Design and synthesis of irreversible inhibitors of foot-and-mouth disease virus 3C protease.

Foot-and-mouth disease virus (FMDV) causes a highly infectious and economically devastating disease of livestock. The FMDV genome is translated as a single polypeptide precursor that is cleaved into functional proteins predominantly by the highly conserved viral 3C protease, making this enzyme an attractive target for antiviral drugs. A peptide corresponding to an optimal substrate has been modified at the C-terminus, by the addition of a warhead, to produce irreversible inhibitors that react as Michael acceptors with the enzyme active site. Further investigation highlighted key structural determinants for inhibition, with a positively charged P2 being particularly important for potency.

Peptidomimetic inhibitors of N-myristoyltransferase from human malaria and leishmaniasis parasites.

N-Myristoyltransferase (NMT) has been shown to be essential in Leishmania and subsequently validated as a drug target in Plasmodium. Herein, we discuss the use of antifungal NMT inhibitors as a basis for inhibitor development resulting in the first sub-micromolar peptidomimetic inhibitors of Plasmodium and Leishmania NMTs. High-resolution structures of these inhibitors with Plasmodium and Leishmania NMTs permit a comparative analysis of binding modes, and provide the first crystal structure evidence for a ternary NMT-Coenzyme A/myristoylated peptide product complex.

A two-step mechanism for the folding of actin by the yeast cytosolic chaperonin.

Actin requires the chaperonin containing TCP1 (CCT), a hexadecameric ATPase essential for cell viability in eukaryotes, to fold to its native state. Following binding of unfolded actin to CCT, the cavity of the chaperone closes and actin is folded and released in an ATP-dependent folding cycle. In yeast, CCT forms a ternary complex with the phosducin-like protein PLP2p to fold actin, and together they can return nascent or chemically denatured actin to its native state in a pure in vitro folding assay. The complexity of the CCT-actin system makes the study of the actin folding mechanism technically challenging. We have established a novel spectroscopic assay through selectively labeling the C terminus of yeast actin with acrylodan and observe significant changes in the acrylodan fluorescence emission spectrum as actin is chemically unfolded and then refolded by the chaperonin. The variation in the polarity of the environment surrounding the fluorescent probe during the unfolding/folding processes has allowed us to monitor actin as it folds on CCT. The rate of actin folding at a range of temperatures and ATP concentrations has been determined for both wild type CCT and a mutant CCT, CCT4anc2, defective in folding actin in vivo. Binding of the non-hydrolysable ATP analog adenosine 5'-(ß,γ-imino)triphosphate to the ternary complex leads to 3-fold faster release of actin from CCT following addition of ATP, suggesting a two-step folding process with a conformational change occurring upon closure of the cavity and a subsequent final folding step involving packing of the C terminus to the native-like state.

A fluorescence-based assay for N-myristoyltransferase activity.

N-myristoylation is the irreversible attachment of a C(14) fatty acid, myristic acid, to the N-terminal glycine of a protein via formation of an amide bond. This modification is catalyzed by myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT), an enzyme ubiquitous in eukaryotes that is up-regulated in several cancers. Here we report a sensitive fluorescence-based assay to study the enzymatic activity of human NMT1 and NMT2 based on detection of CoA by 7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin. We also describe expression and characterization of NMT1 and NMT2 and assay validation with small molecule inhibitors. This assay should be broadly applicable to NMTs from a range of organisms.

Synthesis and incorporation into cyclic peptides of tolan amino acids and their hydrogenated congeners: construction of an array of A-B-loop mimetics of the Cε3 domain of human IgE.

The disruption of the human immunolobulin E-high affinity receptor I (IgE-FcεRI) protein-protein interaction (PPI) is a validated strategy for the development of anti asthma therapeutics. Here, we describe the synthesis of an array of conformationally constrained cyclic peptides based on an epitope of the A-B loop within the Cε3 domain of IgE. The peptides contain various tolan (i.e., 1,2-biarylethyne) amino acids and their fully and partially hydrogenated congeners as conformational constraints. Modest antagonist activity (IC(50) ∼660 µM) is displayed by the peptide containing a 2,2'-tolan, which is the one predicted by molecular modeling to best mimic the conformation of the native A-B loop epitope in IgE.

Synthesis of the C19 methyl ether of aspercyclide A via germyl-Stille macrocyclisation and ELISA evaluation of both enantiomers following optical resolution.

Aspercyclide A (1) is a biaryl ether containing 11-membered macrocyclic natural product antagonist of the human IgE-FcεRI protein-protein interaction (PPI); a key interaction in the signal transduction pathway for allergic disorders such as asthma. Herein we report a novel approach to the synthesis of the C19 methyl ether of aspercyclide A, employing a Pd(0)-catalysed, fluorous-tagged alkenylgermane/arylbromide macrocyclisation (germyl-Stille reaction) as the key step, and evaluation of both enantiomers of this compound via ELISA following optical resolution by CSP-HPLC. A crystal structure for germyl hydride 27 is also reported.

Novel Thienopyrimidine Inhibitors of Leishmania N-Myristoyltransferase with On-Target Activity in Intracellular Amastigotes.

The leishmaniases, caused by Leishmania species of protozoan parasites, are neglected tropical diseases with millions of cases worldwide. Current therapeutic approaches are limited by toxicity, resistance, and cost. N-Myristoyltransferase (NMT), an enzyme ubiquitous and essential in all eukaryotes, has been validated via genetic and pharmacological methods as a promising anti-leishmanial target. Here we describe a comprehensive structure-activity relationship (SAR) study of a thienopyrimidine series previously identified in a high-throughput screen against Leishmania NMT, across 68 compounds in enzyme- and cell-based assay formats. Using a chemical tagging target engagement biomarker assay, we identify the first inhibitor in this series with on-target NMT activity in leishmania parasites. Furthermore, crystal structure analyses of 12 derivatives in complex with Leishmania major NMT revealed key factors important for future structure-guided optimization delivering IMP-105 (43), a compound with modest activity against Leishmania donovani intracellular amastigotes and excellent selectivity (>660-fold) for Leishmania NMT over human NMTs.

Analysis of protein-protein interactions by using droplet-based microfluidics.

Every little drop: The K(D) values of angiogenin (ANG) interactions as shown by FRET analysis of thousands of pL-sized droplets agree with data from bulk-fluorescence polarization measurements. Importantly, the use of fluorophores does not affect the activity of ANG or the binding of anti-ANG antibodies to ANG. Such an experimental platform could be applied to the high-throughput analysis of protein-protein interactions.

Specific N-terminal protein labelling: use of FMDV 3C pro protease and native chemical ligation.

We report an effective strategy for generating N-terminal cysteinyl proteins by proteolytic cleavage using the enzyme 3C pro, suitable for a wide range of applications via native chemical ligation.

Site-specific N-terminal labelling of proteins in vitro and in vivo using N-myristoyl transferase and bioorthogonal ligation chemistry.

N-Myristoyl transferase-mediated modification with azide-bearing substrates is introduced as a highly selective and practical method for in vitro and in vivo N-terminal labelling of a recombinant protein using bioorthogonal ligation chemistry.