Acetyl Pepstatin

The importance of the active site region aspartyl residues 25 and 29 of the mature HIV-1 protease (PR) for the binding of five clinical and three experimental protease inhibitors [symmetric cyclic urea inhibitor DMP323, nonhydrolyzable substrate analog (RPB) and the generic aspartic protease inhibitor acetyl-pepstatin (Ac-PEP)] was assessed by differential scanning calorimetry. DeltaT(m) values, defined as the difference in T(m) for a given protein in the presence and absence of inhibitor, for PR with DRV, ATV, SQV, RTV, APV, DMP323, RPB, and Ac-PEP are 22.4, 20.8, 19.3, 15.6, 14.3, 14.7, 8.7, and 6.5 degrees C, respectively. Binding of APV and Ac-PEP is most sensitive to the D25N mutation, as shown by DeltaT(m) ratios [DeltaT(m)(PR)/DeltaT(m)(PR(D25N))] of 35.8 and 16.3, respectively, whereas binding of DMP323 and RPB (DeltaT(m) ratios of 1-2) is least affected. Binding of the substrate-like inhibitors RPB and Ac-PEP is nearly abolished (DeltaT(m)(PR)/DeltaT(m)(PR(D29N)) > or = 44) by the D29N mutation, whereas this mutation only moderately affects binding of the smaller inhibitors (DeltaT(m) ratios of 1.4-2.2). Of the nine FDA-approved clinical HIV-1 protease inhibitors screened, APV, RTV, and DRV competitively inhibit porcine pepsin with K(i) values of 0.

In order to elucidate the characteristics of different aspartic proteases (Sap) secreted by Candida albicans, the kinetics of the interaction (k(on), k(off)) between Sap1 and Sap2 with acetyl-pepstatin and pepstatin A was determined at different pH by biosensor technology. The enzymes were biotinylated and coupled to a streptavidin-coated sensor chip, whereupon acetyl-pepstatin or pepstatin A was injected and the interaction was measured in real time. Sap2 showed a faster k(on) and a higher affinity for acetyl-pepstatin than Sap1, regardless of pH. The values for both k(on) and k(off) decreased with increased pH from 3.8 to 5.0, except for the k(off) for Sap1, which was only influenced by the pH change from 3.8 to 4.4. Binding of acetyl-pepstatin to Sap1 or Sap2 obviously proceeds by a different mechanism than dissociation of the inhibitor. Association appears to be coupled to protonation of a catalytic aspartic acid residue, consistent with reduced k(on) values at higher pH. In contrast, the stability of the complex is reduced at lower pH due to reduced hydrogen bonding capacity of aspartic acid residues acting as hydrogen bond acceptors. Differences in the number and distribution of charged nonactive site residues in Sap1 and Sap2 evidently result in different electrostatic properties of the binding sites, primarily influencing the association step.

Subtype F wild type HIV protease has been kinetically characterized using six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) commonly used for HIV/AIDS treatment, as well as inhibitor TL-3 and acetyl-pepstatin. We also obtained kinetic parameters for two multi-resistant proteases (one of subtype B and one of subtype F) harboring primary and secondary mutations selected by intensive treatment with ritonavir/nelfinavir. This newly obtained biochemical data shows that all six studied commercially available protease inhibitors are significantly less effective against subtype F HIV proteases than against HIV proteases of subtype B, as judged by increased K(i) and biochemical fitness (vitality) values. Comparison with previously reported kinetic values for subtype A and C HIV proteases show that subtype F wild type proteases are significantly less susceptible to inhibition. These results demonstrate that the accumulation of natural polymorphisms in subtype F proteases yields catalytically more active enzymes with a large degree of cross-resistance, which thus results in strong virus viability.