Discordant Effects of Putative Lysine Acetyltransferase Inhibitors in Biochemical and Living Systems.

Lysine acetyltransferases (KATs) are exquisitely fine-tuned to target specific lysine residues on many proteins, including histones, with aberrant acetylation at distinct lysines implicated in different pathologies. However, researchers face a lack of molecular tools to probe the importance of site-specific acetylation events in vivo. Because of this, there can be a disconnect between the predicted in silico or in vitro effects of a drug and the actual observable in vivo response. We have previously reported on how an in vitro biochemical analysis of the site-specific effects of the compound C646 in combination with the KAT p300 can accurately predict changes in histone acetylation induced by the same compound in cells. Here, we build on this effort by further analyzing a number of reported p300 modulators, while also extending the analysis to correlate the effects of these drugs to developmental and phenotypical changes, utilizing cellular and zebrafish model systems. While this study demonstrates the utility of biochemical models as a starting point for predicting in vivo activity of multi-site targeting KATs, it also highlights the need for the development of new enzyme inhibitors that are more specific to the regulation of KAT activity in vivo.

The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor.

Neurotransmitter:sodium symporters (NSSs) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. Results from many structural, functional, and computational studies on LeuT, a bacterial NSS homolog, have provided critical insight into the mechanism of sodium-coupled transport, but the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of molecular dynamics simulations, single-molecule fluorescence resonance energy transfer (smFRET) imaging, and measurements of Na+ binding and substrate transport that reveals an allosteric substrate specificity mechanism. In this mechanism, residues F259 and I359 in the substrate binding pocket couple the binding of substrate to Na+ release from the Na2 site by allosterically modulating the stability of a partially open, inward-facing state. We propose a model for transport selectivity in which residues F259 and I359 act as a volumetric sensor that inhibits the transport of bulky amino acids.

Synthesis, kinetics and inhibition of Escherichia coli Heptosyltransferase I by monosaccharide analogues of Lipid A.

Gram-negative bacteria comprise the majority of microbes that cause infections that are resistant to pre-existing antibiotics. The complex cell wall architecture contributes to their ability to form biofilms, which are often implicated in hospital-acquired infections. Biofilms promote antibiotic resistance by enabling the bacteria to survive hostile environments such as UV radiation, pH shifts, and antibiotics. The outer membrane of Gram-negative bacteria contains lipopolysaccharide (LPS), which plays a role in adhesion to surfaces and formation of biofilms. The main focus of this work was the synthesis of a library of glycolipids designed to be simplified analogues of the Lipid A, the membrane embedded portion component of LPS, to be tested as substrates or inhibitors of Heptosyltransferase I (HepI or WaaC, a glycosyltransferase enzyme involved in the biosynthesis of LPS). Fourteen analogues were synthesized successfully and characterized. While these compounds were designed to function as nucleophilic substrates of HepI, they all demonstrated mild inhibition of HepI. Kinetic characterization of inhibition mechanism identified that the compounds exhibited uncompetitive and mixed inhibition of HepI. Since both uncompetitive and mixed inhibition result in the formation of an Enzyme-Substrate-inhibitor complex, molecular docking studies (using AutoDock Vina) were performed, to identify potential allosteric binding site for these compounds. The inhibitors were shown to bind to a pocket formed after undergoing a conformational change from an open to a closed active site state. Inhibition of HepI via an allosteric site suggest that disruption of protein dynamics might be a viable mechanism for the inhibition of HepI and potentially other enzymes of the GT-B structural class.

The Stories Tryptophans Tell: Exploring Protein Dynamics of Heptosyltransferase I from Escherichia coli.

Heptosyltransferase I (HepI) catalyzes the addition of l-glycero-ß-d-manno-heptose to Kdo2-Lipid A, as part of the biosynthesis of the core region of lipopolysaccharide (LPS). Gram-negative bacteria with gene knockouts of HepI have reduced virulence and enhanced susceptibility to hydrophobic antibiotics, making the design of inhibitors of HepI of interest. Because HepI protein dynamics are partially rate-limiting, disruption of protein dynamics might provide a new strategy for inhibiting HepI. Discerning the global mechanism of HepI is anticipated to aid development of inhibitors of LPS biosynthesis. Herein, dynamic protein rearrangements involved in the HepI catalytic cycle were probed by combining mutagenesis with intrinsic tryptophan fluorescence and circular dichroism analyses. Using wild-type and mutant forms of HepI, multiple dynamic regions were identified via changes in Trp fluorescence. Interestingly, Trp residues (Trp199 and Trp217) in the C-terminal domain (which binds ADP-heptose) are in a more hydrophobic environment upon binding of ODLA to the N-terminal domain. These residues are adjacent to the ADP-heptose binding site (with Trp217 in van der Waals contact with the adenine ring of ADP-heptose), suggesting that the two binding sites interact to report on the occupancy state of the enzyme. ODLA binding was also accompanied by a significant stabilization of HepI (heating to 95 °C fails to denature the protein when it is in the presence of ODLA). These results suggest that conformational rearrangements, from an induced fit model of substrate binding to HepI, are important for catalysis, and the disruption of these conformational dynamics may serve as a novel mechanism for inhibiting this and other glycosyltransferase enzymes.