A Peptidomimetic HIV‐Entry Inhibitor Directed against the CD4 Binding Site of the Viral Glycoprotein gp120

Abstract

The first step in the infection of a human cell with HIV is interaction between the viral envelope glycoprotein gp120 and the human protein CD4. [1] We used this molecular interaction as a template to design and synthesize a new peptidomimetic compound that has the potential to block the interaction. In HIV infection, binding is followed by a conformational change [2] after which gp120 can interact with a co-receptor (CCR5 or CXCR4). This interaction finally leads to fusion of the membrane of the virus with that of a human T-cell. The fusion process is mediated by viral gp41 and leads to infection of the human cell. Compounds that inhibit this process are referred to as entry inhibitors.[3] We present a new way to inhibit the gp120/CD4 interaction through blocking the binding site on the CD4 protein with a peptidomimetic. Other research groups have targeted the same interaction and have developed gp120-or antibodyrelated peptides, [4] or gp120-binding molecules. [5] In our approach, which involves the development of CD4 ligands, interference with the human immune system or viral resistance are potential problems. However, resistance of the virus to inhibitors that bind to CD4 would require large changes in the viral glycoproteins to allow the virus to utilize a different entry mechanism. Such resistance is therefore not as likely as the evasion of inhibitors of viral proteins whose binding can be circumvented by one or two mutations of the viral amino acids.We started from the CD4-binding decapeptide NMWQKVGTPL, [6] which was derived from an X-ray structure of gp120.[2b] The peptide has a low binding affinity to CD4 (K D = 6 mm, determined by the surface plasmon resonance (SPR) and saturation transfer difference (STD) NMR spectroscopy protocols described herein) but has an antiviral activity that can be detected by a virus neutralization assay. The main targets of the design phase were to lower the molecular weight of the peptide, enhance its binding affinity, and incorporate nonnatural elements to increase proteolytic stability. The docking of peptides generated by molecular modeling studies by substituting each amino acid of the lead sequence in turn with alanine (see the Supporting Information) supported the results of saturation transfer difference (STD) NMR epitope mapping: [7] The docking study results suggest that the amino acids Asn, Met, and Gln do not contribute to the binding to CD4. Therefore, these inactive subunits were removed from the lead structure to reduce the molecular weight. Interactions of the aromatic ring of Trp and the hydrophobic side chain of Leu with CD4 could be replaced by the interaction of numerous other hydrophobic side chains without losing binding energy. Only the core peptide KVGTP was considered crucial for binding. The N and C termini were modified with various hydrophobic residues connected by nonpeptidic bonds. The candidate found from the molecular modeling studies to interact best with CD4 (Figure 1) was synthesized (Scheme 1).We synthesized the peptidomimetic from th...

Chemoenzymatic synthesis of ketomethylene tripeptide isosteres

“…The combined organic layer was washed with brine (60 mL), dried over MgSO 4 , and concentrated to afford 6 as a crude product (5.6 g, 13.9 mmol, 85% in yields, 90-95% UV purity), which was carried over to the next step without further purification. 1 …”

“…75% over two steps from 6) upon chromatographic purification (hexanes/ethyl acetate 95:5) over three steps as a mixture of two diastereomers (1:1). 1 …”

“…The organic layer was separated, washed with 1 M HCl (10 mL), satd NaHCO 3 (10 mL), dried over MgSO 4 , and filtered through a short pad of silica gel to give 1 (216 mg, 0.56 mmol, about 72%, 98% de, 97% ee) upon chromatographic purification (methylene chloride/hexane/IPA = 79/20/3). 1 …”

“…[1][2][3][4][5][6][7][8][9] For example, Rupintrivir TM , which contains ketomethylene tripeptide isostere 1 (Scheme 1), is a lead candidate entering human clinical trials to treat the common cold infection. 7 A number of methods have been reported for the preparation of ketomethylene peptide isosteres with the desired stereochemical configuration.…”

“…7 A number of methods have been reported for the preparation of ketomethylene peptide isosteres with the desired stereochemical configuration. [1][2][3][4][5][6][7][8][9][10] Among them, the shortest approach uses a chiral alkylation strategy to produce both C-and N-terminal masked ketomethylene dipeptide isosteres from amino acids and (R)-2-hydroxy carboxylic esters. 10 Assuming mild deprotection conditions can be identified, these building blocks can then be potentially incorporated into longer peptide sequences such as tripeptide isosteres (e.g., 1) and peptide mimetics.…”