The causative agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus, SARS-CoV. The main proteinase of SARS-CoV, 3CLpro, is an attractive target for therapeutics against SARS owing to its fundamental role in viral replication. We sought to identify novel inhibitors of 3CLpro to advance the development of appropriate therapies in the treatment of SARS. 3CLpro was cloned, expressed, and purified from the Tor2 isolate. A quenched fluorescence resonance energy transfer assay was developed for 3CLpro to screen the proteinase against 50,000 drug-like small molecules on a fully automated system. The primary screen identified 572 hits; through a series of virtual and experimental filters, this number was reduced to five novel small molecules that show potent inhibitory activity (IC50 = 0.5-7 microM) toward SARS-CoV 3CLpro.

The SCF ubiquitin ligases target proteins for degradation by recruitment factors called F-box proteins1, 2. We identified a bi-planar dicarboxylic acid compound, called SCF-I2, as an inhibitor of substrate phosphodegron recognition by the yeast F-box protein Cdc4. SCF-I2 inhibits the binding and ubiquitination of full length phosphorylated substrates by SCFCdc4. A crystal co-structure reveals that SCF-I2 inserts between the β-strands of blades 5 and 6 of the WD40 propeller domain of Cdc4 at a site that is 25 Å remote from the substrate binding site. Long-range transmission of SCF-I2 interactions distorts the substrate binding pocket and impedes recognition of key determinants in the Cdc4 phosphodegron. Mutation of the SCF-I2 binding site abrogates its inhibitory effect and explains specificity in the allosteric inhibition mechanism. Mammalian WD40 domain proteins may exhibit similar allosteric responsiveness and hence represent an extensive new class of druggable target.

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Firefly luciferase (FL) was entrapped in sol-gel-derived silica containing precursors based on covalent linkage of d-gluconolactone or d-maltonolactone to (aminopropyl)triethoxysilane to form N-(3-triethoxysilylpropyl)gluconamide or N-(3-triethoxysilylpropyl)maltonamide. The enzyme was active and stable in this material and showed catalytic constants close to those in solution. As little as 20 amol ATP could be detected with the entrapped FL, and the entrapped enzyme could be used over several cycles.

The biosynthesis of methionine is an attractive antibiotic target given its importance in protein and DNA metabolism and its absence in mammals. We have performed a high-throughput screen of the methionine biosynthesis enzyme cystathionine beta-lyase (CBL) against a library of 50 000 small molecules and have identified several compounds that inhibit CBL enzyme activity in vitro. These hit molecules were of two classes: those that blocked CBL activity with mixed steady-state inhibition and those that covalently interacted with the enzyme at the active site pyridoxal phosphate cofactor with slow-binding inhibition kinetics. We determined the crystal structure of one of the slow-binding inhibitors in complex with CBL and used this structure as a guide in the synthesis of a small, focused library of analogues, some of which had improved enzyme inhibition properties. These studies provide the first lead molecules for antimicrobial agents that target cystathionine beta-lyase in methionine biosynthesis.