Therapeutic Validation of GEF-H1 Using a De Novo Designed Inhibitor in Models of Retinal Disease.

Inflammation and fibrosis are important components of diseases that contribute to the malfunction of epithelia and endothelia. The Rho guanine nucleotide exchange factor (GEF) GEF-H1/ARHGEF-2 is induced in disease and stimulates inflammatory and fibrotic processes, cell migration, and metastasis. Here, we have generated peptide inhibitors to block the function of GEF-H1. Inhibitors were designed using a structural in silico approach or by isolating an inhibitory sequence from the autoregulatory C-terminal domain. Candidate inhibitors were tested for their ability to block RhoA/GEF-H1 binding in vitro, and their potency and specificity in cell-based assays. Successful inhibitors were then evaluated in models of TGFß-induced fibrosis, LPS-stimulated endothelial cell-cell junction disruption, and cell migration. Finally, the most potent inhibitor was successfully tested in an experimental retinal disease mouse model, in which it inhibited blood vessel leakage and ameliorated retinal inflammation when treatment was initiated after disease diagnosis. Thus, an antagonist that blocks GEF-H1 signaling effectively inhibits disease features in in vitro and in vivo disease models, demonstrating that GEF-H1 is an effective therapeutic target and establishing a new therapeutic approach.

Design, synthesis, and evaluation of peptide-imidazo[1,2-a]pyrazine bioconjugates as potential bivalent inhibitors of the VirB11 ATPase HP0525.

Helicobacter pylori (H. pylori) infections have been implicated in the development of gastric ulcers and various cancers: however, the success of current therapies is compromised by rising antibiotic resistance. The virulence and pathogenicity of H. pylori is mediated by the type IV secretion system (T4SS), a multiprotein macromolecular nanomachine that transfers toxic bacterial factors and plasmid DNA between bacterial cells, thus contributing to the spread of antibiotic resistance. A key component of the T4SS is the VirB11 ATPase HP0525, which is a hexameric protein assembly. We have previously reported the design and synthesis of a series of novel 8-amino imidazo[1,2-a]pyrazine derivatives as inhibitors of HP0525. In order to improve their selectivity, and potentially develop these compounds as tools for probing the assembly of the HP0525 hexamer, we have explored the design and synthesis of potential bivalent inhibitors. We used the structural details of the subunit-subunit interactions within the HP0525 hexamer to design peptide recognition moieties of the subunit interface. Different methods (cross metathesis, click chemistry, and cysteine-malemide) for bioconjugation to selected 8-amino imidazo[1,2-a]pyrazines were explored, as well as peptides spanning larger or smaller regions of the interface. The IC50 values of the resulting linker-8-amino imidazo[1,2-a]pyrazine derivatives, and the bivalent inhibitors, were related to docking studies with the HP0525 crystal structure and to molecular dynamics simulations of the peptide recognition moieties.

HIV-1 capsid is involved in post-nuclear entry steps.

BACKGROUND: HIV-1 capsid influences viral uncoating and nuclear import. Some capsid is detected in the nucleus but it is unclear if it has any function. We reported that the antibiotic Coumermycin-A1 (C-A1) inhibits HIV-1 integration and that a capsid mutation confers resistance to C-A1, suggesting that capsid might affect post-nuclear entry steps. RESULTS: Here we report that C-A1 inhibits HIV-1 integration in a capsid-dependent way. Using molecular docking, we identify an extended binding pocket delimited by two adjacent capsid monomers where C-A1 is predicted to bind. Isothermal titration calorimetry confirmed that C-A1 binds to hexameric capsid. Cyclosporine washout assays in Jurkat CD4+ T cells expressing engineered human TRIMCyp showed that C-A1 causes faster and greater escape from TRIMCyp restriction. Sub-cellular fractionation showed that small amounts of capsid accumulated in the nuclei of infected cells and C-A1 reduced the nuclear capsid. A105S and N74D capsid mutant viruses did not accumulate capsid in the nucleus, irrespective of C-A1 treatment. Depletion of Nup153, a nucleoporin located at the nuclear side of the nuclear pore that binds to HIV-1 capsid, made the virus less susceptible to TRIMCyp restriction, suggesting that Nup153 may help maintain some integrity of the viral core in the nucleus. Furthermore C-A1 increased binding of CPSF6, a nuclear protein, to capsid. CONCLUSIONS: Our results indicate that capsid is involved in post-nuclear entry steps preceding integration.

2- and 3-substituted imidazo[1,2-a]pyrazines as inhibitors of bacterial type IV secretion.

A novel series of 8-amino imidazo[1,2-a]pyrazine derivatives has been developed as inhibitors of the VirB11 ATPase HP0525, a key component of the bacterial type IV secretion system. A flexible synthetic route to both 2- and 3-aryl substituted regioisomers has been developed. The resulting series of imidazo[1,2-a]pyrazines has been used to probe the structure-activity relationships of these inhibitors, which show potential as antibacterial agents.

Endothelial C-type natriuretic peptide maintains vascular homeostasis.

The endothelium plays a fundamental role in maintaining vascular homeostasis by releasing factors that regulate local blood flow, systemic blood pressure, and the reactivity of leukocytes and platelets. Accordingly, endothelial dysfunction underpins many cardiovascular diseases, including hypertension, myocardial infarction, and stroke. Herein, we evaluated mice with endothelial-specific deletion of Nppc, which encodes C-type natriuretic peptide (CNP), and determined that this mediator is essential for multiple aspects of vascular regulation. Specifically, disruption of CNP leads to endothelial dysfunction, hypertension, atherogenesis, and aneurysm. Moreover, we identified natriuretic peptide receptor-C (NPR-C) as the cognate receptor that primarily underlies CNP-dependent vasoprotective functions and developed small-molecule NPR-C agonists to target this pathway. Administration of NPR-C agonists promotes a vasorelaxation of isolated resistance arteries and a reduction in blood pressure in wild-type animals that is diminished in mice lacking NPR-C. This work provides a mechanistic explanation for genome-wide association studies that have linked the NPR-C (Npr3) locus with hypertension by demonstrating the importance of CNP/NPR-C signaling in preserving vascular homoeostasis. Furthermore, these results suggest that the CNP/NPR-C pathway has potential as a disease-modifying therapeutic target for cardiovascular disorders.

Suppression of type 1 pilus assembly in uropathogenic Escherichia coli by chemical inhibition of subunit polymerization.

OBJECTIVES: To identify and to characterize small-molecule inhibitors that target the subunit polymerization of the type 1 pilus assembly in uropathogenic Escherichia coli (UPEC). METHODS: Using an SDS-PAGE-based assay, in silico pre-filtered small-molecule compounds were screened for specific inhibitory activity against the critical subunit polymerization step of the chaperone-usher pathway during pilus biogenesis. The biological activity of one of the compounds was validated in assays monitoring UPEC type 1 pilus biogenesis, type 1 pilus-dependent biofilm formation and adherence to human bladder epithelial cells. The time dependence of the in vivo inhibitory activity and the overall effect of the compound on UPEC growth were determined. RESULTS: N-(4-chloro-phenyl)-2-{5-[4-(pyrrolidine-1-sulfonyl)-phenyl]-[1,3,4]oxadiazol-2-yl sulfanyl}-acetamide (AL1) inhibited in vitro pilus subunit polymerization. In bacterial cultures, AL1 disrupted UPEC type 1 pilus biogenesis and pilus-dependent biofilm formation, and resulted in the reduction of bacterial adherence to human bladder epithelial cells, without affecting bacterial cell growth. Bacterial exposure to the inhibitor led to an almost instantaneous loss of type 1 pili. CONCLUSIONS: We have identified and characterized a small molecule that interferes with the assembly of type 1 pili. The molecule targets the polymerization step during the subunit incorporation cycle of the chaperone-usher pathway. Our discovery provides new insight into the design and development of novel anti-virulence therapies targeting key virulence factors of bacterial pathogens.

Molecular fields in ligand discovery.

The discovery of novel biologically active small molecules is now a technologically and economically viable proposition for academic and small biotechnology laboratories wishing to build on their biological research into target proteins. Such small molecules may be useful reagents for further biological research or may form the basis for early-stage drug discovery. The availability of specialized virtual screening software to filter large molecular libraries into manageable numbers of compounds for biological assays is the driving force for finding novel ligands. The main focus of this chapter is the basis and use of molecular field methods to assess the interactions that may be made by small molecules. Molecular field based measures of capability and similarity of interaction may be used to discover novel ligands and expand ligand series for potential use as future therapies.

SIFTS: Structure Integration with Function, Taxonomy and Sequences resource.

The Structure Integration with Function, Taxonomy and Sequences resource (SIFTS; http://pdbe.org/sifts) is a close collaboration between the Protein Data Bank in Europe (PDBe) and UniProt. The two teams have developed a semi-automated process for maintaining up-to-date cross-reference information to UniProt entries, for all protein chains in the PDB entries present in the UniProt database. This process is carried out for every weekly PDB release and the information is stored in the SIFTS database. The SIFTS process includes cross-references to other biological resources such as Pfam, SCOP, CATH, GO, InterPro and the NCBI taxonomy database. The information is exported in XML format, one file for each PDB entry, and is made available by FTP. Many bioinformatics resources use SIFTS data to obtain cross-references between the PDB and other biological databases so as to provide their users with up-to-date information.

Gyrase B inhibitor impairs HIV-1 replication by targeting Hsp90 and the capsid protein.

Chemical genetics is an emerging approach to investigate the biology of host-pathogen interactions. We screened several inhibitors of ATP-dependent DNA motors and detected the gyrase B inhibitor coumermycin A1 (C-A1) as a potent antiretroviral. C-A1 inhibited HIV-1 integration and gene expression from acutely infected cell, but the two activities mapped to distinct targets. Target discovery identified Hsp90 as the C-A1 target affecting viral gene expression. Chromatin immunoprecipitation revealed that Hsp90 associates with the viral promoter and may directly regulate gene expression. Molecular docking suggested that C-A1 binds to two novel pockets at the C terminal domain of Hsp90. C-A1 inhibited Hsp90 dimer formation, suggesting that it impairs viral gene expression by preventing Hsp90 dimerization at the C terminus. The inhibition of HIV-1 integration imposed by C-A1 was independent of Hsp90 and mapped to the capsid protein, and a point mutation at residue 105 made the virus resistant to this block. HIV-1 susceptibility to the integration block mediated by C-A1 was influenced by cyclophilin A. Our chemical genetic approach revealed an unexpected function of capsid in HIV-1 integration and provided evidence for a role of Hsp90 in regulating gene expression in mammalian cells. Both activities were amenable to inhibition by small molecules and represent novel antiretroviral drug targets.

Oxadiazolylindazole sodium channel modulators are neuroprotective toward hippocampal neurones.

We report the discovery of a new class of neuroprotective voltage-dependent sodium channel modulators exemplified by (5-(1-benzyl-1H-indazol-3-yl)-1,2,4-oxadiazol-3-yl)methanamine 11 (CFM1178). The compounds were inhibitors of [(14)C]guanidinium ion flux in rat forebrain synaptosomes and displaced binding of the sodium channel ligand [(3)H]BW202W92. 11 and the corresponding N(2)-benzyl isomer, 38 (CFM6058), demonstrated neuroprotective activity in hippocampal slices comparable to sipatrigine. CYP450 enzyme inhibition observed with 11 was reduced with 38. In electrophysiological experiments on dissociated hippocampal neurons, these two compounds caused use- and voltage-dependent block of sodium currents. Sodium channel isoform profiling against Na(v)1.1-1.8 demonstrated that the standard sodium channel blocker lamotrigine had modest activity against Na(v)1.1, while sipatrigine was generally more potent and less selective. 11 and 38 showed potent activity against Na(v)1.6, pointing to pharmacological block of this isoform being consistent with the neuroprotective effect. 38 also showed use dependent block of Na(v)1.6 in HEK cells.

Discovery of inhibitors of the pentein superfamily protein dimethylarginine dimethylaminohydrolase (DDAH), by virtual screening and hit analysis.

An efficient process for the discovery of inhibitors of DDAH enzymes, without the requirement for high throughput screening, is described. Physicochemical filtering of a 308,000-compound library according to drug likeness followed by reciprocal nearest neighbour selection produced a representative subset of 35,000 compounds. Virtual screening on a dual processor PC using FlexX, followed by biological screening, identified two hit series. Similarity searches of commercial databases and chemical re-synthesis of pure compounds resulted in SR445 as an inhibitor of Pseudomonas aeruginosa DDAH at 2 microM.