Small Molecule Inhibitors of Metabolic Enzymes Repurposed as a New Class of Anthelmintics.

The enormous prevalence of infections caused by parasitic nematodes worldwide, coupled to the rapid emergence of their resistance to commonly used anthelmintic drugs, presents an urgent need for the discovery of new drugs. Herein, we have identified several classes of small molecules with broad spectrum activity against these pathogens. Previously, we reported the identification of carnitine palmitoyltransferases (CPTs) as a representative class of enzymes as potential targets for metabolic chokepoint intervention that was elucidated from a combination of chemogenomic screening and experimental testing in nematodes. Expanding on these previous findings, we have discovered that several chemical classes of known small molecule inhibitors of mammalian CPTs have potent activity as anthelmintics. Cross-clade efficacy against a broad spectrum of adult parasitic nematodes was demonstrated for multiple compounds from different series. Several analogs of these initial hit compounds were designed and synthesized. The compounds we report represent a good starting point for further lead identification and optimization for development of new anthelmintic drugs with broad spectrum activity and a novel mechanism of action.

Structure-based discovery of glycomimetic FmlH ligands as inhibitors of bacterial adhesion during urinary tract infection.

Treatment of bacterial infections is becoming a serious clinical challenge due to the global dissemination of multidrug antibiotic resistance, necessitating the search for alternative treatments to disarm the virulence mechanisms underlying these infections. Uropathogenic Escherichia coli (UPEC) employs multiple chaperone-usher pathway pili tipped with adhesins with diverse receptor specificities to colonize various host tissues and habitats. For example, UPEC F9 pili specifically bind galactose or N-acetylgalactosamine epitopes on the kidney and inflamed bladder. Using X-ray structure-guided methods, virtual screening, and multiplex ELISA arrays, we rationally designed aryl galactosides and N-acetylgalactosaminosides that inhibit the F9 pilus adhesin FmlH. The lead compound, 29ß-NAc, is a biphenyl N-acetyl-ß-galactosaminoside with a Ki of ∼90 nM, representing a major advancement in potency relative to the characteristically weak nature of most carbohydrate-lectin interactions. 29ß-NAc binds tightly to FmlH by engaging the residues Y46 through edge-to-face π-stacking with its A-phenyl ring, R142 in a salt-bridge interaction with its carboxylate group, and K132 through water-mediated hydrogen bonding with its N-acetyl group. Administration of 29ß-NAc in a mouse urinary tract infection (UTI) model significantly reduced bladder and kidney bacterial burdens, and coadministration of 29ß-NAc and mannoside 4Z269, which targets the type 1 pilus adhesin FimH, resulted in greater elimination of bacteria from the urinary tract than either compound alone. Moreover, FmlH specifically binds healthy human kidney tissue in a 29ß-NAc-inhibitable manner, suggesting a key role for F9 pili in human kidney colonization. Thus, these glycoside antagonists of FmlH represent a rational antivirulence strategy for UPEC-mediated UTI treatment.

Inhibitors of HGFA, Matriptase, and Hepsin Serine Proteases: A Nonkinase Strategy to Block Cell Signaling in Cancer.

Hepatocyte growth factor activators (HGFA), matriptase, and hepsin are S1 family trypsin-like serine proteases. These proteases proteolytically cleave the single-chain zymogen precursors, pro-HGF (hepatocyte growth factor), and pro-MSP (macrophage stimulating protein) into active heterodimeric forms. HGF and MSP are activating ligands for the oncogenic receptor tyrosine kinases (RTKs), c-MET and RON, respectively. We have discovered the first substrate-based ketothiazole inhibitors of HGFA, matriptase and hepsin. The compounds were synthesized using a combination of solution and solid-phase peptide synthesis (SPPS). Compounds were tested for protease inhibition using a kinetic enzyme assay employing fluorogenic peptide substrates. Highlighted HGFA inhibitors are Ac-KRLR-kt (5g), Ac-SKFR-kt (6c), and Ac-SWLR-kt (6g) with K is = 12, 57, and 63 nM, respectively. We demonstrated that inhibitors block the conversion of native pro-HGF and pro-MSP by HGFA with equivalent potency. Finally, we show that inhibition causes a dose-dependent decrease of c-MET signaling in MDA-MB-231 breast cancer cells. This preliminary investigation provides evidence that HGFA is a promising therapeutic target in breast cancer and other tumor types driven by c-MET and RON.

Hypersensitivity reactions to intravenous lipid emulsion in swine: relevance for lipid resuscitation studies.

BACKGROUND: Reports in the recent experimental literature have provided contradicting results in different animal species regarding the efficacy of IV lipid emulsion (ILE) in the reversal of cardiovascular and central nervous system symptoms of local anesthetic and other lipophilic drug overdoses. In particular, ILE seemed to be effective in rats, rabbits, dogs, and humans, but not in swine, for which it not only failed to reverse the adverse effects of anesthetics, but the animals also developed a generalized cutaneous mottling or a dusky appearance immediately after ILE, suggestive of another type of toxicity. The latter symptoms arise in complement (C) activation-related pseudoallergy, a hypersensitivity reaction to particulate drugs and agents. METHODS: Ten Yorkshire swine (15-20 kg) were sedated with ketamine and anesthetized with isoflurane. ILE 1.5 and 5 mL/kg 20% was administered via the ear vein while pulmonary arterial pressure, systemic arterial blood pressure, electrocardiogram, and end-tidal CO2 were recorded continuously. Thromboxane was measured in blood collected at baseline and 2 and 10 minutes after injections. Complement activation by lipid emulsion was also assessed in vitro with soluble terminal complement complex (SC5b-9) and sheep red blood cell assays. RESULTS: Significant increases were observed in the pulmonary pressure (median [interquartile range]) within minutes after the administration of ILE, both at doses 1.5 and 5 mL/kg (15 [12-16.5] to 18.5 [16-20] mm Hg, P = 0.0058 and 15.5 [13-17.25] to 39.5 [30.5-48.5], respectively). The systemic arterial blood pressure increased, and the heart rate decreased after both injections. Thromboxane B2 concentration (median [interquartile range]) in the blood plasma increased from a baseline of 617.3 [412.4-920] to 1132 [597.9-1417] pg/mL (P = 0.0055) and from 1276 [1200-2581] to 4046 [2946-8442] pg/mL (P = 0.0017) after the administration of 1.5 and 5 mL/kg ILE, respectively. Intralipid did not cause in vitro complement activation in human serum. CONCLUSIONS: ILE causes clinically significant hemodynamic changes in pigs, in concert with significant increases in the plasma thromboxane concentration. However, the in vitro tests did not confirm involvement of the complement system in human sera, leaving the underlying mechanism of these findings in doubt. Nonetheless, the observed hemodynamic and biochemical effects of ILE serve as a caveat that the pig is not an ideal model for the study of interventions involving ILE.