Non-Hydrolytic ß-Lactam Antibiotic Fragmentation by l,d-Transpeptidases and Serine ß-Lactamase Cysteine Variants.

Enzymes often use nucleophilic serine, threonine, and cysteine residues to achieve the same type of reaction; the underlying reasons for this are not understood. While bacterial d,d-transpeptidases (penicillin-binding proteins) employ a nucleophilic serine, l,d-transpeptidases use a nucleophilic cysteine. The covalent complexes formed by l,d-transpeptidases with some ß-lactam antibiotics undergo non-hydrolytic fragmentation. This is not usually observed for penicillin-binding proteins, or for the related serine ß-lactamases. Replacement of the nucleophilic serine of serine ß-lactamases with cysteine yields enzymes which fragment ß-lactams via a similar mechanism as the l,d-transpeptidases, implying the different reaction outcomes are principally due to the formation of thioester versus ester intermediates. The results highlight fundamental differences in the reactivity of nucleophilic serine and cysteine enzymes, and imply new possibilities for the inhibition of nucleophilic enzymes.

Small Molecule Ligands of the BET-like Bromodomain, SmBRD3, Affect Schistosoma mansoni Survival, Oviposition, and Development.

Schistosomiasis is a disease affecting >200 million people worldwide, but its treatment relies on a single agent, praziquantel. To investigate new avenues for schistosomiasis control, we have conducted the first systematic analysis of bromodomain-containing proteins (BCPs) in a causative species, Schistosoma mansoni. Having identified 29 putative bromodomains (BRDs) in 22 S. mansoni proteins, we selected SmBRD3, a tandem BRD-containing BCP that shows high similarity to the human bromodomain and extra terminal domain (BET) family, for further studies. Screening 697 small molecules identified the human BET BRD inhibitor I-BET726 as a ligand for SmBRD3. An X-ray crystal structure of I-BET726 bound to the second BRD of SmBRD3 [SmBRD3(2)] enabled rational design of a quinoline-based ligand (15) with an ITC Kd = 364 ± 26.3 nM for SmBRD3(2). The ethyl ester pro-drug of compound 15 (compound 22) shows substantial effects on sexually immature larval schistosomula, sexually mature adult worms, and snail-infective miracidia in ex vivo assays.