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Macrocyclic inhibitor 1 {methyl [cyclo-7[(2R)-((N-valyl) amino)-2-(hydroxyl-(1S)-1-methyoxycarbonyl-2-phenylethoxy)phosphinyloxyethyl]-1-naphthaleneacetamide] sodium salt} was designed according to the conformation of the acyclic analogue Iva-l-Val-l-Val-l-LeuP-(O)Phe-OMe [LeuP = the phosphinic acid and analogue of l-leucine; (O)Phe = l-3-phenyllactic acid; OMe = methyl ester] (4) bound to penicillopepsin, by linking the P1 and P3 side chains of the penicillopepsin inhibitor. This compound and its two acyclic derivatives, {methyl (2S)-[1-(((N-Formyl)-l-valyl)amino-2-(2-naphthyl)ethyl)hydroxyphosphinyloxy]-3-phenylpropanoate, sodium salt} (2) and {methyl (2S)-[1-(((N-(1-naphthaleneacetyl))-l-valyl)aminomethyl)hydroxyphosphinyloxy]-3-phenylpropanoate, sodium salt} (3), have been synthesized and evaluated as inhibitors of penicillopepsin. Their binding affinity to the enzyme was found to be inversely related to the predicted degree of conformational flexibility across the series: 3 (Ki = 110 μM), 2 (Ki = 7....

Tuberculosis (TB) infects one-third of the world population. Despite 50 years of available drug treatments, TB continues to increase at a significant rate. The failure to control TB stems in part from the expense of delivering treatment to infected individuals and from complex treatment regimens. Incomplete treatment has fueled the emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (Mtb). Reducing non-compliance by reducing the duration of chemotherapy will have a great impact on TB control. The development of new drugs that either kill persisting organisms, inhibit bacilli from entering the persistent phase, or convert the persistent bacilli into actively growing cells susceptible to our current drugs will have a positive effect. We are taking a multidisciplinary approach that will identify and characterize new drug targets that are essential for persistent Mtb. Targets are exposed to a battery of analyses including microarray experiments, bioinformatics, and genetic techniques to prioritize potential drug targets from Mtb for structural analysis. Our core structural genomics pipeline works with the individual laboratories to produce diffraction quality crystals of targeted proteins, and structural analysis will be completed by the individual laboratories. We also have capabilities for functional analysis and the virtual ligand screening to identify novel inhibitors for target validation. Our overarching goals are to increase the knowledge of Mtb pathogenesis using the TB research community to drive structural genomics, particularly related to persistence, develop a central repository for TB research reagents, and discover chemical inhibitors of drug targets for future development of lead compounds.

The protein toxin produced by Shigella dysenteriae consists of one enzymatically active A subunit of 293 amino acid residues and five B subunits of 69 amino acid residues that are involved with cell attachment. The holotoxin has been purified by blue Sepharose and chromatofocusing column chromatography. Two crystal forms of purified holotoxin have been grown by vapor diffusion. One grows as fine needles, hexagonal in cross-section, which do not diffract well enough to characterize crystallographically. The second grows as thin plates that diffract to at least 3 A resolution. Their space group is P2(1)2(1)2(1) with unit cell dimensions of a = 132.0 A, b = 146.0 A and c = 82.5 A. The asymmetric unit of the crystals is likely to contain two AB5 units.

The design of inhibitors with enhanced potency against proteolytic enzymes has many applications for the treatment of human diseases. In addition to the optimization of chemical interactions between the enzyme and inhibitor, the binding affinity can be increased by constraining the inhibitor to the conformation that is recognized by the enzyme, thus lowering the entropic barrier to complex formation. We have structurally characterized the complexes of a macrocyclic pentapeptide inhibitor and its acyclic analogue with penicillopepsin, an aspartic proteinase, to study the effect of conformational constraint on the binding affinity. The phosphonate-based macrocycle PPi4 (Ki = 0.10 nM) is covalently linked at the P2-Asn and P1'-Phe side chains [nomenclature of Schechter and Berger, Biochim. Biophys. Res. Commun. (1967) 27, 157-162] via an amide bond, relative to the acyclic compound PPi3 (Ki = 42 nM). Comparisons of the high-resolution crystal structures of PPi4-penicillopepsin (0.95 A) and PPi3-penicillopepsin (1.45 A) reveal that the conformations of the inhibitors and their interactions with the enzyme are similar. The 420-fold increase in the binding affinity of PPi4 is attributed to a reduction in its conformational flexibility, thus providing the first rigorous measure of the entropic contribution to the binding energy in a protein-ligand complex and stressing the advantages of the design strategy.