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Journal of Biological and
Chemical Sciences (JBCS)
Sharma et al. (2014) vol.1, no. 1, 1-12
Drug design strategies using non nucleoside reverse transcriptase inhibitors
(NNRTI): current challenges and future perspectives

Ravi Sharma1, Gaurav Kumar2, Preeti Saini3, Hardeep Singh Tuli4*, Shivani Sood4
1Department of Biotechnology, Mata Gujri Collage, Fatehgarh Sahib, India
2Department of Biochemistry, Kurukshetra University, Kurukshetra-136119, India
3Department of Microbiology, PAU, Ludhiana-141004, India
4Department of Biotechnology, Maharishi Markandeshwar University, Mullana-Ambala-133207
*Correspondence at [email protected]
Received: September 13, 2014; Accepted: November 06, 2014
Abstract: The emerging drug resistance towards anti-HIV compounds attracted the attention of
scientific community. Development of potent anti-HIV therapy using non-nucleoside reverse transcriptase inhibitors (NNRTIs) known to be a promising strategy. Although NNRTIs has great importance in HIV-1 treatment and prevention but their mechanism of action has not yet been studied in detail. The present review discusses cellular interactions of HIV-1 followed by the FDA approved anti-HIV-1 chemotherapy. The review highlights the importance of NNRTIs and their mechanism of action in HIV-1 treatment. Also the current challenges and future prospective of NNRTIs to prevent HIV-1infection are well addressed so as to propose the development of novel therapeutic strategies for its treatment. Key words: HIV-1; non-nucleoside reverse transcriptase inhibitors; mechanism; QSAR;
Computer-aided drug design Acquired Immunodeficiency Syndrome (AIDS) is a fatal disease caused by infection with the human immunodeficiency virus (HIV) [1, 2]. In recent years AIDS become a serious global threat to human health and life. HIV harms body's immune system (T-cells) and hence gradually destroys the body's ability to fight against various infections as well as certain types of cancer.

Journal of Biological and
Chemical Sciences (JBCS)
Sharma et al. (2014) vol.1, no. 1, 1-12 Reverse transcriptase (RT) an enzyme that catalyzes the transcription of single stranded viral genomic DNA to double stranded DNA, has been a prominent target in the discovery and development of therapies for HIV treatment. The first anti-HIV agent approved for clinical use was a nucleoside reverse transcriptase inhibitor (NRTI) [3]. However, HIV strains with reduced sensitivity toward NRTI have been isolated from patients receiving prolonged therapy. Further non-nucleoside HIV-1 RT inhibitors (NNRTI's) were recognized as specific inhibitors of HIV-1. These compounds induce a conformational change in the three-dimensional structure of the RT resulted in loss of activity [4]. NNRTIs binding are found to be virus-strain-specific and have been used in combination with two or three other anti-HIV drugs in Highly Active Antiretroviral Therapy (HAART). However, in spite of the remarkable potency of NNRTIs against HIV-1, the rapid clinical development of NNRTIs is hindered due to more susceptibility toward resistance than any other classes of antiretroviral drugs. As single-amino-acid change in NNRTI-binding pocket can result in complete loss of drug activity [5]. Consequently, there is an urgent need to draw attention of scientific community for the development of new RT inhibitors with an alternative mechanism of action. The present review focused on the approaches targeted for HIV-1 therapy with special reference to NNRTI. Cellular interaction of HIV
Human CD4+ T lymphocytes, macrophages, microglial, dendritic and Langerhans cells are considered as potential targets for HIV-1 infection. Endogenous CD4 molecule a 55-KDa protein member of the IgG super family, is a cell surface receptor expressed on helper T lymphocytes, monocytes, B-lymphocytes and several other cells. The replication cycle of HIV begins with the high-affinity binding of the gp120 protein on the host cell surface [6]. This gp120-CD4 complex interacts with a coreceptor on the cell surface and mediates to complete virus entry into the host cell. The whole process is assumed to be accomplished with the help of a cytoplasmic peptidyl- prolyl cis-trans isomerase cyclophilin A (hCyp-18) and appears to be essential for infection by most HIV-1 strains [7, 8]. The exact role of cyclophilins in HIV-1 infection yet to be fully explored but studies indicated that virion associated with CyPA may be involved in an early

Journal of Biological and
Chemical Sciences (JBCS)
Sharma et al. (2014) vol.1, no. 1, 1-12
Reverse transcriptase (RT)
HIV-1 RT was discovered in the year 1970 by two independent groups, a Baltimore group and the Termin and Mizutani group. HIV-1RT, a heterodimer contains subunits of 66 kDa (p66) and 51 kDa (p51). p66 subunit contains two domains, the N-terminal polymerase domain of about 440 residues and the C-terminal RNase H domain with 120 residues. p51 subunit is processed by proteolytic cleavage of p66 and corresponds to the polymerase domain of the p66 subunit. Portions of both p51 and the polymerase domain of p66 can be described as a "right hand" which contains three sub domains: Fingers, palm, and thumb. The connection sub domain connects the hand of the polymerase domain and RNAse H domain of p66. The connection sub domains and the palm sub domains comprise of three-stranded β-sheets with -helices on one side whereas the thumb sub domains include three -helices. There have been 245 structures of HIV-1 RT deposited in the Protein Database so far. Fig. 1: Illustrations of cellular interactions of HIV-1 with Human CD4+ T lymphocytes
Anti-HIV chemotherapy
The treatment of the acquired immunodeficiency syndrome (AIDS) is the most challenging problem worldwide for medical scientist. Anti-HIV compounds that have been approved by FDA for clinical use in the treatment of AIDS are summarized in Table-1. These compounds fall into six categories: nucleoside reverse transcriptase inhibitors (NRTIs: zidovudine, didanosine,

Journal of Biological and
Chemical Sciences (JBCS)
Sharma et al. (2014) vol.1, no. 1, 1-12 zalcitabine, stavudine, lamivudine, abacavir and emtricitabine); nucleotide reverse transcriptase inhibitors (NtRTIs: tenofovir); non-nucleoside reverse transcriptase inhibitors (NNRTIs: nevirapine, delavirdine, efavirenz and etravirine); protease inhibitors (PIs: saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir and darunavir); cell entry inhibitors [fusion inhibitors (FIs: enfuvirtide) and co-receptor inhibitors (CRIs: maraviroc)]; and integrase inhibitors (INIs: raltegravir). These compounds should be used in drug combination regimens (Table 1) to achieve the highest possible benefit, tolerability and compliance and to diminish the risk of resistance development. Fig. 3: mechanism of action of NNRTI's

Journal of Biological and
Chemical Sciences (JBCS)
Sharma et al. (2014) vol.1, no. 1, 1-12 Table 1. Anti-HIV compounds approved by FDA in the treatment of AIDS
Nucleoside
Generic name
approval
Inhibitors
Bristol-Myers Squibb Bristol-Myers Squibb Tenofovir Disoproxil Rescriptor Viiv Healthcare Bristol-Myers Squibb Inhibitors
(NNRTIs)
Pharmaceuticals, Inc. Janssen Pharmaceuticals, Protease
Bristol-Myers Squibb Inhibitors (PIS)
Janssen Pharmaceuticals, Merck & Co., Inc Boehringer Ingelheim Fusion Inhibitors Enfuvirtide
Hoffmann-La Roche Entry Inhibitors
Integrase
Inhibitors
Merck & Co., Inc. Combination
abacavir and lamivudine HIV Medicines
abacavir, dolutegravir, and lamivudine abacavir, lamivudine, and zidovudine efavirenz, emtricitabine, tenofovir disoproxil

Journal of Biological and
Chemical Sciences (JBCS)
Sharma et al. (2014) vol.1, no. 1, 1-12 fumarate elvitegravir, cobicistat†, emtricitabine, and tenofovir disoproxil fumarate emtricitabine, rilpivirine, and tenofovir disoproxil fumarate emtricitabine and tenofovir disoproxil fumarate lamivudine and zidovudine lopinavir and ritonavir Mechanism of NNRTIs
Non-nucleoside HIV-1 RT inhibitors (NNRTI's) were the first to be recognized as specific inhibitors of HIV-1. These have been identified by iterative screening using purified viral enzyme [9] and included a variety of chemical substrates which binds to hydrophobic pocket of p66 subunit of HIV-1 reverse transcriptase. The non nucleoside reverse transcriptase binding pocket (NNRTBP) has been found to a close distance from the active catalytic site of HIV-1 RT and considered as non essential for enzyme function. NNRTIs bind noncompetitively at NNRTBP and induce a conformational change in the three-dimensional (3D) structure of enzyme results in loss of enzyme activity [4]. As a result of NNRTI binding, rate of dNTPs incorporation into synthesizing DNA strand slower down. Drug Design
Computational methods have developed into very useful tools in facilitating new drug discovery [10]. By the use of computational methods, it becomes possible to estimate the biological activity of the candidate molecules even before experimental trials. These methods are simple and non- expensive and expedite to design molecules with desirable biological activity [11]. Quantitative structure-activity relationship (QSAR) and docking procedure are two mostly used computational methods in drug design. In a study, hologram quantitative structure-activity relationship (HQSAR) was applied to three different data sets, such as 70 TIBO, 101 HEPT and 125 dipyridodiazepinone derivatives with Journal of Biological and
Chemical Sciences (JBCS)
Sharma et al. (2014) vol.1, no. 1, 1-12 the inhibitory effect on enzyme activity [12]. Similarly Zhou and Madura (2004) docked 50 TIBO inhibitors and demonstrated that combination of ligand-based and receptor-based modeling as a powerful approach to build 3D-QSAR models [13]. Another group of researcher studied quantitative structure-activity relationship (QSAR) analysis for a set of 82 TIBO using three-layered neural network [14]. Medina-Franco et al. (2004) applied a set of pyridinone derivatives to study the interactions between pyridinone derivatives and binding pocket of non- nucleoside reverse transcriptase inhibitor using CoMFA, CoMSIA and docking experimentation [15]. Also researchers explained the viral resistance to pyridinone derivatives upon mutation of amino acids Tyr181 and Tyr188. In their other study they designed 44 non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs) of the pyridinone derivative [16] and found pyrazolo [3,4-d] pyrimidine and phenothiazine as promising novel compounds for NNRTIs. Another class of HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors includes indolyl aryl sulfones (IASs) have been studied against wild type and resistant strains (Y181C, the double mutant K103N-Y181C, and the K103R-V179D-P225H strain). A predictive 3-D QSAR models have been designed using receptor-based alignment of IASs into the non-nucleoside binding site (NNBS) of RT [17]. Similarly Duchowicz et al. (2006) studied 154 non-nucleoside reverse transcriptase inhibitors (NNRTI) of the wild-type HIV-1 virus which considered as the second generation analogues of Efavirenz [18]. A series of 74 dihydroalkoxybenzyloxopyrimidines (DABOs) have been studied by comparative molecular field analysis (CoMFA) in order to design more potent DABO analogues as anti-HIV/AIDS agents [19]. Hu et al. 2009 carried out molecular modeling of a series of non-nucleoside reverse transcriptase inhibitors (2-amino-6- arylsulfonylbenzonitriles and their thio and sulfinyl congeners) using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) approaches [20]. Study revealed, 2-amino-6-arylsulfonylbenzonitriles with bulky and hydrophobic groups in 3- and 5-position of the B ring showed significant inhibitory activity. Group of researcher investigated symmetric formimidoester disulfides (DSs) as a new class of potent non-nucleoside HIV-1 reverse transcriptase (RT) inhibitors using computational strategy [21]. Liu et al. (2010) discovered and studied a series of 4,6-diamino-1,3,5-triazin-2-ol based Journal of Biological and
Chemical Sciences (JBCS)
Sharma et al. (2014) vol.1, no. 1, 1-12 NNRTIs compounds using SAR approaches [22]. Samuelea et al. (2010) reported Novel benzimidazol-2-one non-nucleoside reverse transcriptase inhibitors (NNRTIs) through rational structure-based molecular modeling and docking approaches [23]. Elena et al. (2011) identified 3,4,5-Trisubstituted-1,2,4-4 H-triazoles (TTs) as a new class of potent non-nucleoside HIV-1 reverse transcriptase (RT) inhibitors [24]. Furthermore they studied 1,3,4,5-tetrasubstituted- pyrazoles (TPs) as NNRTIs by computational strategy based molecular docking approaches [25]. Group of researchers conducted a study on a series of 119 NNRTIs, including 1-(2- hydroxyethoxymethyl)-6-(phenylthio) thymine (HEPT) and dihydroalkoxybenzyloxopyrimidine (DABO) derivatives using computational approaches [26]. Recently a series of 107 1-[(2- hydroxyethoxy)-methyl]-6-(phenylthio) thymine (HEPT) as a non-nucleoside reverse transcriptase inhibitor (NNRTI) has been deliberated [27]. Current challenges
NNRTIs are more susceptible to high-level drug resistance than other classes of antiretroviral drugs because of a single-amino-acid change in the NNRTI-binding pocket. Due to this reason the clinical development of NNRTIs is hindered [28]. The amino acids with which the NNRTIs interact within the NNRTI-binding pocket may be prone to mutate, and this has proven to be the case for, among others. Most of the NNRTI resistance mutations are found in and around the NNIBP. K103N and Y181C are the most frequently observed resistance mutations in patients treated with the approved NNRTIs. When, compared with the ‘older' NNRTIs (e.g. nevirapine), the ‘newer' NNRTIs such as Etravirine and Rilpivirine first described by Janssen, et al. in 2005, have been found to retain sufficient activity against the K103N and Y181C reverse transcriptase (RT) mutants [29]. Other NNRTI resistance mutations that are observed in patients include L100I, K101E, V106A, V179D, Y188L, G190A, and P236L; these NNRTI resistance mutations can occur singly, or in combinations. The most promising third generation NNRTIs are proven to be more effective against HIV strains that carry most common single and double mutations; however, viral strains carrying multiple NNRTI-resistance mutations can exhibit significant levels of drug resistance. Currently there are four NNRTI drugs Nevirapine, Delavirdine (first generation), Efavirenz (second generation), and Etravirine (third generation)) that are approved for treating HIV-1 infections while several other potent NNRTIs that inhibit HIV-1 at nanomolar Journal of Biological and
Chemical Sciences (JBCS)
Sharma et al. (2014) vol.1, no. 1, 1-12 concentrations (EC50) are in clinical trials. In recent years, extensive crystallographic, molecular modeling and biochemical studies have contributed towards understanding NNRTI drug resistance and the development of better NNRTIs with improved drug profile. Conclusion and future perspectives
Reverse transcriptase is known as a key enzyme needed for HIV-1 replication. The present review gives a brief summary of the last decade investigations for designing anti-HIV agents (NNRTIs) using computer-aided approaches. It has been found that NNRTIs could play an important role to design a promising therapy for HIV treatment. Also the development of QSAR based drug designing models with a novel mechanism of actions could improve the pace of new anti-HIV drug inventions. It is essential to study and Understanding the molecular mechanism of NNRTI inhibition to overcome the drug-resistance mutations in RT. Further scientific communities should be more focused on highly specific NNRTI inhibitors with low toxicities and minimal side effects. Conflict of Interest
There is no potential conflict of interest with reference to the current manuscript. All the authors have read the manuscript and agreed to submit the same for publication. The authors would like to acknowledge Mata Gujri Collage (Fatehgarh Sahib) for providing the requisite facilities to carry out this work. References
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