Peptides Derived from Vascular Endothelial Growth Factor B Show Potent Binding to Neuropilin-1.

Vascular endothelial growth factors (VEGFs) regulate significant pathways in angiogenesis, myocardial and neuronal protection, metabolism, and cancer progression. The VEGF-B growth factor is involved in cell survival, anti-apoptotic and antioxidant mechanisms, through binding to VEGF receptor 1 and neuropilin-1 (NRP1). We employed surface plasmon resonance technology and X-ray crystallography to analyse the molecular basis of the interaction between VEGF-B and the b1 domain of NRP1, and developed VEGF-B C-terminus derived peptides to be used as chemical tools for studying VEGF-B - NRP1 related pathways. Peptide lipidation was used as a means to stabilise the peptides. VEGF-B-derived peptides containing a C-terminal arginine show potent binding to NRP1-b1. Peptide lipidation increased binding residence time and improved plasma stability. A crystal structure of a peptide with NRP1 demonstrated that VEGF-B peptides bind at the canonical C-terminal arginine binding site. VEGF-B C-terminus imparts higher affinity for NRP1 than the corresponding VEGF-A165 region. This tight binding may impact on the activity and selectivity of the full-length protein. The VEGF-B167 derived peptides were more effective than VEGF-A165 peptides in blocking functional phosphorylation events. Blockers of VEGF-B function have potential applications in diabetes and non-alcoholic fatty liver disease.

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.

Identification of a potent small-molecule inhibitor of bacterial DNA repair that potentiates quinolone antibiotic activity in methicillin-resistant Staphylococcus aureus.

The global emergence of antibiotic resistance is one of the most serious challenges facing modern medicine. There is an urgent need for validation of new drug targets and the development of small molecules with novel mechanisms of action. We therefore sought to inhibit bacterial DNA repair mediated by the AddAB/RecBCD protein complexes as a means to sensitize bacteria to DNA damage caused by the host immune system or quinolone antibiotics. A rational, hypothesis-driven compound optimization identified IMP-1700 as a cell-active, nanomolar potency compound. IMP-1700 sensitized multidrug-resistant Staphylococcus aureus to the fluoroquinolone antibiotic ciprofloxacin, where resistance results from a point mutation in the fluoroquinolone target, DNA gyrase. Cellular reporter assays indicated IMP-1700 inhibited the bacterial SOS-response to DNA damage, and compound-functionalized Sepharose successfully pulled-down the AddAB repair complex. This work provides validation of bacterial DNA repair as a novel therapeutic target and delivers IMP-1700 as a tool molecule and starting point for therapeutic development to address the pressing challenge of antibiotic resistance.

Fragment-derived inhibitors of human N-myristoyltransferase block capsid assembly and replication of the common cold virus.

Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections.

Using a non-image-based medium-throughput assay for screening compounds targeting N-myristoylation in intracellular Leishmania amastigotes.

We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania N-myristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors.

Structure-based design of potent and selective Leishmania N-myristoyltransferase inhibitors.

Inhibitors of Leishmania N-myristoyltransferase (NMT), a potential target for the treatment of leishmaniasis, obtained from a high-throughput screen, were resynthesized to validate activity. Crystal structures bound to Leishmania major NMT were obtained, and the active diastereoisomer of one of the inhibitors was identified. On the basis of structural insights, enzyme inhibition was increased 40-fold through hybridization of two distinct binding modes, resulting in novel, highly potent Leishmania donovani NMT inhibitors with good selectivity over the human enzyme.

Diverse modes of binding in structures of Leishmania major N-myristoyltransferase with selective inhibitors.

The leishmaniases are a spectrum of global diseases of poverty associated with immune dysfunction and are the cause of high morbidity. Despite the long history of these diseases, no effective vaccine is available and the currently used drugs are variously compromised by moderate efficacy, complex side effects and the emergence of resistance. It is therefore widely accepted that new therapies are needed. N-Myristoyltransferase (NMT) has been validated pre-clinically as a target for the treatment of fungal and parasitic infections. In a previously reported high-throughput screening program, a number of hit compounds with activity against NMT from Leishmania donovani have been identified. Here, high-resolution crystal structures of representative compounds from four hit series in ternary complexes with myristoyl-CoA and NMT from the closely related L. major are reported. The structures reveal that the inhibitors associate with the peptide-binding groove at a site adjacent to the bound myristoyl-CoA and the catalytic α-carboxylate of Leu421. Each inhibitor makes extensive apolar contacts as well as a small number of polar contacts with the protein. Remarkably, the compounds exploit different features of the peptide-binding groove and collectively occupy a substantial volume of this pocket, suggesting that there is potential for the design of chimaeric inhibitors with significantly enhanced binding. Despite the high conservation of the active sites of the parasite and human NMTs, the inhibitors act selectively over the host enzyme. The role of conformational flexibility in the side chain of Tyr217 in conferring selectivity is discussed.