Tuesday, December 01, 2009

World AIDS Day

Recent Development in HIV Therapy (By Bishnu Marasini)
Human immunodeficiency virus (HIV), a lentivirus discovered in 1983 belongs to family Retroviridiae which is responsible for acquired immunodeficiency syndrome (AIDS), needs arduous work to eliminate. It contains two copies of +ssRNA which codes for 9 genes (~9 KB) is bound tightly to reverse transcriptase integrase and nucleocapsid proteins (p6 and p7), which protects RNA preventing digestion by nuclease. this nocleocapsid is surrounded by viral protein, p17 which also cover Vif, Vpr, Nef and viral protease. The envelope of virus contains some of the host cell membrane which is taken out during bulging out from infedted host cell. So, it contains glycoprotein gp120 and gp41 making envelope's spike. This spike structure is important to understand the replication cycle of the virus and drug development strategy (Schubert and Mcclure, 2005).
Due to error prone and rapid replication and hence immediate drug resistance, no successful single drug and neither immunization could be prepared against HIV. Although, some drugs are available are not sufficient to prevent epidemics, no better tolerated, convenient and cheap drugs are available. That’s why combination of drugs against different targets as well as development of new agents have been an exigent in recent days. Majority of these include reverse transcriptase, integrase, protease inhibitors, viral entry blockers, coating of CD4, CCR5, CXCR4, genetic therapy, immunotherapy (IL-7, IFN-α, IFN-γ) etc.

Reverse transcriptase (RT)
The two sub-units p66 (560 amino acids) and p51 (440 amino acids) together which make asymmetric heterodimer of RT. It acts as both DNA polymerage which copy RNA and as RNase H which degrades RNA only if the RNA is a part of an RNA/DNA duplex. This is very potent target for HIV therapy and nearly half of the discovered drug till date is based on its inhibition. Among them, Nucleoside Reverse Transcriptase Inhibitor (NRTI) and Non Nucleoside Reverse Transcriptase Inhibitor (NNRTI) have been catagorised. NRTIs are are structural analogues of the substrate of DNA lacking 3'-OH groups misleading to chain termination during replication. NRTI includes zidovudine, lamivudine, stavudine, didanosine, zalcifabine, abacavir, emtricitabine, tenofovir are already popular in HIV treatment. In addition, racivir, apricitabine, dexelvucitabine, elvucitabine, alovudine, amdoxovir etc. are in the current clinical development stage.
NNRITs are non-competitive inhibitors of RT binds to the pocket other than the active sites and change confirmation and/or inactivate the enzyme. NNRTI includes nevirapine, delavirdine (1st generation), efavirenz (2nd generation), rilpivirine. In addition, combination of these NRTI and NNRTIs has been widely searched for seeking of therapy e.g., Truvada (tenofovir/emtricitabine), Combivir (lamivudine and zidovudine), trizivir, dapivirine (Phase III).
RNAse H inhibitors include BBNH (N-(4-tert-Butylbenzoyl)-2-hydroxynaphthaldehyde hydrazone), DHBNH (dihydroxy benzoyl naphthyl hydrazone), CPHM (4-chlorophenylhydrazone of mesoxalic acid),  Diketo Acid( 4-[5-(Benzoylamino)thien-2-yl]-2,4-dioxobutanoic Acid), N-hydroxyimides, hydroxytropolone etc.

HIV-1 Protease Inhibitors
As already mentioned, HIV contains protease (aspertyl) which cleaves the group specific antigen (Gag) and Gag polymerage (Pol) precursor protein to structural capsid protein (p17, p24, p7 and p1) and functional protein (p11) reverse transcriptase (p66 and p51) and integrase (p32). All of these proteins activated after cleaving by viral protease, so, it has been a potent target for HIV-1 therapy. These includes saquinavir, totanavir, indinavir, nelfinavir, amprenavir, lopinavir, fosamprenavir etc. Side effects such as, lipodistrophy and dosing effect although seen in previous protease inhibitors, better one are atanazvir, brecanavir, darunavir, tipranavir, etc. are extensively under research and in the clinical trial phase which has not exhibited severe side effects as previous drugs.
HIV Integrase Inhibitors
HIV integrase transfer the viral genomic dsDNA into nucleus of host and insert into the host chromosome maintaining its stability, efficient expression and replication. If this enzyme is inhibited then replication and transcription of virus is blocked ultimately reducing viral load in the host body. Inhibitors of HIV integrase can be combined with other inhibitors for best effectiveness. These are efavirenz, raltegravir, elvitegravir etc.

HIV Maturation Inhibitors
At the late stage of the HIV proliferation, capsid precursor p25 (CA-SP1) is converted to mature capsid protein p24 (CA). This conversion can be blocked as HIV therapy. Bevirimat (3-O-(3',3'-dimethylsuccinyl)- betulinic acid) prevent the conversion of p25 to p24 capsid.
Viral Entry Inhibition
The outer spike of virus get attached to the T-cell and macrophase through the receptor and co-receptor (specially CD4, CCR5, CXCR4) present in the cell. Then nucleocapsid is integrated into host cells after attachment and fusion. The inhibition/blocking/coating of these receptor makes virus difficult to fuse with cell and entry. Mobozil, maraviroc, vicriviroc, aplaviroc etc. are the CCR5 and/or CXCR4 antagonist found as HIV therapeutics. However, aplaviroc found to be responsible in increasing liver enzyme and total billirubin. Enfuvirtide, a peptide inhibits the fusion of viral envelope with the host.
Gene Therapy
Although, most of the HIV infected person couldn't afford genetic therapy, significant HIV-infected patients live in setting where they not only afford but also contains sufficient infrastructure to support genetic therapy. Using of anti-sense, ribozymes, optamers, interference (RNAi), proteins (expressed), intrabodies, intrakin, zinc finger nuclease etc. are the the recent strategy for HIV therapy. Ribozymes against the gene tat, rev, and viral U5 has been in the clinical trial stage. Anti-sense RNA transgens pair with HIV RNA making non-functional duplex. Anti-U5 region anti-sensee RNA, envanti-sense RNA is being explored. Homology dependent control of gene activity is triggered by small-double stranded (sds) RNA molecules which is an RNAi regulatory mechanism. This has been applied in viral gene including gag, pol, nef, vif, env, vpr and LTR and showing exhibiting sign in cell line and potentially in HIV therapy in human beings. Another finding is the insertion of gene that express protein resembling mutant form of HIV Rev-protein (M10). It inhibits packing of virus and can be utilized by using retroviral or lentiviral vectors as HIV therapy.

Immunotherapy/ T-Cell Gene Therapy
It has been found that introducing CD28 co-stimulated T-cell reduce the viral infection. Also, genetic change in T-cells that express modified CD4+ significantly lower the virus load. In recent experiment CD4+ lymphocytes, genetically modified to express either protein Rev-M10 or a marking vector with no antiviral payload or both or to express an anti-sense TAR (Trans activating response region) element exhibited good antiviral effects. Gene expression pattern is designed such that it is associated with the acquisition of T-cell memory might be used to reprogram HIV specific T-cells to have TCM (long lived control memory T cell qualities. 

Passive administration of interleukin (IL-2, IL-7) etc. and antibody specific against spike gp120 and gp41 of viral envelope, and other viral proteins. These not only eliminate/reduce virus load but also increase the number of CD4+ T-cells. IL-7 also protects CD4 and CD8 T-cells against apoptosis induced by HIV. Use of snake venom which contains phospholipase A2 (PLA2) protects human primary blood leukocytes from replication of HIV-1 strain, it also block viral entry into cells.
More than 25 drugs of HIV have been approved by FDA within the 20 years of time after first drug discovery. The combination of different specific target against HIV such as integrase inhibitors, reverse transcriptase inhibitors CCR5 inhibitors etc. can be administered as rescue therapy. Although the side effect, cost, availability, resistance acquired by virus is challenging the drug discovery realm. Research priority is being emphasized to vanquish this virus by human being. New better drugs of higher potency, less toxicity, lower risk of drug resistance development etc. are being developed in parallel with the shift point in calender, indicating good hope that the compliance of scientists but also politicians, Samaritans to stymie HIV.
  1. Capitini CM, Chisti AA, Mackall CL (2009) Modulating T-cell homeostasis with IL-7: preclinical and clinical studies (Review), Journal of Internal Medicine, 266: 141-153.
  2. Luke W. Meredith, Haran Sivakumaran, Lee Major, Andreas Suhrbier, David Harrich (2009) Potent Inhibition of HIV-1 Replication by a Tat Mutant, PLoS ONE, 4 (11): e7769.
  3. Olga Latinovic, Janaki Kuruppu, Charles Davis, Nhut Le and Alonso Heredia (2009) Pharmacotherapy of HIV-1 Infection: Focus on CCR5 Antagonist Maraviroc (Review), Clinical Medicine: Therapeutics, 1: 1497-1510.
  4. Paul E. Sax, Camlin Tierney, Ann C. Collier et al, (2009) Abacavir–Lamivudine versus Tenofovir-Emtricitabine for Initial HIV-1 Therapy , New England Journal of Medicine, 361; 2230-2240.
  5. Stefan G. Sarafianos, Bruno Marchand, Kalyan Das, Daniel M. Himmel, Michael A. Parniak, Stephen H. Hughes and Eddy Arnold (2009) Structure and Function of HIV-1 Reverse Transcriptase: Molecular Mechanisms of Polymerization and Inhibition (Review), Journal of Molecular Biology, 385: 693-713.
  6. Ulirich Schubert and Myra Mcclure (2005), Topley and Wilson's Microbiology and Microbial Infection, Virology Vol 2, 10th edition, (Brian WJ Mahay and Volker Ter Meuleu: editors) Edward Arnold Ltd and ASM Press, page no. 1323-1345.
Bishnu Marasini