Investigating Novel Methods of Interaction with Pharmaceutically Relevant Enzymes

Metalloproteins requiring one or more metal ions for normal function make up 30% of all known proteins, and many critical biological pathways contain at least one metallo-enzyme. Di-nuclear metallo-proteins constitute a large class of these proteins yet we currently lack effective methods of inhibiting these enzymes for the development of new medical therapies, particularly for the discovery of new antibiotics. Our work has focused on developing novel functionalities that selectively interact with di-nuclear catalytic centers, and we are targeting three separate di-zinc-metallo-enzymes that are unique to bacteria and play key roles in their growth and development. These enzymes are DapE, AiiA, and NDM-1. DapE is involved in biosynthesis of lysine and meso-diaminopimelic acid, essential precursors in the production of bacterial cell walls. AiiA is a di-Zn-dependent lactonase involved in bacterial cell-cell communication, and NDM-1 is a di-metallo-beta-lactamase capable of deactivating the most commonly administered antibiotics, gaining international attention for this enzyme as a clinically-relevant pharmaceutical target, yet drug development efforts have proven ineffective due to a lack of effective inhibitors. As part of our ongoing studies to functionally annotate the Gcn5-related N-acetyltransferase (GNAT) PA4794 from Pseudomonas aeruginosa with unknown functions, we have used PA4794 as a model system for exploring efficient formation of bisubstrate complexes to enhance our success rate in obtaining co-crystal structures of GNATs with ligands bound in their acceptor sites. We have synthesized and tested substrate analogs of the previously identified N-phenylacetyl glycine lysine (NPAcGK) enabling two separate three-dimensional structures of PA4794 with NPAcGK analog-derived bisubstrates formed through direct reaction with CoA—the first through direct alkylation with a reactive substrate, and the second through X-ray induced radical-mediated process. We have also performed docking and molecular dynamics simulations of the reverse reaction pathway from the NPAcGK product back to formation of the tetrahedral intermediate/transition state to complement our structural work and to explore the key ligand-protein interactions within the active site of PA4794, guiding mutant synthesis and kinetics to explore the role of key residues in the active site

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

Indoline‐6‐Sulfonamide Inhibitors of the Bacterial Enzyme DapE

Inhibitors of the bacterial enzyme dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18) hold promise as antibiotics with a new mechanism of action. Herein we describe the discovery of a new series of indoline sulfonamide DapE inhibitors from a high-throughput screen and the synthesis of a series of analogs. Inhibitory potency was measured by a ninhydrin-based DapE assay recently developed by our group. Molecular docking experiments suggest active site binding with the sulfonamide acting as a zinc-binding group (ZBG)

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

First Contact: 7-Phenyl-2-Aminoquinolines, Potent and Selective Neuronal Nitric Oxide Synthase Inhibitors That Target an Isoform-Specific Aspartate.

Inhibition of neuronal nitric oxide synthase (nNOS), an enzyme implicated in neurodegenerative disorders, is an attractive strategy for treating or preventing these diseases. We previously developed several classes of 2-aminoquinoline-based nNOS inhibitors, but these compounds had drawbacks including off-target promiscuity, low activity against human nNOS, and only modest selectivity for nNOS over related enzymes. In this study, we synthesized new nNOS inhibitors based on 7-phenyl-2-aminoquinoline and assayed them against rat and human nNOS, human eNOS, and murine and (in some cases) human iNOS. Compounds with a meta-relationship between the aminoquinoline and a positively charged tail moiety were potent and had up to nearly 900-fold selectivity for human nNOS over human eNOS. X-ray crystallography indicates that the amino groups of some compounds occupy a water-filled pocket surrounding an nNOS-specific aspartate residue (absent in eNOS). This interaction was confirmed by mutagenesis studies, making 7-phenyl-2-aminoquinolines the first aminoquinolines to interact with this residue

Practical spectrophotometric assay for the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase, a potential antibiotic target.

A new enzymatic assay for the bacterial enzyme succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) is described. This assay employs N6-methyl-N2-succinyl-L,L-diaminopimelic acid (N6-methyl-L,L-SDAP) as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N6-methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono-N6-methylated-L,L-SDAP as an alternate substrate for the assay, given binding in the active site of DapE predicted to be comparable to the endogenous substrate. The alternate substrate for the assay, N6-methyl-L,L-SDAP, was synthesized from the tert-butyl ester of Boc-L-glutamic acid employing a Horner-Wadsworth-Emmons olefination followed by an enantioselective reduction employing Rh(I)(COD)(S,S)-Et-DuPHOS as the chiral catalyst. Validation of the new ninhydrin assay was demonstrated with known inhibitors of DapE from Haemophilus influenza (HiDapE) including captopril (IC50 = 3.4 [± 0.2] μM, 3-mercaptobenzoic acid (IC50 = 21.8 [±2.2] μM, phenylboronic acid (IC50 = 316 [± 23.6] μM, and 2-thiopheneboronic acid (IC50 = 111 [± 16] μM. Based on these data, this assay is simple and robust, and should be amenable to high-throughput screening, which is an important step forward as it opens the door to medicinal chemistry efforts toward the discovery of DapE inhibitors that can function as a new class of antibiotics

“Click” Chemistry-Mediated Phenylene-Cored Carborane Dendrimers

Phenylene-cored small dendrimers containing three to nine peripheral <i>o</i>-carborane clusters were synthesized via Cu­(I)-catalyzed Huisgen-type azide alkyne cycloaddition reactions. The resulting dendrimers have been characterized by IR and NMR spectroscopy and MALDI-TOF mass spectral analysis. The biological evaluation of branched dendrimer <b>16</b> containing nine carborane cages has been carried out using human liver cancer cells (SK-Hep1). Dendrimer <b>16</b> was accumulated in the SK-Hep1 cancer cells in a concentration-dependent manner. The highest boron accumulation up to 2540 ng of boron/5 × 10⁵ cells was observed at a 50 μM concentration of <b>16</b> over a period of 20 h. The high accumulation of <b>16</b> into the tumor cell lines indicates that such dendritic boron drug delivery platforms could be possible for application in boron neutron capture therapy in cancer treatment

Circular dichroism thermal denaturation study of <i>Hi</i>DapE.

<p>The α-helical structures are represented in red and β-sheets are represented in blue with (A) percent secondary structure observed over the course of heating from 20–80 <i>°</i>C, and (B) percent secondary structure remaining with continued heating at 80 <i>°</i>C.</p

Asymmetric synthesis of <i>N</i>⁶-methyl-L,L-SDAP 1b.

<p>Synthetic route for preparation of monomethyl substrate analog as the hydrochloride salt (<b>1b.HCl</b>) via the methyl ester or the trifluoroacetate salt (<b>1b.TFA</b>) via the benzyl ester.</p