Synthesis and Evaluation of diaryl ether modulators of the leukotriene A4 hydrolase aminopeptidase activity.

Activation of the aminopeptidase (AP) activity of leukotriene A4 hydrolase (LTA4H) presents a potential therapeutic strategy for resolving chronic inflammation. Previously, ARM1 and derivatives were found to activate the AP activity using the alanine-p-nitroanilide (Ala-pNA) as a reporter group in an enzyme kinetics assay. As an extension of this previous work, novel ARM1 derivatives were synthesized using a palladium-catalyzed Ullmann coupling reaction and screened using the same assay. Analogue 5, an aminopyrazole (AMP) analogue of ARM1, was found to be a potent AP activator with an AC50 of 0.12 µM. An X-ray crystal structure of LTA4H in complex with AMP was refined at 2.7 Å. Despite its AP activity with Ala-pNA substrate, AMP did not affect hydrolysis of the previously proposed natural ligand of LTA4H, Pro-Gly-Pro (PGP). This result highlights a discrepancy between the hydrolysis of more conveniently monitored chromogenic synthetic peptides typically employed in assays and endogenous peptides. The epoxide hydrolase (EH) activity of AMP was measured in vivo and the compound significantly reduced leukotriene B4 (LTB4) levels in a murine bacterial pneumonia model. However, AMP did not enhance survival in the murine pneumonia model over a 14-day period. A liver microsome stability assay showed metabolic stability of AMP. The results suggested that accelerated Ala-pNA cleavage is not sufficient for predicting therapeutic potential, even when the full mechanism of activation is known.

A Review of Omadacycline for Potential Utility in the Military Health System for the Treatment of Wound Infections.

INTRODUCTION: Combat-related wound infections complicate the recovery of wounded military personnel, contributing to overall morbidity and mortality. Wound infections in combat settings present unique challenges because of the size and depth of the wounds, the need to administer emergency care in the field, and the need for subsequent treatment in military facilities. Given the increase in multidrug-resistant pathogens, a novel, broad-spectrum antibiotic is desired across this continuum of care when the standard of care fails. Omadacycline was FDA-approved in 2018 for treatment of adults with acute bacterial skin and skin structure infections (ABSSSI), as well as community-acquired bacterial pneumonia (CABP). It is a broad-spectrum antibiotic with activity against gram-positive, gram-negative, and atypical bacterial pathogens, including multidrug-resistant species. Omadacycline can overcome commonly reported tetracycline resistance mechanisms, ribosomal protection proteins, and efflux pumps, and is available in once-daily intravenous or oral formulations. In this review, we discuss the potential role of omadacycline, which is included in the Department of Defense Formulary, in the context of combat wound infections. MATERIALS AND METHODS: A literature review was undertaken for manuscripts published before July 21, 2023. This included a series of publications found via PubMed and a bibliography made publicly available on the Paratek Pharmaceuticals, Inc. website. Publications presenting primary data published in English on omadacycline in relation to ESKAPEE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and Enterobacter species) pathogens and Clostridioides difficile, including in vitro, in vivo, and clinical data were included. RESULTS: Of 260 identified records, 66 were included for evidence review. Omadacycline has in vitro activity against almost all the ESKAPEE pathogens, apart from P. aeruginosa. Importantly, it has activity against the four most prevalent bacterial pathogens that cause wound infections in the military healthcare system: S. aureus, including methicillin-resistant S. aureus, A. baumannii, K. pneumoniae, and E. coli. In vivo studies in rats have shown that omadacycline is rapidly distributed in most tissues, with the highest tissue-to-blood concentration ratios in bone mineral. The clinical efficacy of omadacycline has been assessed in three separate Phase 3 studies in patients with ABSSSI (OASIS-1 and OASIS-2) and with CABP (OPTIC). Overall, omadacycline has an established safety profile in the treatment of both ABSSSI and CABP. CONCLUSIONS: Omadacycline has broad-spectrum activity, the option to be orally administered and an established safety profile, making it a potentially attractive replacement for moxifloxacin in the military individual first aid kit, especially when accounting for the increasing resistance to fluoroquinolones. Further studies and clinical evaluation are warranted to support broader use of omadacycline to treat combat wound infections in the military healthcare system.

Substrate-dependent modulation of the leukotriene A4 hydrolase aminopeptidase activity and effect in a murine model of acute lung inflammation.

The aminopeptidase activity (AP) of the leukotriene A4 hydrolase (LTA4H) enzyme has emerged as a therapeutic target to modulate host immunity. Initial reports focused on the benefits of augmenting the LTA4H AP activity and clearing its putative pro-inflammatory substrate Pro-Gly-Pro (PGP). However, recent reports have introduced substantial complexity disconnecting the LTA4H modulator 4-methoxydiphenylmethane (4MDM) from PGP as follows: (1) 4MDM inhibits PGP hydrolysis and subsequently inhibition of LTA4H AP activity, and (2) 4MDM activates the same enzyme target in the presence of alternative substrates. Differential modulation of LTA4H by 4MDM was probed in a murine model of acute lung inflammation, which showed that 4MDM modulates the host neutrophilic response independent of clearing PGP. X-ray crystallography showed that 4MDM and PGP bind at the zinc binding pocket and no allosteric binding was observed. We then determined that 4MDM modulation is not dependent on the allosteric binding of the ligand, but on the N-terminal side chain of the peptide. In conclusion, our study revealed that a peptidase therapeutic target can interact with its substrate and ligand in complex biochemical mechanisms. This raises an important consideration when ligands are designed to explain some of the unpredictable outcomes observed in therapeutic discovery targeting LTA4H.

Characterization and Inhibition of 1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase: A Promising Drug Target in Acinetobacter baumannii and Klebsiella pneumoniae.

The ESKAPE pathogens comprise a group of multidrug-resistant bacteria that are the leading cause of nosocomial infections worldwide. The prevalence of antibiotic resistant strains and the relative ease by which bacteria acquire resistance genes highlight the continual need for the development of novel antibiotics against new drug targets. The methylerythritol phosphate (MEP) pathway is an attractive target for the development of new antibiotics. The MEP pathway governs the synthesis of isoprenoids, which are key lipid precursors for vital cell components such as ubiquinone and bacterial hopanoids. Additionally, the MEP pathway is entirely distinct from the corresponding mammalian pathway, the mevalonic acid (MVA) pathway, making the first committed enzyme of the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC), an attractive target for antibiotic development. To facilitate drug development against two of the ESKAPE pathogens, Acinetobacter baumannii and Klebsiella pneumoniae, we cloned, expressed, purified, and characterized IspC from these two Gram-negative bacteria. Enzyme inhibition assays using IspC from these two pathogens, and compounds fosmidomycin and FR900098, indicate IC50 values ranging from 19.5-45.5 nM. Antimicrobial susceptibility tests with these inhibitors reveal that A. baumannii is susceptible to FR900098, whereas K. pneumoniae is susceptible to both compounds. Finally, to facilitate structure-based drug design of inhibitors targeting A. baumannii IspC, we determined the 2.5 Å crystal structure of IspC from A. baumannii in complex with inhibitor FR900098, and cofactors NADPH and magnesium.

Inhibition of the Yersinia pestis Methylerythritol Phosphate Pathway of Isoprenoid Biosynthesis by α-Phenyl-Substituted Reverse Fosmidomycin Analogues.

Fosmidomycin inhibits IspC (1-deoxy-d-xylulose 5-phosphate reductoisomerase), the first committed enzyme in the methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis. The MEP pathway of isoprenoid biosynthesis is essential to the causative agent of the plague, Yersinia pestis, and is entirely distinct from the corresponding mammalian pathway. To further drug development, we established structure-activity relationships of fosmidomycin analogues by assessing a suite of 17 α-phenyl-substituted reverse derivatives of fosmidomycin against Y. pestis IspC. Several of these compounds showed increased potency over fosmidomycin with IC50 values in the nanomolar range. Additionally, we performed antimicrobial susceptibility testing with Y. pestis A1122 (YpA1122). The bacteria were susceptible to several compounds with minimal inhibitory concentration (MIC) values ranging from 128 to 512 µg/mL; a correlation between the IC50 and MIC values was observed.

Effect of Modifier Structure on the Activation of Leukotriene A4 Hydrolase Aminopeptidase Activity.

Activation of the leukotriene A4 hydrolase (LTA4H) aminopeptidase (AP) activity with 4-methoxydiphenylmethane (4MDM) promoted resolution of neutrophil infiltration in a murine cigarette smoke-induced model for emphysematous chronic obstructive pulmonary disease. Recently, 4-(4-benzylphenyl)thiazol-2-amine (ARM1) was published as a ligand for LTA4H with potential anti-inflammatory properties. To investigate the effect of modifier structure on enzyme kinetics of LTA4H, a series of analogues bearing structural features of ARM1 and 4MDM were synthesized using trifluoroborate Suzuki coupling reactions. Following, the 2.8 Å X-ray crystal structure of LTA4H complexed with 4-OMe-ARM1, a 4MDM-ARM1 hybrid molecule, was determined. Kinetic analysis showed that ARM1 and related analogues lowered affinity for the enzyme-substrate complex, resulting in a change of mechanism from hyperbolic mixed predominately catalytic activation (HMx(Sp < Ca)A) as observed for 4MDM to a predominately specific activation (HMx(Sp > Ca)A) mechanism. 4-OMe-ARM1 was then shown to dose responsively reduce LTB4 production in human neutrophils.

Discovery of novel inhibitors of multidrug-resistant Acinetobacter baumannii.

The recent emergence of multidrug-resistant Acinetobacter baumannii strains and the non-efficacy of currently available antibiotics against such infections have led to an urgent need for the development of novel antibacterials. In an effort to address this problem, we have identified three novel inhibitors, namely, D5, D12 and D6 using in silico screening with a homology model of the outer membrane protein W2 (OmpW2) from A. baumannii, as the proposed new drug target. OmpW is an eight-stranded ß-barrel protein involved in the transport of hydrophobic molecules across the outer membrane and maintenance of homeostasis under cellular stress. The antimicrobial activities of compounds D5, D12 and D6 were evaluated against a panel of clinical isolates of A. baumannii strains. These compounds inhibited the growth of the strains with minimum inhibitory concentration (MIC) ranges of 1-32µg/mL. Time-kill kinetic studies with the highly virulent and multidrug-resistant strain, A. baumannii 5075, indicated that D6 exhibited the highest bactericidal activity asa≥3log10 CFU/mL (99.9%) reduction in colony count from the initial inoculum was observed after 30min incubation. D5 and D12 reduced at least 1log10 CFU/mL (90%) of the initial inoculum after 24h. In conclusion, these three lead inhibitors have provided two distinct chemical scaffolds for further analog design and optimizations, using chemical synthesis, to develop more potent inhibitors of the pathogen.

Identification of catalytic cysteine, histidine, and lysine residues in Escherichia coli homoserine transsuccinylase.

Homoserine transsuccinylase catalyzes the succinylation of homoserine in several bacterial species, the first unique step in methionine biosynthesis in these organisms. The enzyme from Escherichia coli is reported to be a dimer and uses a ping-pong catalytic mechanism involving transfer of succinate from succinyl-CoA to an enzyme nucleophile, followed by transfer to homoserine to form O-succinylhomoserine. Site-directed mutagenesis and steady-state kinetics were used to identify three amino acids that participate in catalysis. Mutation of cysteine-142 to serine or alanine eliminated all measurable activity, suggesting this amino acid acts as the catalytic nucleophile. Cysteine nucleophiles are often deprotonated by histidine residues, and histidine-235 was identified as the sole absolutely conserved histidine residue among family members. This residue was mutated to both alanine and asparagine, and no activity was observed with either mutant. Lysine-47 had been previously identified as an essential residue. Mutation of this amino acid to arginine reduced catalytic activity by greater than 90%, while mutation to alanine yielded an enzyme with <1% of wild-type activity. A pH-rate profile of the K47R mutant demonstrated that this amino acid participates in the first half reaction. The data presented here provide the first detailed description of the homoserine transsuccinylase active site and provide a framework for additional mechanistic characterization of this enzyme.

Human PXR forms a tryptophan zipper-mediated homodimer.

The human nuclear receptor pregnane X receptor (PXR) responds to a wide variety of potentially harmful chemicals and coordinates the expression of genes central to xenobiotic and endobiotic metabolism. Structural studies reveal that the PXR ligand binding domain (LBD) uses a novel sequence insert to form a homodimer unique to the nuclear receptor superfamily. Terminal beta-strands from each monomeric LBD interact in an ideal antiparallel fashion to bury potentially exposed surface beta-strands, generating a 10-stranded intermolecular beta-sheet. Conserved tryptophan and tyrosine residues lock across the dimer interface and provide the first tryptophan-zipper (Trp-Zip) interaction observed in a native protein. We show using analytical ultracentrifugation that the PXR LBD forms a homodimer in solution. We further find that removal of the interlocking aromatic residues eliminates dimer formation but does not affect PXR's ability to interact with DNA, RXRalpha, or ligands. Disruption of the homodimer significantly reduces receptor activity in transient transfection experiments, however, and effectively eliminates the receptor's recruitment of the transcriptional coactivator SRC-1 both in vitro and in vivo. Taken together, these results suggest that the unique Trp-Zip-mediated PXR homodimer plays a role in the function of this nuclear xenobiotic receptor.

2.1 A crystal structure of human PXR in complex with the St. John's wort compound hyperforin.

The nuclear xenobiotic receptor PXR is activated by a wide variety of clinically used drugs and serves as a master regulator of drug metabolism and excretion gene expression in mammals. St. John's wort is used widely in Europe and the United States to treat depression. This unregulated herbal remedy leads to dangerous drug-drug interactions, however, in patients taking oral contraceptives, antivirals, or immunosuppressants. Such interactions are caused by the activation of the human PXR by hyperforin, the psychoactive agent in St. John's wort. In this study, we show that hyperforin induces the expression of numerous drug metabolism and excretion genes in primary human hepatocytes. We present the 2.1 A crystal structure of hyperforin in complex with the ligand binding domain of human PXR. Hyperforin induces conformational changes in PXR's ligand binding pocket relative to structures of human PXR elucidated previously and increases the size of the pocket by 250 A(3). We find that the mutation of individual aromatic residues within the ligand binding cavity changes PXR's response to particular ligands. Taken together, these results demonstrate that PXR employs structural flexibility to expand the chemical space it samples and that the mutation of specific residues within the ligand binding pocket of PXR tunes the receptor's response to ligands.

Effects of molecular crowding by saccharides on alpha-chymotrypsin dimerization.

Given the importance of protein complexes as therapeutic targets, it is necessary to understand the physical chemistry of these interactions under the crowded conditions that exist in cells. We have used sedimentation equilibrium to quantify the enhancement of the reversible homodimerization of alpha-chymotrypsin by high concentrations of the osmolytes glucose, sucrose, and raffinose. In an attempt to rationalize the osmolyte-mediated stabilization of the alpha-chymotrypsin homodimer, we have used models based on binding interactions (transfer-free energy analysis) and steric interactions (excluded volume theory) to predict the stabilization. Although transfer-free energy analysis predicts reasonably well the relatively small stabilization observed for complex formation between cytochrome c and cytochrome c peroxidase, as well as that between bobtail quail lysozyme and a monoclonal Fab fragment, it underestimates the sugar-mediated stabilization of the alpha-chymotrypsin dimer. Although predictions based on excluded volume theory overestimate the stabilization, it would seem that a major determinant in the observed stabilization of the alpha-chymotrypsin homodimer is the thermodynamic nonideality arising from molecular crowding by the three small sugars.

Structural insights into the promiscuity and function of the human pregnane X receptor.

The pregnane X receptor (PXR) is a promiscuous nuclear receptor that responds to a wide variety of drugs, xenobiotics and endogenous compounds, and plays a critical role in mediating drug-drug interactions in humans. PXR is the master regulator of the expression of the CYP3A4 gene, which encodes for the most abundant and promiscuous drug-metabolizing enzyme in humans. PXR also regulates the expression of other genes involved in xenobiotic metabolism, including CYP2C8, CYP2C9, CYP2B6, GSTA2 and MDR1, as well as genes critical to bile acid metabolism. While PXR functions as a xenobiotic sensor in numerous vertebrates, its relatively low sequence conservation across species causes the PXRs from different organisms to respond to distinct subsets of xenobiotics. Thus, PXR promiscuity is directed and not random. The recent determination of crystal structures of the ligand binding domain of human PXR has provided the first detailed molecular view of this promiscuous receptor, and has advanced our understanding of its varied biological functions. We review the evidence establishing the binding promiscuity of PXR and its directed specificity in different species, and analyze the structural determinants of these characteristics. In addition, we examine the relationship between the interaction of PXR with ligands and the manner in which CYP3A4 is thought to bind to substrate molecules. The accumulating structural and functional data on PXR may facilitate the development of improved methods for in vitro, in vivo and in silico screening for PXR activation.