virologic failure to maraviroc was associated with emergence of CXCR4-using virus in 57% of subjects in which a repeat tropism test was obtained at the failure timepoint [37, 40-42]. Favorable clinical efficacy with an antagonizing effect on viral entry and relatively low adverse toxicity profile leads maraviroc to be considered for including in anti-retroviral treatment intensification strategies and have shown some promising results for immune restoration [43-45]. Maraviroc intensification also showed a decrease in proviral latent HIV-1 reservoir size in memory T cells, but no change in residual, low-level viremia was observed [44]. Since the R5 virus is primarily responsible for establishing acute and early HIV-1 infection, there has been interest in using maraviroc as PrEP, and some promising results have been shown in a humanized mice model study [46]. Cenicriviroc (TBR-652, formerly TAK-652) is an investigational small-molecule CCR5 antagonist currently under clinical development [47]. Cenicriviroc demonstrated a longer halflife than maraviroc and is dosed once daily. It also inhibits CCR2, a receptor for monocyte chemoattractant protein-1 that has been linked to various inflammatory diseases. In phase-1 and 2a studies, cenicriviroc treatment resulted in up to a 1.8 log10 reduction in plasma HIV-1 RNA levels and was safe and well tolerated without any significant adverse events [48, 49]. 5.2. CXCR4 Antagonists In contrast to CCR5, there are no known naturally occurring mutations that select for CXCR4 deletion. Hence, the development of CXCR4 inhibitors has been a challenging task. A variety of CXCR4 antagonists that exhibit potent anti-HIV activity in vitro against X4 viruses have been developed. However, administration of these drugs in vivo results in mobilization of hematopoietic stem cell from the bone marrow to the peripheral blood as CXCR4 is important in hematopoietic stem-cell homing and these effects limit their use in HIV-infected individuals [50]. Since X4 viruses are usually present as mixtures together with R5 viruses [45, 51], targeting the X4 component of the virus alone may not lead to decline in overall plasma viremia. Preliminary data with CXCR4 antagonist AMD3100 showed inhibition of the X4 component of the virus population [52]. However, homing of myeloid and plurioptent (CD34+) stem cells from the bone marrow into the blood due to CXCR4 blockade appears to be a major drawback of this approach [53]. However, the safety of long-term CXCR4 blockade during HIV infection is currently unknown. 5.3. Fusion Inhibitors Enfuvirtide (T-20), approved for clinical use in 2003, is a polypeptide with 36 amino acids derived from the HIV-1 envelope subunit gp41 C-terminal heptad repeat (HR-2) and provides a novel treatment strategy for HIV-1 therapy. Enfuvirtide acts by binding to the trimeric HR-1 complex of gp41 and thereby blocking the interactions of HR-1 with HR-2. This fusion inhibition leads to blockade of virus entry [54].
A phase-3 clinical trial with enfuvirtide demonstrated a greater efficacy when combined with an optimized background regimen [55, 56]. The clinical efficacy of enfuvirtide was associated with minimal systemic toxicity and adverse reactions. Co-administration of enfuvirtide along with newer agents such as tirapanavir, darunavir, and maraviroc in clinical trials demonstrated significant improvement in response rates to these drugs than when used alone in highly treatment-experienced patients [37, 57, 58]. However, rapid resistance to enfuvirtide has been a major obstacle for its wide usage [59]. Sifuvirtide, a third generation fusion inhibitor has entered early phase human clinical studies in Asia as a microbicide candidate in PrEP [60, 61]. Compared to enfuvirtide, sifuvirtide has a longer plasma half-life and greater in vitro antiviral activity as a result of improved helical structure stability and high affinity for its target N-terminal HR peptide. It also demonstrated activity against enfuvirtideresistant HIV-1 strains [62-64]. Other HR-1-based and small molecule fusion inhibitors are under development and clinical data are yet to be generated [65]. 6. INHIBITORS OF HIV INTEGRATION WITH HOST DNA 6.1. Integrase Inhibitors One of the enzymes that HIV-1 uses to establish infection in the host is the enzyme integrase which catalyzes the integration of the proviral DNA into the host genome. This is a crucial step in the HIV pathogenesis and therefore preventing this integration has become the focus of developing new therapeutic strategies over the years. There are quite a number of integrase inhibitors, some like raltegravir (RAL), which have been approved by the FDA and are now used successfully. Nevertheless, the development of resistance and persistent toxicities warrants development of additional drugs targeting unique and constitutive steps in the HIV-1 viral life cycle. Integrase is an enzyme responsible for catalyzing the integration of HIV to the host genome which is vital for HIV replication. Integrase inhibitors have become the focus of immense research in the past few years primarily due to the fact that they have no homologs in humans. Integrase, encoded by the pol gene is comprised of 288 amino acids. After entry into CD4+ T cells, transcription of viral RNA into DNA occurs by HIV reverse transcriptase. Integrase enzyme forms a preintegration complex (PIC) following interaction between integrase and the viral cDNA. This is followed by insertion of the viral DNA into the chromosomal DNA which is referred to as the strand transfer step [66, 67]. After this step, host enzymes repair gaps between the host and viral DNA and subsequently infection is established. Raltegravir and other integrase inhibitors target this strand transfer step and are also referred to as integrase strand transfer inhibitors. Raltegravir was the first US FDA-approved integrase inhibitor in 2007, and since then other drugs like elvitegravir have taken their place in the list of drugs that are able to inhibit the strand transfer step. In this section we will review some of these inhibitors, both that are approved for clinical use and those in clinical trials or under development (Table 1).
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Table 1.
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Patents for HIV entry and Integration Inhibitors. Patent Title
Drug Class/Mode of Action
Patent No.
Authors / Year
Reference
Prodrugs of piperazine and substituted piperidine antiviral agents
Entry inhibitor: Small molecule
US8168615
Ueda et al. / 2012
[116]
HIV fusion inhibitor peptides with improved biological properties
Entry inhibitor/fusion inhibitor: HIV fusion inhibitor peptide
US8034899
Dwyer et al. / 2012
[117]
Heterocyclic antiviral compounds
Entry inhibitor: CCR5 antagonists
US7714018
Lee et al. / 2010
[118]
Diketopiperidine derivatives as HIV attachment inhibitor.
HIV/entry attachment inhibitors: diketopiperidine derivatives Small molecule
US8242124
Regueiro-Ren et al. / 2012
[119]
Methods of inhibiting human immunodeficiency virus infection through the administration of synergistic combinations of antiCCR5 monoclonal antibodies, fusion inhibitors, and CD4-GP120 binding inhibitors
HIV/entry attachment inhibitors: combinations of anti-CCR5 monoclonal antibodies, fusion inhibitors, and CD4GP120 binding inhibitors.
US7736649
Olson et al. / 2010
[120]
Antiviral cell-penetrating peptides
HIV/entry attachment inhibitors: peptides
US8324153
Debnath et al. / 2012
[121]
CXCR4 antagonists for the treatment of HIV infection
HIV/entry attachment inhibitors: chemokine CXCR4 receptor antagonist.
US8008312
Shim et al. / 2011
[122]
Highly potent synergistic combinations of human immunodeficiency virus (HIV) fusion inhibitors
Entry inhibitor/fusion inhibitor: combinations of HIV fusion/entry inhibitors
US7919101
Jiang et al. (2011)
[123]
Commensal strain of E. coli encoding an HIV GP41 protein
Entry inhibitor/fusion inhibitor: E.coli encoding an HIV GP41 protein
US8349586
Hamer (2013)
[124]
Long lasting fusion peptide inhibitors for HIV infection
fusion peptide inhibitors
US7741453
Erickson et al. (2010)
[125]
Inhibitors of human immunodeficiency virus replication
Integration: inhibitors of HIV replication
US8377960
Tsantrizos et al. (2013)
[126]
HIV integrase inhibitors
Integration: inhibit HIV integrase and prevent viral integration into human DNA
US8383639
Naidu et al. (2013)
[127]
4-Oxoquinoline derivatives
Integration: HIV integrase inhibitor, such as elvitegravir
US7994194
Harbeson (2011)
[128]
Pyridinone diketo acids: inhibitors of HIV replication
Inhibitors of HIV replication through inhibition of HIV integrase
US7888375
Nair et al. (2011)
[129]
6.1.1. First-Generation Integrase Inhibitors 6.1.1.1. Raltegravir (MK-0518) Raltegravir, is a diketo acid (DKA) that inhibits the strand transfer step of integration. Developed by Merck &Co., Inc, it was officially approved by the FDA for clinical use in 2007 and until recently, was the only integrase inhibitor employed in treatment naïve and treatment experienced subjects. Raltegravir is shown to be rapidly absorbed within one hour (Cmax) of administering. Stable levels are achieved within 48hr and bioavailability is reportedly around 32%. In a multi-centre double blinded study, subjects were administered with varying doses (100, 200, 400 or 600 mg twice daily) of raltegravir monotherapy versus placebo for 10 days. These HIV-1 infected subjects were treatment naïve and had viral loads of 5000 copies/mL at entry. More than 50 % of subjects suppressed viral loads to < 400 copies/mL. The drug was fairly well tolerated. The STARTMRK study, a double-
blinded, non-inferiority study included patients with HIV1loads of > 5000 copies/mL and with no resistance at baseline to efavirenz, tenofovir, or emtricitabine [68-70]. The patients were randomized to receive RAL or efavirenz. At week 96, 81% of raltegravir treated subjects were able to bring down viral loads to
