AbstractThe 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. ΔTm values, defined as the difference in Tm 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°C, respectively. Binding of APV and Ac‐PEP is most sensitive to the D25N mutation, as shown by ΔTm ratios [ΔTm(PR)/ΔTm(PRD25N)] of 35.8 and 16.3, respectively, whereas binding of DMP323 and RPB (ΔTm ratios of 1–2) is least affected. Binding of the substrate‐like inhibitors RPB and Ac‐PEP is nearly abolished (ΔTm(PR)/ΔTm(PRD29N) ≥ 44) by the D29N mutation, whereas this mutation only moderately affects binding of the smaller inhibitors (ΔTm 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 Ki values of 0.3, 0.6, and 2.14 μM, respectively. DSC results were consistent with this relatively weak binding of APV (ΔTm 2.7°C) compared with the tight binding of Ac‐PEP (ΔTm ≥ 17°C). Comparison of superimposed structures of the PR/APV complex with those of PR/Ac‐PEP and pepsin/pepstatin A complexes suggests a role for Asp215, Asp32, and Ser219 in pepsin, equivalent to Asp25, Asp25′, and Asp29 in PR in the binding and stabilization of the pepsin/APV complex. Proteins 2009. Published 2008 Wiley‐Liss, Inc.