Alaa M. Hayallah

Slit2-Robo4 signalling promotes vascular stability by blocking Arf6 activity.

Slit–Roundabout (Robo) signalling has a well-understood role in axon guidance. Unlike in the nervous system, however, Slit-dependent activation of an endothelial-specific Robo, Robo4, does not initiate a guidance program. Instead, Robo4 maintains the barrier function of the mature vascular network by inhibiting neovascular tuft formation and endothelial hyperpermeability induced by pro-angiogenic factors. In this study, we used cell biological and biochemical techniques to elucidate the molecular mechanism underlying the maintenance of vascular stability by Robo4. Here, we demonstrate that Robo4 mediates Slit2-dependent suppression of cellular protrusive activity through direct interaction with the intracellular adaptor protein paxillin and its paralogue, Hic-5. Formation of a Robo4–paxillin complex at the cell surface blocks activation of the small GTPase Arf6 and, consequently, Rac by recruitment of Arf-GAPs (ADP-ribosylation factor- directed GTPase-activating proteins) such as GIT1. Consistent with these in vitro studies, inhibition of Arf6 activity in vivo phenocopies Robo4 activation by reducing pathologic angiogenesis in choroidal and retinal vascular disease and VEGF-165 (vascular endothelial growth factor-165)-induced retinal hyperpermeability. These data reveal that a Slit2–Robo4–paxillin–GIT1 network inhibits the cellular protrusive activity underlying neovascularization and vascular leak, and identify a new therapeutic target for ameliorating diseases involving the vascular system.

Affinity-Based Labeling of Cytohesins with a Bifunctional SecinH3 Photoaffinity Probe†

We recently identified the 1,2,4-triazole derivative SecinH3 (see Scheme 1) as the first small-molecule inhibitor of a class of cytoplasmic regulatory proteins called cytohesins by using an aptamer-displacement assay. 2] Cytohesins are small guanine nucleotide exchange factors (GEFs) that stimulate ADP (adenosine diphosphate) ribosylation factors (Arfs), ubiquitously expressed Ras-like GTPases which control various cellular regulatory networks ranging from vesicle trafficking to integrin activation. The four highly homologous cytohesins known in mammals share the same domains: a Sec7 domain, which harbors the GEF activity, a pleckstrinhomology (PH) domain, and a coiled-coil (CC) domain. SecinH3 targets the Sec7 domain of cytohesins 1, 2 (also known as “Arf nucleotide-binding-site opener” (ARNO)), and 3, and inhibits their guanine-nucleotide-exchange activity. Its application in human liver cells, flies, and mice led to impaired insulin signaling in each case. Thus, cytohesins associated with the insulin-receptor complex appear to be essential components of this central cellular signaling pathway. Two classes of Arf-GEFs are known and can be distinguished by their size. Cytohesins belong to the small 47 kDa Arf-GEFs; the large Arf-GEFs, which also contain a Sec7 domain, have a mass of about 200 kDa. Unlike the large Arf-GEFs, the small SecinH3-responsive cytohesins are insensitive to the fungal metabolite brefeldin A (BFA). The specificity of SecinH3 for cytohesin GEFs has so far been tested only for a limited set of proteins by means of isothermal titration calorimetry (ITC). A powerful method for assessing the specificity of smallmolecule modulators for their targets is photoaffinity labeling, as it enables the analysis of a large number of individual proteins or protein mixtures in parallel. The combination of photoaffinity labeling and mass spectrometry has been established as an efficient approach for the identification of the binding site of modulator compounds to their protein ligands. Reporter-tagged derivatives of small molecules can also serve as probes for activity-based protein profiling (ABPP). Herein we report the design, synthesis, and application of SecinH3 photoaffinity probes. By applying them to a wide range of GEFs and their small GTPase substrates, we demonstrate that Secin derivatives, which enable the direct detection of the covalent linkage between the photoaffinity probe and the cytohesin Sec7 domain, exhibit high specificity for cytohesins. Preliminary structure–activity-relationship (SAR) studies of SecinH3 showed that the terminal thiophenyl group is critical for inhibitory activity. The deletion of this residue in XH1009 (Scheme 1) resulted in a dramatic loss of inhibition

Versatile, convenient synthesis of pyrimido[1,2,3-cd]purinediones

Abstract The alkylation of 3-substituted cycloalkylcarboxamido-6-aminouracil derivatives with 3-bromo-1-propanol followed by ring closure yields 1,3,8-trisubstituted xanthine derivatives bearing a polar hydroxyl group. Use of the more reactive 1,3-dibromopropane or homologous dibromoalkanes for the alkylation reaction results in simultaneous alkylation at N1 and the exocyclic amino group (N 6 ) yielding imidazo-, pyrimido- and diazepino-pyrimidine derivatives. The pyrimidopyrimidine derivatives can subsequently be cyclised using hexamethyldisilazane at high temperature affording an easy, convenient and general access to tricyclic pyrimido[1,2,3- cd ]purinediones. Alternatively, 3-substituted 6-amino-5-benzylideneaminouracil derivatives can be reacted with 1,3-dibromopropane followed by an oxidative cyclisation using thionyl chloride to obtain the desired tricyclic pyrimido[1,2,3- cd ]purinediones, which are sterically fixed analogues of pharmacologically active purine derivatives.

Design and synthesis of some new theophylline derivatives with bronchodilator and antibacterial activities.

Methylxanthines especially theophylline have been recognized as potent bronchodilators for the relief of acute asthma for over 65 years. Recently, it was found that bacterial infection plays a role in asthma pathogenesis. Accordingly, the present work involves the synthesis of 6-(4-(un)substituted phenyl)thiazolo[2,3-f]theophyllines 2a–g and different series of 8-(1,2,4-triazol-3-ylmethylthio)theophyllines 6–9. The chemical structures of the target compounds were proved by IR, 1H NMR, 13C NMR, EI-MS and HRMS spectroscopic techniques along with elemental analyses. The bronchodilator activity of fifteen compounds was determined in vivo by acetylcholine induced bronchospasm in anaesthetized guinea pigs. Results revealed that all compounds showed moderate to good activity; in addition, five compounds exhibited a bronchodilator activity nearly similar to that of aminophylline as a standard. The antibacterial activity of all the target compounds was investigated in vitro against both Gram-positive and Gram-negative bacterial strains. Results revealed that some compounds showed more potent antibacterial activity than ampicillin as a standard. Acute toxicity study for four target compounds revealed that none of these derivatives showed significant toxicity up to 300 mg/kg. It was found that compound 8c combined both promising bronchodilator and antibacterial activities. This compound could be subjected for further investigations as a new possible candidate in the treatment of bronchial asthma.

Design and Synthesis of New (SecinH3) Derivatives as Potential Cytohesin Inhibitors

Cytohesins are small guanine nucleotide exchange factors that stimulate ADP ribosylation factors, Ras-like GTPases, which control various cellular regulatory networks ranging from vesicle trafficking to integrin activation. A small molecule SecinH3 (1,2,4-triazole derivative) in an aptamer displacement assay as a pan-cytohesin Sec7-domain inhibitor was previously identified. Here a series of different SecinH3-analogues was designed and synthesised as potential cytohesin Sec7-domain inhibitors. All final synthesized compounds 6-8, 43-58 and their intermediates were confirmed by 1H NMR, 13C NMR and high resolution Mass. Preliminary biological screening of target compounds indictaed that some of the new synthesized secinH3 derivatives showed higher potency and promising activity more than secinH3 itself (unpublished results). Compund 9 and 10 were approximate equal to secinH3 activity as cytohesin antagonist activity. Furthermore compound 52 showed twice inhibition potency if compared to secinH3.

Design and synthesis of new 8-anilide theophylline derivatives as bronchodilators and antibacterial agents

Theophylline derivatives have long been recognized as potent bronchodilators for the relief of acute asthma. Recently, it was found that bacterial infection has a role in asthma pathogenesis. The present work involves the design and synthesis of 8-substituted theophylline derivatives as bronchodilators and antibacterial agents. The chemical structures of these compounds were elucidated by IR, 1H-NMR, mass spectrometry, and elemental analyses. The bronchodilator activity was evaluated using acetylcholine-induced bronchospasm in guinea pigs, and most of the compounds showed significant anti-bronchoconstrictive activity in comparison with standard aminophylline. In addition, the antibacterial activity of all the target compounds was investigated in vitro against Gram-positive and Gram-negative bacteria using ampicillin as a reference drug. Results showed that some of the tested compounds possessed significant antibacterial activity. A pharmacophore model was computed to obtain useful insight into the essential structural features of bronchodilator activity. A structure activity relationship was also discussed.