Synthesis of GNAT PA3944 Substrate Analogs

The Gcn5-related N-acetyltransferase (GNAT) superfamily is responsible for diverse biological functions and is critically important in cellular and metabolic processes in all kingdoms of life. GNATs transfer an acetyl-group from an active donor, typically acetyl-coenzyme A (AcCoA), to a primary amine of an acceptor substrate. Members of this family are well known for their roles in aminoglycoside antibiotic resistance, histone modification, protein acetylation, xenobiotic metabolism, and other cellular processes.1, 2 A small subset of bacterial GNAT enzymes have been studied and characterized both structurally and functionally, but the function of the vast majority remains unknown. Most of the reported 3D crystallographic structures of GNATs contain no acceptor substrate bound in their active sites. We previously screened the PA3944 protein against a panel of potential substrates and found the enzyme exhibited the highest activity toward aspartame, polymyxin B and colistin (polymyxin E).3 Our project involves the synthesis of molecular analogs of previously identified functionally relevant acceptor substrates that will be co-crystallized with GNAT-PA3944, in particular simplified derivatives of polymyxin B including NANMO and AAB, and we have shown that NANMO is as efficient as polymyxin B as a substrate. The ligand-bound crystallographic structures will provide insight into the structural features of the active site that are involved in substrate recognition and advance our understanding of types of substrates recognized by this enzyme of unknown function. Syntheses of NANMO and AAB will be described, along with modeling and substrate efficiency

In Silico Binding of 2-Aminocyclobutanones to SARS-CoV-2 Nsp13 Helicase and Demonstration of Antiviral Activity

The landscape of viral strains and lineages of SARS-CoV-2 keeps changing and is currently dominated by Delta and Omicron variants. Members of the latest Omicron variants, including BA.1, are showing a high level of immune evasion, and Omicron has become a prominent variant circulating globally. In our search for versatile medicinal chemistry scaffolds, we prepared a library of substituted α-aminocyclobutanones from an α-aminocyclobutanone synthon (11). We performed an in silico screen of this actual chemical library as well as other virtual 2-aminocyclobutanone analogs against seven SARS-CoV-2 nonstructural proteins to identify potential drug leads against SARS-CoV-2, and more broadly against coronavirus antiviral targets. Several of these analogs were initially identified as in silico hits against SARS-CoV-2 nonstructural protein 13 (Nsp13) helicase through molecular docking and dynamics simulations. Antiviral activity of the original hits as well as α-aminocyclobutanone analogs that were predicted to bind more tightly to SARS-CoV-2 Nsp13 helicase are reported. We now report cyclobutanone derivatives that exhibit anti-SARS-CoV-2 activity. Furthermore, the Nsp13 helicase enzyme has been the target of relatively few target-based drug discovery efforts, in part due to a very late release of a high-resolution structure accompanied by a limited understanding of its protein biochemistry. In general, antiviral agents initially efficacious against wild-type SARS-CoV-2 strains have lower activities against variants due to heavy viral loads and greater turnover rates, but the inhibitors we are reporting have higher activities against the later variants than the wild-type (10–20X). We speculate this could be due to Nsp13 helicase being a critical bottleneck in faster replication rates of the new variants, so targeting this enzyme affects these variants to an even greater extent. This work calls attention to cyclobutanones as a useful medicinal chemistry scaffold, and the need for additional focus on the discovery of Nsp13 helicase inhibitors to combat the aggressive and immune-evading variants of concern (VOCs)

Cyclobutanone Inhibitor of Cobalt-Functionalized Metallo-γ-Lactonase AiiA with Cyclobutanone Ring Opening in the Active Site

An α-amido cyclobutanone possessing a C10 hydrocarbon tail was designed as a potential transition-state mimetic for the quorum-quenching metallo-γ-lactonase autoinducer inactivator A (AiiA) with the support of in-house modeling techniques and found to be a competitive inhibitor of dicobalt(II) AiiA with an inhibition constant of Ki = 0.007 ± 0.002 mM. The catalytic mechanism of AiiA was further explored using our product-based transition-state modeling (PBTSM) computational approach, providing substrate-intermediate models arising during enzyme turnover and further insight into substrate–enzyme interactions governing native substrate catalysis. These interactions were targeted in the docking of cyclobutanone hydrates into the active site of AiiA. The X-ray crystal structure of dicobalt(II) AiiA cocrystallized with this cyclobutanone inhibitor unexpectedly revealed an N-(2-oxocyclobutyl)decanamide ring-opened acyclic product bound to the enzyme active site (PDB 7L5F). The C10 alkyl chain and its interaction with the hydrophobic phenylalanine clamp region of AiiA adjacent to the active site enabled atomic placement of the ligand atoms, including the C10 alkyl chain. A mechanistic hypothesis for the ring opening is proposed involving a radical-mediated process

Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too

Enzymes in the Gcn5-related N-acetyltransferase (GNAT) superfamily are widespread and critically involved in multiple cellular processes ranging from antibiotic resistance to histone modification. While acetyl transfer is the most widely catalyzed reaction, recent studies have revealed that these enzymes are also capable of performing succinylation, condensation, decarboxylation, and methylcarbamoylation reactions. The canonical chemical mechanism attributed to GNATs is a general acid/base mechanism; however, mounting evidence has cast doubt on the applicability of this mechanism to all GNATs. This study shows that the Pseudomonas aeruginosa PA3944 enzyme uses a nucleophilic serine residue and a hybrid ping-pong mechanism for catalysis instead of a general acid/base mechanism. To simplify this enzyme’s kinetic characterization, we synthesized a polymyxin B substrate analog and performed molecular docking experiments. We performed site-directed mutagenesis of key active site residues (S148 and E102) and determined the structure of the E102A mutant. We found that the serine residue is essential for catalysis toward the synthetic substrate analog and polymyxin B, but the glutamate residue is more likely important for substrate recognition or stabilization. Our results challenge the current paradigm of GNAT mechanisms and show that this common enzyme scaffold utilizes different active site residues to accomplish a diversity of catalytic reactions

Synthesis of a Protected 2-Aminocyclobutanone as a Modular Transition State Synthon for Medicinal Chemistry

The hydrochloride salt of ɑ-aminocyclobutanone protected as its dimethyl acetal 2,2-dimethoxycyclobutan-1-aminium chloride (3) has been prepared as a modular synthon for convenient access to cyclobutanone-containing lead inhibitors of hydrolase enzymes including serine proteases and metalloproteases. Protected ɑ-aminocyclobutanone 3 was converted to representative amide and sulfonamide-functionalized 2-aminocyclobutanone derivatives. Reaction of the amino acetal 3 with phenyl isothiocyanate afforded the bicyclic urea 1-hydroxyl-2,4-diazabicyclo[3.2.0]heptane-3-thione (9) as confirmed by a single crystal X-ray structure

Atomic-Resolution 1.3 Å Crystal Structure, Inhibition by Sulfate, and Molecular Dynamics of the Bacterial Enzyme DapE

We report the atomic-resolution (1.3 Å) X-ray crystal structure of an open conformation of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE, EC 3.5.1.18) from Neisseria meningitidis. This structure [Protein Data Bank (PDB) entry 5UEJ] contains two bound sulfate ions in the active site that mimic the binding of the terminal carboxylates of the N-succinyl-l,l-diaminopimelic acid (l,l-SDAP) substrate. We demonstrated inhibition of DapE by sulfate (IC50 = 13.8 ± 2.8 mM). Comparison with other DapE structures in the PDB demonstrates the flexibility of the interdomain connections of this protein. This high-resolution structure was then utilized as the starting point for targeted molecular dynamics experiments revealing the conformational change from the open form to the closed form that occurs when DapE binds l,l-SDAP and cleaves the amide bond. These simulations demonstrated closure from the open to the closed conformation, the change in RMS throughout the closure, and the independence in the movement of the two DapE subunits. This conformational change occurred in two phases with the catalytic domains moving toward the dimerization domains first, followed by a rotation of catalytic domains relative to the dimerization domains. Although there were no targeting forces, the substrate moved closer to the active site and bound more tightly during the closure event