Fragment-based lead discovery is gaining momentum in drug development. Typically, a hierarchical cascade of several screening techniques is consulted to identify fragment hits which are then analyzed by crystallography. Because crystal structures with bound fragments are essential for the subsequent hit-to-lead-to-drug optimization, the screening process should distinguish reliably between binders and non-binders. We therefore investigated whether different screening methods would reveal similar collections of putative binders. First we used a biochemical assay to identify fragments that bind to endothiapepsin, a surrogate for disease-relevant aspartic proteases. In a comprehensive screening approach, we then evaluated our 361-entry library by using a reporter-displacement assay, saturation-transfer difference NMR, native mass spectrometry, thermophoresis, and a thermal shift assay. While the combined results of these screening methods retrieve 10 of the 11 crystal structures originally predicted by the biochemical assay, the mutual overlap of individual hit lists is surprisingly low, highlighting that each technique operates on different biophysical principles and conditions.

Successful lead optimization in structure-based drug discovery depends on the correct deduction and interpretation of the underlying structure-activity relationships (SAR) to facilitate efficient decision-making on the next candidates to be synthesized. Consequently, the question arises, how frequently a binding mode (re)-validation is required, to ensure not to be misled by invalid assumptions on the binding geometry. We present an example in which minor chemical modifications within one inhibitor series lead to surprisingly different binding modes. X-ray structure determination of eight inhibitors derived from one core scaffold resulted in four different binding modes in the aspartic protease endothiapepsin, a well-established surrogate for e.g. renin and β-secretase. In addition, we suggest an empirical metrics that might serve as an indicator during lead optimization to qualify compounds as candidates for structural revalidation.

HTLV-1 protease (HTLV-1 PR) is an aspartic protease which represents a promising drug target for the discovery of novel anti-HTLV-1 drugs. The X-ray structure of HTLV-1 PR in complex with the well-known and approved HIV-1 PR inhibitor Indinavir was determined at 2.40 Å resolution. In this contribution, we describe the first crystal structure in complex with a nonpeptidic inhibitor that accounts for rationalizing the rather moderate affinity of Indinavir against HTLV-1 PR and provides the basis for further structure-guided optimization strategies.

3,4-disubstituted pyrrolidines originally designed to inhibit the closely related HIV-1 protease were evaluated as privileged structures against HTLV-1 protease (HTLV-1 PR). The most potent inhibitor of this series exhibits two-digit nanomolar affinity and represents, to the best of our knowledge, the most potent nonpeptidic inhibitor of HTLV-1 PR described so far. The X-ray structures of two representatives bound to HTLV-1 PR were determined, and the structural basis of their affinity is discussed.

In low- and normal- renin hypertensive patients, but not in high-renin patients, the acute antihypertensive response to the angiotensin-converting enzyme (ACE) inhibitor captopril was completely blocked by aprotinin-induced kallikrein inhibition. Blood pressure reduction with long-term ACE inhibition could be overcome only in part by aprotinin. It is proposed that in low- and normal-renin hypertension the vasodepressor effect of acute ACE inhibition is mainly due to kinin accumulation. Conversely, in high-renin patients a fall in angiotensin II concentration accounts for the hypotensive response to captopril. From the pressor effect of aprotinin in chronically captopril treated patients it appears that kinins are also involved in the blood pressure reduction with long-term ACE inhibition. The finding that ACE inhibition and kallikrein blockade produced predictable and opposite effects on blood pressure suggests broad participation of changes in depressor kinin production in the control of vascular tone in essential hypertension.