Design of small-sized libraries by combinatorial assembly of linkers and functional groups to a given scaffold: application to the structure-based optimization of a phosphodiesterase 4 inhibitor.

Combinatorial chemistry and library design have been reconciled by applying simple medicinal chemistry concepts to virtual library design. The herein reported "Scaffold-Linker-Functional Group" (SLF) approach has the aim to maximize diversity while minimizing the size of a scaffold-focused library with the aid of simple molecular variations in order to identify critical pharmacophoric elements. Straightforward rules define the way of assembling three building blocks: a conserved scaffold, a variable linker, and a variable functional group. By carefully selecting a limited number of functional groups not overlapping in pharmacophoric space, the size of the library is kept to a few hundred. As an application of the SLF approach, a small-sized combinatorial library (320 compounds) was derived from the scaffold of the known phosphodiesterase 4 inhibitor zardaverine. The most interesting analogues were further prioritized for synthesis and enzyme inhibition by FlexX docking to the X-ray structure of the enzyme, leading to a 900-fold increased affinity within nine synthesized compounds and a single screening round.

Inhibition of proteinase 3 by [alpha]1-antitrypsin in vitro predicts very fast inhibition in vivo.

Neutrophil proteinase 3 (Pr3) cleaves elastin and other matrix proteins, and is thought to cause lung tissue destruction in emphysema and cystic fibrosis. Its deleterious action is theoretically prevented by alpha1-antitrypsin, a serpin present in lung secretions. We have evaluated the anti-Pr3 activity of this inhibitor to decide whether it may play a physiologic proteolysis-preventing function in vivo. We show that (i). the oxidized inhibitor does not inhibit Pr3; (ii). the inhibitor competes favorably with elastin for the binding of Pr3, but is less efficient for inhibiting elastin-bound proteinase than for complexing free enzyme; and (iii). the inhibition takes place in at least two steps: the enzyme and the inhibitor first form a high-affinity reversible inhibitory complex EI* with an equilibrium dissociation constant K*i of 38 nM; EI* subsequently transforms into an irreversible complex EI with a first-order rate constant k2 of 0.04 s-1. Because the alpha1-antitrypsin concentration in the epithelial lining fluid is much higher than K*i, any Pr3 molecule released from neutrophils will be taken up as an EI* complex within much less than 1 s, indicating very efficient inhibition in vivo.

Conformational change in elastase following complexation with alpha1-proteinase inhibitor: a CD investigation.

The CD spectrum of porcine pancreatic elastase in complex with alpha1-proteinase inhibitor (alpha1-PI) was calculated by subtracting the CD spectrum of the proteolytically cleaved inhibitor from that of the elastase-alpha1-PI complex. Elastase undergoes a moderate secondary structure change: its beta-structure is partially disordered while its alpha-helix content is poorly affected. In contrast, its tertiary structure undergoes a significant structural loosening upon complexation. These alterations have been compared with those following chemical and thermal unfolding of free elastase. Inhibitor-bound elastase and the denaturation intermediate of free elastase share secondary but not tertiary structural features. On the other hand, both free and complexed elastases undergo a single-step transition in tertiary structure upon thermal unfolding. These data are discussed in terms of the inhibition and structural modification of elastase induced by alpha1-PI observed by previous investigators.