UDP‐3‐O‐(R‐3‐hydroxymyristoyl)‐N‐acetylglucosamine deacetylase (LpxC) is a Zn2+ deacetylase that is essential for the survival of most pathogenic Gram‐negative bacteria. ACHN‐975 (N‐((S)‐3‐amino‐1‐(hydroxyamino)‐3‐methyl‐1‐oxobutan‐2‐yl)‐4‐(((1R,2R)‐2‐(hydroxymethyl)cyclopropyl)buta‐1,3‐diyn‐1‐yl)benzamide) was the first LpxC inhibitor to reach human clinical testing and was discovered to have a dose‐limiting cardiovascular toxicity of transient hypotension without compensatory tachycardia. Herein we report the effort beyond ACHN‐975 to discover LpxC inhibitors optimized for enzyme potency, antibacterial activity, pharmacokinetics, and cardiovascular safety. Based on its overall profile, compound 26 (LPXC‐516, (S)‐N‐(2‐(hydroxyamino)‐1‐(3‐methoxy‐1,1‐dioxidothietan‐3‐yl)‐2‐oxoethyl)‐4‐(6‐hydroxyhexa‐1,3‐diyn‐1‐yl)benzamide) was chosen for further development. A phosphate prodrug of 26 was developed that provided a solubility of >30 mg mL−1 for parenteral administration and conversion into the active drug with a t1/2 of approximately two minutes. Unexpectedly, and despite our optimization efforts, the prodrug of 26 still possesses a therapeutic window insufficient to support further clinical development.

Modulating disease-relevant protein–protein interactions (PPIs) using pharmacological tools is a critical step toward the design of novel therapeutic strategies. Over the years, however, targeting PPIs has proven a very challenging task owing to the large interfacial areas. Our recent efforts identified possible novel routes for the design of potent and selective inhibitors of PPIs using a structure-based design of covalent inhibitors targeting Lys residues. In this present study, we report on the design, synthesis, and characterizations of the first Lys-covalent BH3 peptide that has a remarkable affinity and selectivity for hMcl-1 over the closely related hBfl-1 protein. Our structural studies, aided by X-ray crystallography, provide atomic-level details of the inhibitor interactions that can be used to further translate these discoveries into novel generation, Lys-covalent pro-apoptotic agents.

Human Cellular Retinol Binding Protein II (hCRBPII), a member of the intracellular Lipid binding protein family, is a monomeric protein responsible for the intracellular transport of retinol and retinal. Herein we report that hCRBPII forms an extensive domain swapped dimer during bacterial expression. The domain swapped region encompasses almost half of the protein. The dimer represents a novel structural architecture with the mouths of the two binding cavities facing each other, producing a new binding cavity that spans the length of the protein complex. Though wild-type hCRBPII forms the dimer, the propensity for dimerization can be substantially increased via mutation at Tyr60. The monomeric form of the wild type protein represents the thermodynamically more stable species, making the domain swapped dimer a kinetically trapped entity. Hypothetically, the wild type protein has evolved to minimize dimerization of the folding intermediate through a critical hydrogen bond (Tyr60-Glu72) that disfavors the dimeric form.

Pancreatic cancer cells are characterized by deregulated metabolic programs that facilitate growth and resistance to oxidative stress. Among these programs, pancreatic cancers preferentially utilize a metabolic pathway through the enzyme aspartate aminotransferase 1 [also known as glutamate oxaloacetate transaminase 1 (GOT1)] to support cellular redox homeostasis. As such, small molecule inhibitors that target GOT1 could serve as starting points for the development of new therapies for pancreatic cancer. We ran a high-throughput screen for inhibitors of GOT1 and identified a small molecule, iGOT1-01, with in vitro GOT1 inhibitor activity. Application in pancreatic cancer cells revealed metabolic and growth inhibitory activity reflecting a promiscuous inhibitory profile. We then performed an in silico docking analysis to study inhibitor—GOT1 interactions with iGOT1-01 analogues that possess improved solubility and potency properties. These results suggested that the GOT1 inhibitor competed for binding to the pyridoxal 5-phosphate (PLP) cofactor site of GOT1. To analyze how the GOT1 inhibitor bound to GOT1, a series of GOT1 mutant enzymes that abolished PLP binding were generated. Application of the mutants in X-ray crystallography and thermal shift assays again suggested but were unable to formally conclude that the GOT1 inhibitor bound to the PLP site. Mutational studies revealed the relationship between PLP binding and the thermal stability of GOT1 while highlighting the essential nature of several residues for GOT1 catalytic activity. Insight into the mode of action of GOT1 inhibitors may provide leads to the development of drugs that target redox balance in pancreatic cancer.

Protein conformational switches or allosteric proteins play a key role in the regulation of many essential biological pathways. Nonetheless, the implementation of protein conformational switches in protein design applications has proven challenging, with only a few known examples that are not derivatives of naturally occurring allosteric systems. We have discovered that the domainswapped (DS) dimer of hCRBPII undergoes a large and robust conformational change upon retinal binding, making it a potentially powerful template for the design of protein conformational switches. Atomic resolution structures of the apo-and holo-forms illuminate a simple, mechanical movement involving sterically driven torsion angle flipping of two residues that drive the motion. We further demonstrate that the conformational "readout" can be altered by addition of crossdomain disulfide bonds, also visualized at atomic resolution. Finally, as a proof of principle, we have created an allosteric metal binding site in the DS dimer, where ligand binding results in a reversible 5-fold loss of metal binding affinity. The high resolution structure of the metal-bound variant illustrates a well-formed metal binding site at the interface of the two domains of the DS dimer and confirms the design strategy for allosteric regulation.