International cooperation and the challenge of internet accessibility in Caribbean territories: example of a collaborative platform between the University Hospital of Martinique and Ramón González Coro Hospital in Cuba, through the French PRPH-3 program.

BACKGROUND: Martinique shares with the other Caribbean countries specific public health issues, particularly in the diagnostic and therapeutic management of cancer patients. Mutualization of human and material resources by promoting cooperation is the most appropriate response to the challenges of the health systems of the Caribbean territories. Through the French PRPH-3 program, we propose to set up a collaborative digital platform adapted to the specificities of the Caribbean to strengthen professional links and skills in oncofertility and oncosexology and reduce inequalities in access to reproductive and sexual health care for cancer patients. METHODS: Within the context of this program, we have developed of an open-source platform based on a Learning Content Management System (LCMS), with an operating system developed by UNFM for low speed internet. LO libraries have been created and interaction between trainers and learners were done in asynchronous mode. This training management platform is based on: a TCC learning system (Training, Coaching, Communities); a web-hosting with pedagogical engineering appropriate to low bandwidth; a reporting system and a responsibility for processing. RESULTS: We have carried out a flexible, multilingual and accessible digital learning strategy functionality called e-MCPPO according to low-speed internet ecosystem. In close connection with the e-learning strategy we conceived (i) a multidisciplinary team; (ii) an appropriate training program for expert health professionals and (iii) a responsive design. DISCUSSION AND CONCLUSION: This low-speed web-based infrastructure allows communities of experts to cooperate in creating, validating, publishing and managing academic learning content. The self-learning modules provide the digital layer for each learner to extend their skills. Learners, as well as trainers, would gradually take ownership of this platform and encourage its promotion. Innovation in this context is both technological (low-speed Internet broadcasting, free interactive software) and organizational (moderating educational resources). This collaborative digital platform is unique in its form and content. This challenge could contribute to the digital transformation of the Caribbean ecosystem for capacity building in this specifics topics.

Large-Scale Virtual Screening Against the MET Kinase Domain Identifies a New Putative Inhibitor Type.

By using an ensemble-docking strategy, we undertook a large-scale virtual screening campaign in order to identify new putative hits against the MET kinase target. Following a large molecular dynamics sampling of its conformational space, a set of 45 conformers of the kinase was retained as docking targets to take into account the flexibility of the binding site moieties. Our screening funnel started from about 80,000 chemical compounds to be tested in silico for their potential affinities towards the kinase binding site. The top 100 molecules selected-thanks to the molecular docking results-were further analyzed for their interactions, and 25 of the most promising ligands were tested for their ability to inhibit MET activity in cells. F0514-4011 compound was the most efficient and impaired this scattering response to HGF (Hepatocyte Growth Factor) with an IC 50 of 7.2 µ M. Interestingly, careful docking analysis of this molecule with MET suggests a possible conformation halfway between classical type-I and type-II MET inhibitors, with an additional region of interaction. This compound could therefore be an innovative seed to be repositioned from its initial antiviral purpose towards the field of MET inhibitors. Altogether, these results validate our ensemble docking strategy as a cost-effective functional method for drug development.

New structural insights into the apelin receptor: identification of key residues for apelin binding.

Apelin is the endogenous ligand of the orphan 7-transmembrane domain GPCR APJ, now named the apelin receptor (ApelinR). Apelin plays a prominent role in body fluid and cardiovascular homeostasis. To better understand the structural organization of the ApelinR, we built 3 homology 3-dimensional (3D) models of the human ApelinR using the validated cholecystokinin receptor-1 3D model or the X-ray structures of the ß2-adrenergic and CXCR4 receptors as templates. Docking of the pyroglutamyl form of apelin 13 (pE13F) into these models revealed the conservation at the bottom of the binding site of a hydrophobic cavity in which the C-terminal Phe of pE13F was embedded. In contrast, at the top of the binding site, depending on the model, different interactions were visualized between acidic residues of the ApelinR and the basic residues of pE13F. Using site-directed mutagenesis, we showed that Asp 92, Glu 172, and Asp 282 of rat ApelinR are key residues in apelin binding by interacting with Lys 8, Arg 2, and Arg 4 of pE13F, respectively. These residues are only seen in the CXCR4-based ApelinR 3D model, further validating this model. These findings bring new insights into the structural organization of the ApelinR and the mode of apelin binding.

Recent trends and applications in 3D virtual screening.

Virtual screening (VS) is becoming an increasingly important approach for identifying and selecting biologically active molecules against specific pharmaceutically relevant targets. Compared to conventional high throughput screening techniques, in silico screening is fast and inexpensive, and is increasing in popularity in early-stage drug discovery endeavours. This paper reviews and discusses recent trends and developments in three-dimensional (3D) receptor-based and ligand-based VS methodologies. First, we describe the concept of accessible chemical space and its exploration. We then describe 3D structural ligand-based VS techniques, hybrid approaches, and new approaches to exploit additional knowledge that can now be found in large chemogenomic databases. We also briefly discuss some potential issues relating to pharmacokinetics, toxicity profiling, target identification and validation, inverse docking, scaffold-hopping and drug re-purposing. We propose that the best way to advance the state of the art in 3D VS is to integrate complementary strategies in a single drug discovery pipeline, rather than to focus only on theoretical or computational improvements of individual techniques. Two recent 3D VS case studies concerning the LXR-ß receptor and the CCR5/CXCR4 HIV co-receptors are presented as examples which implement some of the complementary methods and strategies that are reviewed here.

Exploring c-Met kinase flexibility by sampling and clustering its conformational space.

It is now widely recognized that the flexibility of both partners has to be considered in molecular docking studies. However, the question how to handle the best the huge computational complexity of exploring the protein binding site landscape is still a matter of debate. Here we investigate the flexibility of c-Met kinase as a test case for comparing several simulation methods. The c-Met kinase catalytic site is an interesting target for anticancer drug design. In particular, it harbors an unusual plasticity compared with other kinases ATP binding sites. Exploiting this feature may eventually lead to the discovery of new anticancer agents with exquisite specificity. We present in this article an extensive investigation of c-Met kinase conformational space using large-scale computational simulations in order to extend the knowledge already gathered from available X-ray structures. In the process, we compare the relevance of different strategies for modeling and injecting receptor flexibility information into early stage in silico structure-based drug discovery pipeline. The results presented here are currently being exploited in on-going virtual screening investigations on c-Met.

Identification of new aminoacid amides containing the imidazo[2,1-b]benzothiazol-2-ylphenyl moiety as inhibitors of tumorigenesis by oncogenic Met signaling.

The Met receptor tyrosine kinase is a promising target in anticancer therapies for its role during tumor evolution and resistance to treatment. It is characterized by an unusual structural plasticity as its active site accepts different inhibitor binding modes. Such feature can be exploited to identify distinct agents targeting tumor dependence and/or resistance by oncogenic Met. Here we report the identification of bioactive agents, featuring a new 4-(imidazo[2,1-b]benzothiazol-2-yl)phenyl moiety, targeting cancer cells dependent on oncogenic Met. One of these compounds (7c; Triflorcas) impairs survival, anchorage-independent growth, and in vivo tumorigenesis, without showing side effects. Our medicinal chemistry strategy was based on an in-house Met-focused library of aminoacid-amide derivatives enriched through structure-based computer modeling, taking into account the Met multiple-binding-mode feature. Altogether, our findings show how a rational structure-based drug design approach coupled to cell-based drug evaluation strategies can be applied in medicinal chemistry to identify new agents targeting a given oncogenic-dependency setting.

By interacting with the C-terminal Phe of apelin, Phe255 and Trp259 in helix VI of the apelin receptor are critical for internalization.

Apelin is the endogenous ligand of the orphan seven-transmembrane domain (TM) G protein-coupled receptor APJ. Apelin is involved in the regulation of body fluid homeostasis and cardiovascular functions. We previously showed the importance of the C-terminal Phe of apelin 17 (K17F) in the hypotensive activity of this peptide. Here, we show either by deleting the Phe residue (K16P) or by substituting it by an Ala (K17A), that it plays a crucial role in apelin receptor internalization but not in apelin binding or in Gα(i)-protein coupling. Then we built a homology three-dimensional model of the human apelin receptor using the cholecystokinin receptor-1 model as a template, and we subsequently docked K17F into the binding site. We visualized a hydrophobic cavity at the bottom of the binding pocket in which the C-terminal Phe of K17F was embedded by Trp(152) in TMIV and Trp(259) and Phe(255) in TMVI. Using molecular modeling and site-directed mutagenesis studies, we further showed that Phe(255) and Trp(259) are key residues in triggering receptor internalization without playing a role in apelin binding or in Gα(i)-protein coupling. These findings bring new insights into apelin receptor activation and show that Phe(255) and Trp(259), by interacting with the C-terminal Phe of the pyroglutamyl form of apelin 13 (pE13F) or K17F, are crucial for apelin receptor internalization.

Identification and pharmacological properties of E339-3D6, the first nonpeptidic apelin receptor agonist.

Apelin plays a prominent role in body fluid and cardiovascular homeostasis. To explore further upstream the role played by this peptide, nonpeptidic agonists and antagonists of the apelin receptor are required. To identify such compounds that do not exist to date, we used an original fluorescence resonance energy transfer-based assay to screen a G-protein-coupled receptor-focused library of fluorescent compounds on the human EGFP-tagged apelin receptor. This led to isolated E339-3D6 that displayed a 90 nM affinity and behaved as a partial agonist with regard to cAMP production and as a full agonist with regard to apelin receptor internalization. Finally, E339-3D6 induced vasorelaxation of rat aorta precontracted with noradrenaline and potently inhibited systemic vasopressin release in water-deprived mice when intracerebroventricularly injected. This compound represents the first nonpeptidic agonist of the apelin receptor, the optimization of which will allow development of a new generation of vasodilator and aquaretic agents.

Analysis of c-Met kinase domain complexes: a new specific catalytic site receptor model for defining binding modes of ATP-competitive ligands.

The receptor tyrosine kinase c-Met have multiple roles during cancer development and is currently considered as an important target for molecularly targeted therapies. Structural knowledge of how compounds interact on c-Met catalytic site could guide structure-based drug design strategies towards more effective and selective anticancer drug candidates. However, although 17 crystal structures of c-Met complexed with adenosine triphosphate (ATP)-competitive kinase inhibitors are publicly available (August 2009), there are still open questions regarding the prediction of ligand binding modes. We have applied molecular modeling and molecular mechanics to analyze the distribution of ligands interaction energy on c-Met residues, and deduced a new model of the active site allowing for an unambiguous identification of ligand binding modes. We demonstrate that the binding of known ligands on the c-Met catalytic site involves seven identified structurally-distinct areas. Five of these match the generic kinase ATP binding site model built by Novartis scientists in the 1990s, while the two others are distinct allosteric regions that can be exploited by second generation kinase inhibitors such as Gleevec. We show here that c-Met can accept both such kinds of allosteric inhibitors, a very unusual feature in the kinase family that opens new grounds for highly specific drug design.

A new Met inhibitory-scaffold identified by a focused forward chemical biological screen.

The receptor tyrosine kinase Met is crucial for the genetic program causing cancer progression and metastasis. Its nodal function during aggressiveness and resistance acquisition poses Met inhibition as an obligatory step in anti-cancer targeted therapy. Here, we applied a "Met-focussed" forward chemical biological screen to discover new agents antagonizing Met-triggered biological functions. The identified new scaffold, JLK1360, has a dual mechanism of action towards Met: it impairs Met signalling and also prevents its restoration after degradation. Docking and molecular dynamics provide evidences on the interacting mode of JLK1360 within the Met ATP-binding pocket. Moreover, computational and biochemical studies also highlighted that JLK1360 has a good degree of selectivity towards Met than other RTKs tested. Altogether, these findings demonstrate that the approach we have applied is a powerful strategy to identify compounds with combined properties towards a chosen target. Our studies show how integration of chemistry, biology and computational analysis can provide robust strategies to identify new inhibitory scaffolds suitable for further development of anti-cancer targeted therapies.