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β-Lactam antibiotics have long been a treatment of choice for bacterial infections since they bind irreversibly to Penicillin-Binding Proteins (PBPs), enzymes that are vital for cell wall biosynthesis. Many pathogens express drug-insensitive PBPs rendering β-lactams ineffective, revealing a need for new types of PBP inhibitors active against resistant strains. We have identified alkyl boronic acids that are active against pathogens including methicillin-resistant S. aureus (MRSA). The crystal structures of PBP1b complexed to 11 different alkyl boronates demonstrate that in vivo efficacy correlates with the mode of inhibitor side chain binding. Staphylococcal membrane analyses reveal that the most potent alkyl boronate targets PBP1, an autolysis system regulator, and PBP2a, a low β-lactam affinity enzyme. This work demonstrates the potential of boronate-based PBP inhibitors for circumventing β-lactam resistance and opens avenues for the development of novel antibiotics that target Gram-positive pathogens.

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The Src Homology 3 (SH3) domains are small protein-protein interaction domains that bind proline-rich sequences and mediate a wide range of cell-signaling and other important biological processes. Since deregulated signaling pathways form the basis of many human diseases, the SH3 domains have been attractive targets for novel therapeutics. High-affinity ligands for SH3 domains have been designed; however, these have all been peptide-based and no examples of entirely nonpeptide SH3 ligands have previously been reported. Using the mouse Tec Kinase SH3 domain as a model system for structure-based ligand design, we have identified several simple heterocyclic compounds that selectively bind to the Tec SH3 domain. Using a combination of nuclear magnetic resonance chemical shift perturbation, structure-activity relationships, and site-directed mutagenesis, the binding of these compounds at the proline-rich peptide-binding site has been characterized. The most potent of these, 2-aminoquinoline, bound with Kd = 125 microM and was able to compete for binding with a proline-rich peptide. Synthesis of 6-substituted-2-aminoquinolines resulted in ligands with up to 6-fold improved affinity over 2-aminoquinoline and enhanced specificity for the Tec SH3 domain. Therefore, 2-aminoquinolines may potentially be useful for the development of high affinity small molecule ligands for SH3 domains.

Penicillin binding proteins (PBPs) catalyze steps in the biosynthesis of bacterial cell walls and are the targets for the beta-lactam antibiotics. Non-beta-lactam based antibiotics that target PBPs are of interest because bacteria have evolved resistance to the beta-lactam antibiotics. Boronic acids have been developed as inhibitors of the mechanistically related serine beta-lactamases and serine proteases; however, they have not been explored extensively as PBP inhibitors. Here we report aromatic boronic acid inhibitors of the D,D-carboxypeptidase R39 from Actinomadura sp. strain. Analogues of an initially identified inhibitor [3-(dihydroxyboryl)benzoic acid 1, IC(50) 400 microM] were prepared via routes involving pinacol boronate esters, which were deprotected via a two-stage procedure involving intermediate trifluorborate salts that were hydrolyzed to provide the free boronic acids. 3-(Dihydroxyboryl)benzoic acid analogues containing an amide substituent in the meta, but not ortho position were up to 17-fold more potent inhibitors of the R39 PBP and displayed some activity against other PBPs. These compounds may be useful for the development of even more potent boronic acid based PBP inhibitors with a broad spectrum of antibacterial activity.

The Src homology 3 (SH3) domains are small protein-protein interaction domains that mediate a range of important biological processes and are considered valuable targets for the development of therapeutic agents. We have been developing 2-aminoquinolines as ligands for SH3 domains--so far the only reported examples of entirely small-molecule ligands for the SH3 domains. The highest affinity 2-aminoquinolines so far identified are 6-substituted compounds. In this article, the synthesis of several new 2-aminoquinolines, including 5-, 6- and 7-substituted compounds, for Tec SH3 domain ligand binding studies is presented. As a part of the synthetic investigation, the utility of different methods for the synthesis of 2-aminoquinolines was explored and potentially powerful methods were identified for the synthesis of 2-aminoquinolines with diverse functionality. Of the compounds prepared, the 5-substituted-2-aminoquinolines generally bound with similar affinities to unsubstituted 2-aminoquinoline, whilst the 7-substituted compounds generally bound with similar or lower affinity than unsubstituted 2-aminoquinoline. However, the 6-substituted-2-aminoquinolines generally bound with significantly higher affinity than unsubstituted 2-aminoquinoline. In addition, one 6-substituted-N-benzylated-2-aminoquinoline was also tested for SH3 binding and some evidence for the formation of additional contacts at other regions of the SH3 domain was found. These results provide new and useful SAR information that should greatly assist with the challenge of developing high affinity small-molecule ligands for the SH3 domains.

Following from the evaluation of different types of electrophiles, combined modeling and crystallographic analyses are used to generate potent boronic acid based inhibitors of a penicillin binding protein. The results suggest that a structurally informed approach to penicillin binding protein inhibition will be useful for the development of both improved reversibly binding inhibitors, including boronic acids, and acylating inhibitors, such as β-lactams.