H333N] was significantly more resistant to both antibiotics than FabB and had an affinity for TLM an order of magnitude less than the wild-type enzyme, illustrating that the two-histidine active site architecture is critical to protein-antibiotic interaction. These data provide a structural framework for understanding antibiotic sensitivity within this group of enzymes.

The sulfonamide antibiotics inhibit dihydropteroate synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. Here, we report structural, computational and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an SN1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active site residues, and reveals how sulfonamide resistance arises.

In Escherichia coli, the expression of fatty acid metabolic genes is controlled by the transcription factor, FadR

The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. These mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containing the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. Using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitorresistant influenza strains.

Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis-targeting chimera (PRO-TAC) approaches,o wingt of avorable drug-like properties of CRBN ligands,i mmunomodulatory imide drugs (IMiDs). However,I MiDs are knownt ob ei nherently unstable,r eadily undergoing hydrolysis in body fluids.H ere we showt hat IMiDs and IMiD-based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy.W ed esigned novel CRBN binders,p henyl glutarimide (PG) analogues,a nd showed that they retained affinity for CRBN with high ligand efficiency (LE > 0.48) and displayed improved chemical stability.O ur efforts led to the discovery of PG PROTAC 4c (SJ995973), au niquely potent degrader of bromodomain and extra-terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4-11 cells at lowp icomolar concentrations (IC 50 = 3pM; BRD4 DC 50 = 0.87 nM). These findings strongly support the utility of PG derivatives in the design of CRBN-directed PROTACs.