Katie M. Cergol

Peptide Ligation–Desulfurization Chemistry at Arginine

The utility of a new β-thiol arginine building block in ligation-desulfurization chemistry has been demonstrated through reactions and kinetic studies with a range of peptide thioesters. Application of the method is highlighted by a one-pot, kinetically controlled, rapid ligation to generate a 7 kDa MUC1 glycopeptide.

One-Pot Peptide Ligation–Desulfurization at Glutamate

An efficient methodology for ligation at glutamate (Glu) is described. A γ-thiol-Glu building block was accessed in only three steps from protected glutamic acid and could be incorporated at the N-terminus of peptides. The application of these peptides in one-pot ligation-desulfurization chemistry is demonstrated with a range of peptide thioesters, and the utility of this methodology is highlighted through the synthesis of the osteoporosis peptide drug teriparatide (Forteo).

Chemoselective sulfenylation and peptide ligation at tryptophan

Peptide ligation–desulfurization chemistry at 2-thiol tryptophan (Trp) is described for the first time. Installation of a thiol auxiliary was achieved through late-stage chemoselective sulfenylation chemistry at the 2-position of the indole ring of Trp either in solution or on solid support, thus abrogating the need for the preparation of a pre-formed thiolated amino acid. Peptides possessing the 2-thiol Trp functionality on the N-terminus were shown to facilitate high yielding ligation reactions with a variety of C-terminal peptide thiophenyl thioesters. Efficient removal of the 2-thiol Trp auxiliary following the ligation reactions was achieved via reductive desulfurization and provided native peptide products in excellent yields. The utility of the methodology was demonstrated in the synthesis of a glycosylated fragment of the N-terminal extracellular domain of the chemokine receptor CXCR1.

Inhibitors of an essential mycobacterial cell wall lipase (Rv3802c) as tuberculosis drug leads

The first targeted inhibitors of an essential M. tuberculosis cell wall lipase, Rv3802c, are described. Lead compounds exhibited nanomolar inhibition of the enzyme, and encouraging antibacterial activity against M. tuberculosis in vitro, supporting Rv3802c as a novel TB drug target.

Assessment of Myeloperoxidase Activity by the Conversion of Hydroethidine to 2-Chloroethidium

Background: Myeloperoxidase activity is commonly assessed in vivo by the accumulation of 3-chlorotyrosine. Results: Myeloperoxidase-derived chlorinating species specifically converted hydroethidine to 2-chloroethidium with efficiency superior to that of the corresponding conversion of tyrosine to 3-chlorotyrosine. Conclusion: Hydroethidine is useful to assess myeloperoxidase activity in vivo, in parallel with its simultaneous use to detect superoxide. Significance: 2-Chloroethidium is a useful additional marker of myeloperoxidase activity. Oxidants derived from myeloperoxidase (MPO) contribute to inflammatory diseases. In vivo MPO activity is commonly assessed by the accumulation of 3-chlorotyrosine (3-Cl-Tyr), although 3-Cl-Tyr is formed at low yield and is subject to metabolism. Here we show that MPO activity can be assessed using hydroethidine (HE), a probe commonly employed for the detection of superoxide. Using LC/MS/MS, 1H NMR, and two-dimensional NOESY, we identified 2-chloroethidium (2-Cl-E+) as a specific product when HE was exposed to hypochlorous acid (HOCl), chloramines, MPO/H2O2/chloride, and activated human neutrophils. The rate constant for HOCl-mediated conversion of HE to 2-Cl-E+ was estimated to be 1.5 × 105 m−1s−1. To investigate the utility of 2-Cl-E+ to assess MPO activity in vivo, HE was injected into wild-type and MPO-deficient (Mpo−/−) mice with established peritonitis or localized arterial inflammation, and tissue levels of 2-Cl-E+ and 3-Cl-Tyr were then determined by LC/MS/MS. In wild-type mice, 2-Cl-E+ and 3-Cl-Tyr were detected readily in the peritonitis model, whereas in the arterial inflammation model 2-Cl-E+ was present at comparatively lower concentrations (17 versus 0.3 pmol/mg of protein), and 3-Cl-Tyr could not be detected. Similar to the situation with 3-Cl-Tyr, tissue levels of 2-Cl-E+ were decreased substantially in Mpo−/− mice, indicative of the specificity of the assay. In the arterial inflammation model, 2-Cl-E+ was absent from non-inflamed arteries and blood, suggesting that HE oxidation occurred locally in the inflamed artery. Our data suggest that the conversion of exogenous HE to 2-Cl-E+ may be a useful selective and sensitive marker for MPO activity in addition to 3-Cl-Tyr.

Synthesis and evaluation of M. tuberculosis salicylate synthase (MbtI) inhibitors designed to probe plasticity in the active site

Mycobacterium tuberculosis salicylate synthase (MbtI) catalyses the first committed step in the biosynthesis of mycobactin T, an iron-chelating siderophore essential for the virulence and survival of M. tuberculosis. Co-crystal structures of MbtI with members of a first generation inhibitor library revealed large inhibitor-induced rearrangements within the active site of the enzyme. This plasticity of the MbtI active site was probed via the preparation of a library of inhibitors based on a 2,3-dihydroxybenzoate scaffold with a range of substituted phenylacrylate side chains appended to the C3 position. Most compounds exhibited moderate inhibitory activity against the enzyme, with inhibition constants in the micromolar range, while several dimethyl ester variants possessed promising anti-tubercular activity in vitro.

Synthesis of gallinamide A analogues as potent falcipain inhibitors and antimalarials.

Analogues of the natural product gallinamide A were prepared to elucidate novel inhibitors of the falcipain cysteine proteases. Analogues exhibited potent inhibition of falcipain-2 (FP-2) and falcipain-3 (FP-3) and of the development of Plasmodium falciparum in vitro. Several compounds were equipotent to chloroquine as inhibitors of the 3D7 strain of P. falciparum and maintained potent activity against the chloroquine-resistant Dd2 parasite. These compounds serve as promising leads for the development of novel antimalarial agents.

Cyclic Peptides Incorporating Phosphotyrosine Mimetics as Potent and Specific Inhibitors of the Grb7 Breast Cancer Target

The Grb7 adaptor protein is a therapeutic target for both TNBC and HER2+ breast cancer. A nonphosphorylated cyclic peptide 1 (known as G7-18NATE) inhibits Grb7 via targeting the Grb7-SH2 domain, but requires the presence of phosphate ions for both affinity and specificity. Here we report the discovery of malonate bound in the phosphotyrosine binding pocket of the apo-Grb7-SH2 structure. Based on this, we carried out the rational design and synthesis of two analogues of peptide 1 that incorporate carboxymethylphenylalanine (cmF) and carboxyphenylalanine (cF) as mimics of phosphotyrosine (pY). Binding studies using SPR confirmed that affinity for Grb7-SH2 domain is improved up to 9-fold over peptide 1 under physiological phosphate conditions (KD = 2.1-5.7 μM) and that binding is specific for Grb7-SH2 over closely related domains (low or no detectable binding to Grb2-SH2 and Grb10-SH2). X-ray crystallographic structural analysis of the analogue bearing a cmF moiety in complex with Grb7-SH2 has identified the precise contacts conferred by the pY mimic that underpin this improved molecular interaction. Together this study identifies and characterizes the tightest specific inhibitor of Grb7 to date, representing a significant development toward a new Grb7-targeted therapeutic.

Rapid assembly of potent type II dehydroquinase inhibitors via “Click” chemistry

The rapid synthesis of a library of potent type II dehydroquinase inhibitors is described. Inhibitors were prepared via a key quinate-derived ene-yne intermediate using Cu(I)-catalysed azide-alkyne cycloaddition (CuAAC) chemistry with a variety of aryl- and heteroaryl-azides.

Synthesis and evaluation of potent ene-yne inhibitors of type II dehydroquinases as tuberculosis drug leads.

E-9: You sunk my battleship! Potent ene-yne-based inhibitors of typeII dehydroquinase are described. The majority of the compounds exhibited nanomolar inhibition of the Streptomyces coelicolor, Helicobacter pylori and Mycobacterium tuberculosis typeII dehydroquinases. The inhibitors also possessed moderate activity against the growth of M.tuberculosis in vitro.