* Design & Synthesis  of HIV Protease Inhibitor for AIDS

Darunavir  (TMC-114)

 Ki (HIV-1) = 16 pM

 ID90 = 4.1 nM (MT-cells)
 

    We have designed & synthesized GRL-94017 (TMC114), a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing a 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) and a sulfonamide isostere which is extremely potgent against laboratory HIV-1 strains and primary clinical isolates (50% inhibitory concentration [IC₅₀], ~0.003μM; IC_90, ~0.009μM) with minimal cytotoxicity (50% cytotoxic concentration for CD4⁺ MT-2 cells, 74μM). 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). GRL-94017 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)

Virus

GRL-PI (TMC-126)

 Misuya, 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, 687-90;

Weber, Ghosh, et. al. J. Mol. Biol. 2004, 338, 341-352

Activities of selected anti-HIV ageents

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

* Structure-based Design of Potent and Selective

Memapsin 2 (b-Secretase) Inhibitors for Alzheimer抯 Disease

       Memapsin 2 (b-secretase) is one of two proteases that cleave the b-amyloid precursor protein (APP) to produce the 40-42 residue amyloid-b peptide (Ab) in the human brain, a key event in the progression of Alzheimer抯 disease. In collaboration with Jordan Tang, Head of the Protein Research group at the Oklahoma Medical Research Foundation, we have designed and synthesized two potent transition-state inhibitors. The strategy for the synthesis of the inhibitors was to first synthesize Leu*Ala dipeptide isostere with protections of a-N-Leu with a Fmoc group and hydroxyl group of the isostere with a silyl group. The Leu*Ala isostere was used in solid-state peptide synthesis of the peptides. Two HPLC purified inhibitors were shown to be highly potent  against recombinant memapsin 2 with K_i values of 6.8 nM and 1.6 nM respectively. This work represents the first case of designed potent inhibitors for this important pharmaceutical target related to Alzheimer抯 disease. Subsequently, the crystal structure of the protease domain of human memapsin 2 complexed to one of the inhibitors at 1.9 angstrom resolution was determined  by Jordan Tang and Lin Hong  at  the  Oklahoma  Medical  Research  Foundation.    This   crystal   structure   provided   invaluable  information  regarding the specific

ligand-binding site interactions in the active site of memapsin 2. Based upon this X-ray crystal structure bound to memapsin,  we have reduced the molecular weight and designed potent and selective memapsin inhibitors. Furthermore, a series of novel macrocyclic amide-urethanes was designed and synthesized based upon the X-ray crystal structure of our lead inhibitor bound to memapsin 2.  Ring size and substituent effects have been investigated.  Cycloamide-urethanes

Cyclic inhibitor-bound memapsin 2  X-ray crystal structure (resolution of 2 Å)

 containing a 16-membered ring exhibited low nanomolar inhibitory potencies against memapsin 2. From a therapeutic point of view, the selectivity of memapsin 2 inhibitors over other human aspartic proteases is expected to   be  important,  especially  memapsin 1 and cathepsin D. Memapsin 1, which has specificity similarity with   memapsin 2, has independent physiological functions. Cathepsin D, which is abundant in all cells, plays an important role in cellular protein catabolism.  Its inhibition would likely consume inhibitor drugs as well as lead to probable toxicity.  our structure-based design            

led to the development of very potent and highly selective memapsin 2 inhibitors.  Furthermore, our X-ray structural analysis of protein-inhibitor complexes has uncovered potentially important molecular interactions useful in the design of selectivity against other aspartyl proteases. Additional studies to further elucidate the role of these and other interactions important for selectivity are in progress.

Key references:

Ghosh, A. K., et. al. J. Am. Chem. Soc. 2006, 128, 5310-11;

Ghosh, A. K., Nagaswamy, K., Tang, J.   Curr. Med. Chem. 2005, 16, 1609-22;

Ghosh, A. K., Tang, J. et. al.   Bioorg. Med. Chem. Lett.  2005, 15, 15-20.

Tang, J., Ghosh, A. K., et. al. J.  Mol.  Neuroscience  2003,  20,  299-304;

Ghosh, A. K., Hong, A. K.; Tang. J. Curr. Med. Chem. 2002,  9, 1135-44;

Ghosh, A. K., Tang, J. et. al. J. Med. Chem. 2001, 44, 2865-68;

Tang, J., Ghosh, A. K., et. al.  Biochemistry 2001, 40, 10001-06;

Hong, L., Tang, J., Ghosh, A. K., et. al.  Science, 2000, 290 , 150-53;

Ghosh, A. K., Tang, J. et. al. J. Am. Chem. Soc. 2000, 122, 3522-23;