Development of New Generation Treatment for HIV/AIDS

Patients with Multidrug-Resistant HIV-1 Variants

Darunavir  (TMC-114)

K_i (HIV-1_WT) = 16 pM

ID₉₀ (Antiviral) = 4.1 nM (MT-cells)

    We have designed & synthesized Darunavir (GRL-94017 or, TMC114), a novel nonpeptidic HIV-1 protease inhibitor (PI) containing a 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) ligand and a sulfonamide isostere. It is extremely potent against laboratory HIV-1 strains and primary clinical isolates (50% inhibitory concentration, IC₅₀ = ~0.003 μM; IC₉₀ = ~0.009 μM) with minimal cytotoxicity (50% cytotoxic concentration for CD4⁺ MT-2 cells = 74 μM). GRL-94017 blocked the infectivity and replication of each of HIV-1_NL4-3 variants exposed to and selected for resistance to saquinavir, indinavir, nelfinavir, or ritonavir at concentrations up to 5μM (IC₅₀s, 0.003 to 0.029 μM), although it was less active against HIV-1_NL4-3 variants selected for resistance to amprenavir (IC₅₀ = 0.22 μM). It was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after having received a variety of antiviral agents.

X-Ray structure of Darunavir-Bound HIV-1 Protease (1.23Å).

Ghosh et. al. Acc. Chem. Res. 2008, 41, 78; J. Mol. Biol.  2004, 338, 341.

Koh, Ghosh, Mitsuya, et al. Antimicrob. Agents  Chemotherapy, 2003, 47, 3123.

Virus

GRL-PI (TMC-126)

 Mitsuya, H. Ghosh, A. K. et al. J. Virology 2002, 76, 1349; Ghosh, A. K. Duzguiness, N. et. al. Antiviral Res. 2002, 54, 29; Ghosh, et al. Bioorg. Med. Chem. Lett. 1998, 8, 687; Weber, Ghosh, et. al. J. Mol. Biol. 2004, 338, 341.

Activities of selected anti-HIV agents

against HIV-1_Ba-L, HIV-2_ROD, and HIV-2_EHO^a

     ^a For HIV-1_Ba-L, the IC₅₀s were determined by using PHA-PBMCs and inhibition of p24 Gag protein production by the drug as an endpoint. For HIV-2_ROD and HIV-2_EHO , MT-2 cells were exposed to the virus and cultured and the IC₅₀s were determined by the MTT assay. All assays were conducted in duplicate or triplicate,  and the data shown represent means ? standard deviation derived from the results of three independent experiments.

 Koh, Ghosh, Mitsuya, et. al. Antimicrobial Agents and Chemotherapy, 2003, 47, 3123; Ghosh, A. K., Chapsal, B. D., Mitsuaya, H. Darunavir, a New PI with Dual Mechanism: From a Novel Drug Design Concept to New Hope Against Drug-Resistant HIV. Aspartic Acid Proteases as Therapeutic Targets, Edited by Ghosh, A. K., Wiley-VCH, 2010, Ch.8, 205-235.

Unique Dual Mode of Action: Darunavir also Inhibits Dimerization of HIV-1 Protease

      The dimerization of two 99-amino acid HIV-1 protease monomers is an essential process for enzyme maturation and proteolytic activity. Inhibition of this mechanism is logically an attractive therapeutic option to fend off HIV replication. The two monomers bind through four stranded antiparallel β-sheets at the N- and C-termini of each unit.

FRET-based HIV-1 expression system

        Through our studies employing an intermolecular fluorescence resonance energy transfer (FRET)-based HIV-1 expression assay, conducted with fluorescent protein-tagged HIV protease monomers, we have demonstrated that Darunavir inhibits the protease dimerization process at concentrations as low as 0.01 µM. Inhibition of protease dimerization is considered to only occur at the nascent stage and once dimerization occurs, inhibition is deemed infeasible.  However, the subsequent inhibition of the protease catalytic activity by DRV confers to the inhibitor a unique dual mode of action. This may explain DRV’s superb antiviral potency and durability against the emergence of drug resistance.

Koh, Y., Ghosh, A. K., Mitsuya, H.  et al.   J. Biol. Chem. 2007, 282, 28709-28720; Mitsuya, H., Ghosh, A. K. Development of HIV-1 Protease Inhibitors, Antiretroviral Resistance, and Current Challenges of HIV/AIDS Management. Aspartic Acid Proteases as Therapeutic Targets, Edited by Ghosh, A. K., Wiley-VCH, 2010, Ch.9, 245-259.

Enhancing ‘Protein Backbone Binding’: An Intriguing Molecular Design Concept for Combating Drug-Resistant HIV/AIDS

      While HIV protease inhibitors are critical for current antiretroviral treatment regimens, the rapid onset of drug resistance limits the effectiveness of the majority of approved drugs.  In our efforts to combat drug resistance, we developed a useful concept of ‘backbone binding’ for designing inhibitors to withstand drug-resistance.  On the basis of X-ray structural evidence that the active site backbone conformation of mutant proteases is minimally distorted, we developed a strategy of maximizing ‘backbone binding’ particularly hydrogen bonding with the protein backbone to retain potency against mutant strains.  Furthermore, our molecular design efforts led to structurally unprecedented molecular templates and ligands inspired by polyether natural products.  The combination of the ‘backbone binding’ strategy and molecular design novelty culminated in the development of Darunavir, GRL-0519, and a variety of exceedingly potent HIV-1 protease inhibitors with intriguing features.

     GRL-0519 maintained remarkable potency against various MDR HIV-1 variants examined.  It displayed IC₅₀ values ranging from 0.6 nM to 4.3 nM, consistently 10-fold more potent than DRV.  Furthermore, GRL-0519 exhibited 10-fold improved dimerization inhibitory activity compared to Darunavir.  A protein-ligand X-ray structure of inhibitor-bound HIV-1 protease, revealed that indeed, the inhibitor formed a network of hydrogen bonds with Asp-29 and Asp-30 amide NH’s similar to Darunavir.  In addition, the third THF ring appeared to make a water mediated interaction with Arg-8’ as well as fill in the hydrophobic pocket more effectively than Darunavir.

The numbers in parentheses represent the fold changes of EC₅₀ values for each isolate compared to the IC₅₀ values for wild-type HIV-1_ERS104pre

X-ray Crystal Structure of GRL-0519A

Ghosh et al. ChemMedChem 2010, 5, 1850-1854; Bioog. Med. Chem. Lett. 2010, 20, 1241-46; J. Med. Chem. 2009, 52, 2163-76; J. Med. Chem. 2009, 52, 2992-03.