Discovery of Nanomolar Melanocortin-3 Receptor (MC3R)-Selective Small Molecule Pyrrolidine Bis-Cyclic Guanidine Agonist Compounds Via a High-Throughput "Unbiased" Screening Campaign.

The central melanocortin-3 and melanocortin-4 receptors (MC3R, MC4R) are key regulators of body weight and energy homeostasis. Herein, the discovery and characterization of first-in-class small molecule melanocortin agonists with selectivity for the melanocortin-3 receptor over the melanocortin-4 receptor are reported. Identified via "unbiased" mixture-based high-throughput screening approaches, pharmacological evaluation of these pyrrolidine bis-cyclic guanidines resulted in nanomolar agonist activity at the melanocortin-3 receptor. The pharmacological profiles at the remaining melanocortin receptor subtypes tested indicated similar agonist potencies at both the melanocortin-1 and melanocortin-5 receptors and antagonist or micromolar agonist activities at the melanocortin-4 receptor. This group of small molecules represents a new area of chemical space for the melanocortin receptors with mixed receptor pharmacology profiles that may serve as novel lead compounds to modulate states of dysregulated energy balance.

Identification of a small molecule inhibitor of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] using mixture-based combinatorial libraries.

The aminoglycoside, 6'-N-acetyltransferase type Ib [AAC(6')-Ib] is the most widely distributed enzyme among AAC(6')-I-producing Gram-negative pathogens and confers resistance to clinically relevant aminoglycosides, including amikacin. This enzyme is therefore an ideal target for enzymatic inhibitors that could overcome resistance to aminoglycosides. The search for inhibitors was carried out using mixture-based combinatorial libraries, the scaffold ranking approach, and the positional scanning strategy. A library with high inhibitory activity had pyrrolidine pentamine scaffold and was selected for further analysis. This library contained 738,192 compounds with functionalities derived from 26 different amino acids (R1, R2 and R3) and 42 different carboxylic acids (R4) in four R-group functionalities. The most active compounds all contained S-phenyl (R1 and R3) and S-hydromethyl (R2) functionalities at three locations and differed at the R4 position. The compound containing 3-phenylbutyl at R4 (compound 206) was a robust enzymatic inhibitor in vitro, in combination with amikacin it potentiated the inhibition of growth of three resistant bacteria in culture, and it improved survival when used as treatment of Galleria mellonella infected with aac(6')-Ib-harboring Klebsiella pneumoniae and Acinetobacter baumannii strains.

High-Throughput Flow Cytometry Screening of Multidrug Efflux Systems.

The resistance nodulation cell division (RND) family of proteins are inner membrane transporters that associate with periplasmic adaptor proteins and outer membrane porins to affect substrate transport from the cytosol and periplasm in Gram-negative bacteria. Various structurally diverse compounds are substrates of RND transporters. Along with their notable role in antibiotic resistance, these transporters are essential for niche colonization, quorum sensing, and virulence as well as for the removal of fatty acids and bile salts. As such, RNDs are an attractive target for antimicrobial development. However, while enhancing the utility of antibiotics with an RND inhibitor is an appealing concept, only a small core of chemotypes has been identified as efflux pump inhibitors (EPIs). Thus, our key objective is the development and validation of an efflux profiling and discovery strategy for RND model systems. Here we describe a flow cytometric dye accumulation assay that uses fluorescein diacetate (FDA) to interrogate the model Gram-negative pathogens Escherichia coli, Franscisella tularensis, and Burkholderia pseudomallei. Fluorochrome retention is increased in the presence of known efflux inhibitors and in RND deletion strains. The assay can be used in a high-throughput format to evaluate efflux of dye-substrate candidates and to screen chemical libraries for novel EPIs. Triaged compounds that inhibit efflux in pathogenic strains are tested for growth inhibition and antibiotic potentiation using microdilution culture plates in a select agent Biosafety Level-3 (BSL3) environment. This combined approach demonstrates the utility of flow cytometric analysis for efflux activity and provides a useful platform in which to characterize efflux in pathogenic Gram-negative bacteria. Screening small molecule libraries for novel EPI candidates offers the potential for the discovery of new classes of antibacterial compounds.

Potent antimicrobial small molecules screened as inhibitors of tyrosine recombinases and Holliday junction-resolving enzymes.

Holliday junctions (HJs) are critical intermediates in many recombination-dependent DNA repair pathways. Our lab has previously identified several hexameric peptides that target HJ intermediates formed in DNA recombination reactions. One of the most potent peptides, WRWYCR, is active as a homodimer and has shown bactericidal activity partly because of its ability to interfere with DNA repair proteins that act upon HJs. To increase the possibility of developing a therapeutic targeting DNA repair, we searched for small molecule inhibitors that were functional surrogates of the peptides. Initial screens of heterocyclic small molecule libraries resulted in the identification of several N-methyl aminocyclic thiourea inhibitors. Like the peptides, these inhibitors trapped HJs formed during recombination reactions in vitro, but were less potent than the peptides in biochemical assays and had little antibacterial activity. In this study, we describe the screening of a second set of libraries containing somewhat larger and more symmetrical scaffolds in an effort to mimic the symmetry of a WRWYCR homodimer and its target. From this screen, we identified several pyrrolidine bis-cyclic guanidine inhibitors that also interfere with processing of HJs in vitro and are potent inhibitors of Gram-negative and especially Gram-positive bacterial growth. These molecules are proof-of-principle of a class of compounds with novel activities, which may in the future be developed into a new class of antibiotics that will expand the available choices for therapy against drug-resistant bacteria.