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Over the past several decades, the frequency of antibacterial resistance in hospitals, including multidrug resistance (MDR) and its association with serious infectious diseases, has increased at alarming rates. Pseudomonas aeruginosa is a leading cause of nosocomial infections, and resistance to virtually all approved antibacterial agents is emerging in this pathogen. To address the need for new agents to treat MDR P. aeruginosa, we focused on inhibiting the first committed step in the biosynthesis of lipid A, the deacetylation of uridyldiphospho-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine by the enzyme LpxC. We approached this through the design, synthesis, and biological evaluation of novel hydroxamic acid LpxC inhibitors, exemplified by 1, where cytotoxicity against mammalian cell lines was reduced, solubility and plasma-protein binding were improved while retaining potent anti-pseudomonal activity in vitro and in vivo.

Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate the C(55) undecaprenyl pyrophosphate (UPP). It has been demonstrated that tetramic acids (TAs) are selective and potent inhibitors of UPPS, but the mode of inhibition was unclear. In this work, we used a fluorescent FPP probe to study possible TA binding at the FPP binding site. A photosensitive TA analogue was designed and synthesized for the study of the site of interaction of TA with UPPS using photo-cross-linking and mass spectrometry. The interaction of substrates with UPPS and with the UPPS.TA complex was investigated by protein fluorescence spectroscopy. Our results suggested that tetramic acid binds to UPPS at an allosteric site adjacent to the FPP binding site. TA binds to free UPPS enzyme but not to substrate-bound UPPS. Unlike Escherichia coli UPPS which follows an ordered substrate binding mechanism, Streptococcus pneumoniae UPPS appears to follow a random-sequential substrate binding mechanism. Only one substrate, FPP or IPP, is able to bind to the UPPS.TA complex, but the quaternary complex, UPPS.TA.FPP.IPP, cannot be formed. We propose that binding of TA to UPPS significantly alters the conformation of UPPS needed for proper substrate binding. As the result, substrate turnover is prevented, leading to the inhibition of UPPS catalytic activity. These probe compounds and biophysical assays also allowed us to quickly study the mode of inhibition of other UPPS inhibitors identified from a high-throughput screening and inhibitors produced from a medicinal chemistry program.

Total syntheses of the cytotoxic marine natural products bengamides B and E are described. Both bengamides are prepared via amide coupling of a protected polyhydroxylated lactone intermediate 9 with a suitably substituted aminocaprolactam intermediate. Lactone 9 is prepared in five steps from commercially available alpha-D-glucoheptonic gamma-lactone. The key reactions are a selective deprotection of a 1,2-acetonide in the presence of a 1,3-acetonide and an (E)-selective olefination of an unstable aldehyde using a gem-dichromium reagent. The bengamide B lactam intermediate 10 is prepared in seven steps from commercially available (5R)-5-hydroxy-L-lysine (12). The desired S-configuration at the gamma-OH lactam position is established using the Mitsunobu reaction.

The structural chemistry and biological activity of the bengamide class of compounds have been further characterized. Extracts prepared from recollected Jaspis cf. coriacea from five sites in Fiji were pooled. Six new bengamides, M (7b), N (8a), O (8b), P (9a), Q (9b), and R (10), were identified, accompanied by the known bengamides A (1a), B (1b), E (3a), F (3b), Y (5), Z (6), L (7a), G (11a), H (11b), and I (12). The structures of the new compounds were determined from spectroscopic data, and some were additionally confirmed by semisynthesis. Cytotoxicity screening data were obtained from the NCI-DTP 60 cell screen for bengamides A, B, and P. Bengamides A and B were more potent than bengamide P, with average IC(50) values of 0.046, 0.011, and 2.70 FM, respectively. The in vitro antitumor activity against MDA-MB-435 human mammary carcinoma was also determined for natural bengamides A, B, E, F, P, M, O, and Z and for synthetic samples of B and O. The best activity was observed for the natural bengamides A (IC(50) = 1 nM) and O (IC(50) = 0.3 nM).