SUMMARY LpxC is an essential enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria. Several promising antimicrobial lead compounds targeting LpxC have been reported, though they typically display a large variation in potency against different Gram-negative pathogens. We report that inhibitors with a diacetylene scaffold effectively overcome the resistance caused by sequence variation in the LpxC substrate-binding passage. Compound binding is captured in complex with representative LpxC orthologs, and structural analysis reveals large conformational differences that mostly reflect inherent molecular features of distinct LpxC orthologs, whereas ligand-induced structural adaptations occur at a smaller scale. These observations highlight the need for a molecular understanding of inherent structural features and conformational plasticity of LpxC enzymes for optimizing LpxC inhibitors as broad-spectrum antibiotics against Gram-negative infections.

␤-lactam antibiotics inhibit penicillin binding proteins (PBPs) involved in peptidoglycan synthesis. Although inhibition of peptidoglycan biosynthesis is generally thought to induce cell lysis, the pattern and mechanism of cell lysis can vary substantially. ␤-lactams that inhibit FtsI, the only division specific PBP, block cell division and result in growth as filaments. These filaments ultimately lyse through a poorly understood mechanism. Here we find that one such ␤-lactam, cephalexin, can, under certain conditions, lead instead to rapid lysis at nascent division sites through a process that requires the complete and ordered assembly of the divisome, the essential machinery involved in cell division. We propose that this assembly process (in which the localization of cell wall hydrolases depends on properly targeted FtsN, which in turn depends on the presence of FtsI) ensures that the biosynthetic machinery to form new septa is in place before the machinery to degrade septated daughter cells is enabled. ␤-lactams that target FtsI subvert this mechanism by inhibiting FtsI without perturbing the normal assembly of the cell division machinery and the consequent activation of cell wall hydrolases. One seemingly paradoxical implication of our results is that ␤-lactam therapy may be improved by promoting active cell division.amidase ͉ cell wall hydrolase ͉ ftsN ͉ penicillin-binding proteins ͉ peptidoglycan

The Kriging method is an interpolation scheme that can be used for modeling deterministic computer analyses as the realization of a stochastic process. The technique has been recognized as an alternative to the traditional Response Surface method in generating approximation models of computationally expensive CFD analyses. This is due to its ability to interpolate sample data and to model a function with multiple local extrema. To fully exploit the advantage of the Kriging method, however, a large number of sample data points should be spread out to fill the design space. This can be very costly and even impractical in high-dimensional design optimization. In this work, the Cokriging method, an extension of Kriging, which can incorporate secondary information such as values of gradients in addition to primary function values of the sample points has been utilized for constructing approximation models in a realistic design optimization process. This approach improves on the accuracy and efficiency of using the Kriging method for highdimensional design problems. Provided that gradient information is available through inexpensive algorithms such as the adjoint method, Cokriging significantly reduces the large computational cost needed for the original Kriging method to accurately capture multiple local extrema of the unknown response function within a relatively large design space. After validating the feasibility of the Cokriging method using simple one-and two-dimensional analytic functions, the approach is applied to the aerodynamic design of a supersonic business jet. The results of these 2-and 5-variable test design problems indicate that great improvements on the efficiency and applicability of the Kriging method in high-dimensional design optimization problems can be achieved.

Compounds inhibiting LpxC in the lipid A biosynthetic pathway are promising leads for novel antibiotics against multidrug-resistant Gram-negative pathogens. We report the syntheses and structural and biochemical characterizations of LpxC inhibitors based on a diphenyl-diacetylene (1,4-diphenyl-1,3-butadiyne) threonylhydroxamate scaffold. These studies provide a molecular interpretation for the differential antibiotic activities of compounds with a substituted distal phenyl ring as well as the absolute stereochemical requirement at the C2, but not C3, position of the threonyl group.

Background:LpxC is an essential enzyme in the biosynthesis of lipid A of the Gram-negative bacterial outer membrane. Results: Mutations that confer resistance to LpxC inhibitors in ThrS slow the cellular growth and in FabZ unexpectedly reduce the cellular LpxC activity. Conclusion: Alteration of fatty acid and protein biosyntheses compensates for impaired lipid A biosynthesis. Significance: Our results reveal an important role of cellular homeostasis in antibiotic resistance.