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International audience; The kinase insert domain (KID) of RTK KIT is the key recruitment region for downstream signalling proteins. KID, studied by molecular dynamics simulations as a cleaved polypeptide and as a native domain fused to KIT, showed intrinsic disorder represented by a set of heterogeneous conformations. The accurate atomistic models showed that the helical fold of KID is mainly sequence dependent. However, the reduced fold of the native KID suggests that its folding is allosterically controlled by the kinase domain. The tertiary structure of KID represents a compact array of highly variable α- and 310-helices linked by flexible loops playing a principal role in the conformational diversity. The helically folded KID retains a collapsed globule-like shape due to non-covalent interactions associated in a ternary hydrophobic core. The free energy landscapes constructed from first principles—the size, the measure of the average distance between the conformations, the amount of helices and the solvent-accessible surface area—describe the KID disorder through a collection of minima (wells), providing a direct evaluation of conformational ensembles. We found that the cleaved KID simulated with restricted N- and C-ends better reproduces the native KID than the isolated polypeptide. We suggest that a cyclic, generic KID would be best suited for future studies of KID f post-transduction effects.

Receptor tyrosine kinases (RTKs) are key regulators of normal cellular processes and have a critical role in the development and progression of many diseases. RTK ligand-induced stimulation leads to activation of the cytoplasmic kinase domain that controls the intracellular signalling. Although the kinase domain of RTKs has been extensively studied using X-ray analysis, the kinase insert domain (KID) and the C-terminal are partially or fully missing in all reported structures. We communicate the first structural model of the full-length RTK KIT cytoplasmic domain, a crucial target for cancer therapy. This model was achieved by integration of ab initio KID and C-terminal probe models into an X-ray structure, and by their further exploration through molecular dynamics (MD) simulation. An extended (2-µs) MD simulation of the proper model provided insight into the structure and conformational dynamics of the full-length cytoplasmic domain of KIT, which can be exploited in the description of the KIT transduction processes.

BACKGROUND In spite of intensive use of bromadiolone, rodent control was inefficient on a farm infested by rats in Zaragoza, Spain. While metabolic resistance was previously described in this rodent species, the observation of a target resistance to antivitamin K rodenticides had been poorly documented in Rattus rattus. RESULTS From rats trapped on the farm, cytochrome b and Vkorc1 genes were amplified by PCR and sequenced in order to identify species and detect potential Vkorc1 mutations. VKORC1-deduced amino acid sequences were thus expressed in Pichia pastoris, and inhibition constants towards various rodenticides were determined. The ten rats trapped on the farm were all identified as R. rattus. They were found to be homozygous for the g.74A>T nucleotide replacement in exon 1 of the Vkorc1 gene, leading to p.Y25F mutation. This mutation led to increased apparent inhibition constants towards various rodenticides, probably caused by a partial loss of helical structure of TM4. CONCLUSION The p.Y25F mutation detected in the Vkorc1 gene in R. rattus trapped on the Spanish farm is associated with the resistance phenotype to bromadiolone that has been observed. It is the first evidence of target resistance to antivitamin K anticoagulants in R. rattus. © 2015 Society of Chemical Industry

HIV-1 IN is a pertinent target for the development of AIDS chemotherapy. The first IN-specific inhibitor approved for the treatment of HIV/AIDS, RAL, was designed to block the ST reaction. We characterized the structural and conformational features of RAL and its recognition by putative HIV-1 targets - the unbound IN, the vDNA, and the IN•vDNA complex - mimicking the IN states over the integration process. RAL binding to the targets was studied by performing an extensive sampling of the inhibitor conformational landscape and by using four different docking algorithms: Glide, Autodock, VINA, and SurFlex. The obtained data evidenced that: (i) a large binding pocket delineated by the active site and an extended loop in the unbound IN accommodates RAL in distinct conformational states all lacking specific interactions with the target; (ii) a well-defined cavity formed by the active site, the vDNA, and the shortened loop in the IN•vDNA complex provide a more optimized inhibitor binding site in which RAL chelates Mg(2+) cations; (iii) a specific recognition between RAL and the unpaired cytosine of the processed DNA is governed by a pair of strong H-bonds similar to those observed in DNA base pair G-C. The identified RAL pose at the cleaved vDNA shed light on a putative step of RAL inhibition mechanism. This modeling study indicates that the inhibition process may include as a first step RAL recognition by the processed vDNA bound to a transient intermediate IN state, and thus provides a potentially promising route to the design of IN inhibitors with improved affinity and selectivity.

Highlights • Rotavirus cores: biochemical and biophysical characterisation. • Packaging of single stranded RNA into rotavirus cores. • Competitive packaging assays. • Non-specificity of RNAs packaging into rotavirus cores. • Infectivity of subviral rotavirus particles., Rotavirus (RV) cores were released from double-layered particles (DLPs) by high concentrations of CaCl2, purified and ‘opened’ by treatment with EDTA or EGTA. Under appropriate in vitro conditions DLPs have been shown to have transcriptase and ‘open cores’ replicase activity. Furthermore, it has been demonstrated that transcriptase activity and infectivity of native cores can be restored by transcapsidation with VP6, VP7 and VP4. The missing link for particle reconstitution in vitro has been the manipulation of ‘open cores’ to become functionally active cores again. The experiments described here were undertaken with the aim of exploring packaging of RV RNAs into opened cores in vitro. Rotavirus cores were opened by approximately 200 μM EGTA, leading to the release of genomic dsRNA. Conversely, RV cores were found to be stable in the presence of minimum concentrations of Ca2+, Mg2+, spermidine3+ and cobalthexamine3+ of between 40 and 300 μM. Aggregates of purified cores were resolved in the presence of 0.3 mM deoxycholate (minimum concentration). Core shells opened with EGTA were reconstituted by the addition of di- or trivalent cations within 2 min of the opening procedure. Addition of purified, baculovirus recombinant-expressed VP6 to native and reconstituted cores led to the formation of DLPs or DLP-like particles, which upon transfection into MA104 cells were infectious. The rescued infectivity likely originated in part from unopened and in part from reconstituted cores. Radiolabelled RV (+) ssRNAs could be packaged into reconstituted cores and DLPs, as indicated by resistance to RNase I digestion. The packaging reaction was, however, not RV RNA sequence-specific, since unrelated ssRNAs, such as those transcribed from HIV-2 cDNAs, were also packaged. The kinetics of packaging of homologous and heterologous RNAs were similar, as evidenced by competitive packaging assays. None of the packaged in vitro engineered RNA segments has so far been rescued into infectious virus.

International audience; The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors' sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib.

International audience; The fundamental oncology-related research is required for a deeper understanding of the molecular mechanisms associated with the normal and/or abnormal protein functions, which are closely related with structure and dynamics of the macromolecules involved in these process. The most common origin of oncogenic events is related to missense mutations. Mutation-induced structural effects promoted by oncogenic mutations in receptor tyrosine kinases (RTKs), are not yet fully characterized. Computational biology completes and enriches experimental data, producing a novel vision of molecular mechanisms governing RTKs activity. In series of our papers, we studied the structural and dynamical features of native and mutated RTKs from III family (KIT and CSF-1R), yielding a detailed description of their mechanisms of activation, ligand-depend for the native proteins and constitutive for the distinct mutants. The mechanisms of RTKs activation are described in terms of allosteric regulation between coupled regulating fragments of the protein, juxta-membrane region (JMR) and activation (A-) loop. As some mutations promote resistance to the clinically-used drugs, we analyzed the affinity of imatinib to these therapeutic targets. The computationally-obtained (in silico) data were correlated with in vivo and in vitro observations, thus validating our numerically-based accounts. Going forward, clinical validation of cancer-related models and simulations are cornerstones key of translation of in silico data into biomedical research, at clinical and pharmacological levels.

Integrase (IN), the HIV-1 enzyme responsible for the integration of the viral genome into the chromosomes of infected cells, is the target of the recently approved antiviral raltegravir (RAL). Despite this drug's activity against viruses resistant to other antiretrovirals, failures of raltegravir therapy were observed, in association with the emergence of resistance due to mutations in the integrase coding region. Two pathways involving primary mutations on residues N155 and Q148 have been characterized. It was suggested that mutations at residue Y143 might constitute a third primary pathway for resistance. The aims of this study were to investigate the susceptibility of HIV-1 Y143R/C mutants to raltegravir and to determine the effects of these mutations on the IN-mediated reactions. Our observations demonstrate that Y143R/C mutants are strongly impaired for both of these activities in vitro . However, Y143R/C activity can be kinetically restored, thereby reproducing the effect of the secondary G140S mutation that rescues the defect associated with the Q148R/H mutants. A molecular modeling study confirmed that Y143R/C mutations play a role similar to that determined for Q148R/H mutations. In the viral replicative context, this defect leads to a partial block of integration responsible for a weak replicative capacity. Nevertheless, the Y143 mutant presented a high level of resistance to raltegravir. Furthermore, the 50% effective concentration (EC 50 ) determined for Y143R/C mutants was significantly higher than that obtained with G140S/Q148R mutants. Altogether our results not only show that the mutation at position Y143 is one of the mechanisms conferring resistance to RAL but also explain the delayed emergence of this mutation.

Integration of the HIV-1 viral DNA generated by reverse transcription of the RNA genome into the host cell chromosomes is a key step of viral replication, catalyzed by the viral integrase. In October 2007, the first integrase inhibitor, raltegravir, was approved for clinical use under the name of Isentress™. The results of the various clinical trials that have evaluated raltegravir have been very encouraging with regard to the immunological and virological efficacy and tolerance. However, as observed for other anti-retrovirals, specific resistance mutations have been identified in patients failing to respond to treatment with raltegravir. Although knowledge of the integrase structural biology remains fragmentary, the structures and modeling data available might provide relevant clues on the origin of the emergence of these resistance mutations. In this review, we describe the mechanism of action of this drug and the main data relating to its use in vivo, together with recent structural data important to our understanding of the origin of viral resistance.

Raltegravir (MK-0518) is a potent inhibitor of human immunodeficiency virus (HIV) integrase and is clinically effective against viruses resistant to other classes of antiretroviral agents. However, it can select mutations in the HIV integrase gene. Nine heavily pretreated patients who received salvage therapy including raltegravir and who subsequently developed virological failure under raltegravir therapy were studied. For each patient, the sequences of the integrase-coding region were determined and compared to that at the beginning of the treatment. Four different mutation profiles were identified in these nine patients: E92Q, G140S Q148H, N155H, and E157Q mutations. For four patients, each harboring a different profile, the wild-type and mutated integrases were produced, purified, and assayed in vitro. All the mutations identified altered the activities of integrase protein: both 3′ processing and strand transfer activities were moderately affected in the E92Q mutant; strand transfer was markedly impaired in the N155H mutant; both activities were strongly impaired in the G140S Q148H mutant; and the E157Q mutant was almost completely inactive. The sensitivities of wild-type and mutant integrases to raltegravir were compared. The E92Q and G140S Q148H profiles were each associated with a 7- to 8-fold decrease in sensitivity, and the N155H mutant was more than 14-fold less sensitive to raltegravir. At least four genetic profiles (E92Q, G140S Q148H, N155H, and E157Q) can be associated with in vivo treatment failure and resistance to raltegravir. These mutations led to strong impairment of enzymes in vitro in the absence of raltegravir: strand transfer activity was affected, and in some cases 3′ processing was also impaired.

The classic model of tumor suppression implies that malignant transformation requires full “two-hit” inactivation of a tumor-suppressor gene. However, more recent work in mice has led to the proposal of a “continuum” model that involves more fluid concepts such as gene dosage-sensitivity and tissue specificity. Mutations in the tumor-suppressor gene von Hippel-Lindau (VHL) are associated with a complex spectrum of conditions. Homozygotes or compound heterozygotes for the R200W germline mutation in VHL have Chuvash polycythemia, whereas heterozygous carriers are free of disease. Individuals with classic, heterozygous VHL mutations have VHL disease and are at high risk of multiple tumors (e.g., CNS hemangioblastomas, pheochromocytoma, and renal cell carcinoma). We report here an atypical family bearing two VHL gene mutations in cis (R200W and R161Q), together with phenotypic analysis, structural modeling, functional, and transcriptomic studies of these mutants in comparison with classical mutants involved in the different VHL phenotypes. We demonstrate that the complex pattern of disease manifestations observed in VHL syndrome is perfectly correlated with a gradient of VHL protein (pVHL) dysfunction in hypoxia signaling pathways. Thus, by studying naturally occurring familial mutations, our work validates in humans the “continuum” model of tumor suppression. Cancer Res; 74(22); 6554–64. ©2014 AACR.

International audience; β-Thalassaemia major (β-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates. Despite extensive knowledge of the molecular defects causing β-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation. Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation. Here we show in vitro that during the maturation of human β-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of β-TM erythroblasts, which may provide a rationale for new targeted therapies of β-TM

The colony stimulating factor-1 receptor (CSF-1R) and the stem cell factor receptor KIT, type III receptor tyrosine kinases (RTKs), are important mediators of signal transduction. The normal functions of these receptors can be compromised by gain-of-function mutations associated with different physiopatological impacts. Whereas KIT D816V/H mutation is a well-characterized oncogenic event and principal cause of systemic mastocytosis, the homologous CSF-1R D802V has not been identified in human cancers. The KIT D816V oncogenic mutation triggers resistance to the RTK inhibitor Imatinib used as first line treatment against chronic myeloid leukemia and gastrointestinal tumors. CSF-1R is also sensitive to Imatinib and this sensitivity is altered by mutation D802V. Previous in silico characterization of the D816V mutation in KIT evidenced that the mutation caused a structure reorganization of the juxtamembrane region (JMR) and facilitated its departure from the kinase domain (KD). In this study, we showed that the equivalent CSF-1R D802V mutation does not promote such structural effects on the JMR despite of a reduction on some key H-bonds interactions controlling the JMR binding to the KD. In addition, this mutation disrupts the allosteric communication between two essential regulatory fragments of the receptors, the JMR and the A-loop. Nevertheless, the mutation-induced shift towards an active conformation observed in KIT D816V is not observed in CSF-1R D802V. The distinct impact of equivalent mutation in two homologous RTKs could be associated with the sequence difference between both receptors in the native form, particularly in the JMR region. A local mutation-induced perturbation on the A-loop structure observed in both receptors indicates the stabilization of an inactive non-inhibited form, which Imatinib cannot bind.

Receptor tyrosine kinase KIT controls many signal transduction pathways and represents a typical allosterically regulated protein. The mutation-induced deregulation of KIT activity impairs cellular physiological functions and causes serious human diseases. The impact of hotspots mutations (D816H/Y/N/V and V560G/D) localized in crucial regulatory segments, the juxtamembrane region (JMR) and the activation (A-) loop, on KIT internal dynamics was systematically studied by molecular dynamics simulations. The mutational outcomes predicted in silico were correlated with in vitro and in vivo activation rates and drug sensitivities of KIT mutants. The allosteric regulation of KIT in the native and mutated forms is described in terms of communication between the two remote segments, JMR and A-loop. A strong correlation between the communication profile and the structural and dynamical features of KIT in the native and mutated forms was established. Our results provide new insight on the determinants of receptor KIT constitutive activation by mutations and resistance of KIT mutants to inhibitors. Depiction of an intra-molecular component of the communication network constitutes a first step towards an integrated description of vast communication pathways established by KIT in physiopathological contexts., Author Summary Receptor tyrosine kinase KIT plays a crucial role in the regulation of cell signaling. This allosterically controlled activity may be affected by gain-of-function mutations that promote the development of several cancers. Identification of the molecular basis of KIT constitutive activation and allosteric regulation has inspired computational study of KIT hotspot mutations. In the present contribution, we investigated the mutation-induced effects on KIT conformational dynamics and intra-protein communication conditionally on the mutation location and the nature of the substituting amino acid. Our data elucidate that all studied mutations stabilize an inactive non-autoinhibited state of KIT over the inactive auto-inhibited state prevalent for the native protein. This shift in the protein conformational landscape promotes KIT constitutive activation. Our in silico analysis established correlations between the structural and dynamical effects induced by oncogenic mutations and the mutants auto-activation rates and drug sensitivities measured in vitro and in vivo. Particularly, the A-loop mutations stabilize the drug-resistant forms, while the JMR mutations may facilitate inhibitors binding to the active site. Cross-correlations established between local and long-range structural and dynamical effects demonstrate the allosteric character of the gain-of-function mutations mode of action.

A new dihydrochalcone, two new dihydroflavanones and eight new biflavonoids have been isolated together with cryptocaryone from the cytotoxic methanol extract of Cryptocarya infectoria. The absolute structure of cryptocaryone was established by X-ray analysis of its 8-bromo derivative. The structure of the new compounds were elucidated by spectroscopic means and their absolute stereochemistry was deduced from chemical correlation, circular dichroism data and NOESY experiments. Several compounds displayed significant cytotoxicity and cryptocaryone was shown to possess cytotoxicity towards multi-drug resistant K562-DOX cells.

Four new Fe(III) catecholate complexes, [(bispicMe2en)FeIII(DBC)]+, [(bispicCl2Me2en)FeIII(DBC)]+, [(trispicMeen)FeIII(DBC)]+, and [(BQPA)FeIII(DBC)]+, which all contain aminopyridine ligands, were synthesized. The structure of [(bispicMe2en)FeIII(DBC)]+ was determined by X-ray diffraction. It crystallizes in the triclinic space group P1 with a = 10.666(3) A, b = 13.467(5) A, c = 17.685(2) A, alpha = 93.46(2) degrees, beta = 93.68(2) degrees, gamma = 109.0(3) degrees, V = 2387.4 A3, and Z = 2. All of these complexes were found to be active toward oxidation of catechol by O2 in DMF at 20 degrees C to afford intradiol cleavage products. The catechol was quantitatively oxidized, mainly (90%) into 3,5-di-tert-butyl-5-(carboxymethyl)-2-furanone. Reaction rates were measured, and for the first three (topologically similar) complexes, a correlation of the second-order kinetic constants k with the optical parameters of the two LMCT O(DBC)--Fe(III) bands was found. In particular, k increases with the epsilon max of the charge-transfer bands. The k value of the complex [(BQPA)FeIII(DBC)]+, containing a tripodal ligand, is smaller than expected on the basis of these correlations. This discrepancy could be related to steric hindrance induced by the BQPA ligand. However, the much lower activity of the bispicen-Fe(III)-type complexes compared to that of the [(TPA)FeIII(DBC)]+ complex synthesized by Jang et al. (J. Am. Chem. Soc. 1991, 113, 9200-9204), despite similar epsilon max values, shows that a knowledge of optical and NMR parameters values is not sufficient to explain the dioxygenase activity rate. In their study of protocatechuate 3,4-dioxygenase, Orville et al. (Biochemistry 1997, 36, 10052-10066) suggested that asymmetric chelation of the catecholate to Fe(III) is of great importance in the efficiency of the intradiol dioxygenase reaction. Indeed, a comparison of the X-ray structures of [(TPA)FeIII(DBC)]+ and [(bispicMe2en)FeIII(DBC)]+ shows that the Fe(III)-O bonds differ by 0.019 A in the former and are identical in the latter. Asymmetry could also play a role in the model complexes. An alternative explanation is the possible existence of a low-spin state for [(TPA)FeIII(DBC)]+, as recently identified in [(TPA)FeIII(cat)]+ by Simaan et al.

A new ferric complex, [(bispic(PA)2en)Fe(III)Cl2]·[PF6]3 (bispic(PA)2en=N,N′-bis(2-pyridylmethyl)-N,N′-bis(5-trimethylammoniumpentyl)-1,2-diaminoethane) has been synthesized, and its structure has been determined by X-ray crystallography. The cation adopts a cis-α-dichloro configuration with the six-coordinated iron(III) in a pseudo octahedral geometry. Its relative catecholato adduct [(bispic(PA)2en)Fe(III)(DBC)]·[PF6]3 has been synthesized and found to be active toward oxidation of catechol by O2 to afford intradiol cleavage products.

The new disymmetric ligand N,N,N′ -tris(2-pyridylmethyl)- N′ -(2-salicylideneethyl)ethane-1,2-diamine (LH) has been synthesized in the search of a novel type of manganese complex to mimic the active site of the water-oxidizing enzyme in photosystem II. The complex [Mn(II)L]ClO 4 has been obtained and characterized by X-ray diffraction techniques. It crystallizes in the monoclinic space group Pn with the following unit cell parameters: a =10.164(3), b =10.122(4), c =14.166(5) A, β =93.48(2)° and Z =2. The manganese ion is heptacoordinated with the coordination being achieved by only one ligand; it is bonded to the oxygen atom of the phenolate group in an axial position, the imino and the three pyridine nitrogen atoms in an equatorial position and the two amine atoms in a pseudo-axial position. The coordination polyhedron is best described as a distorted monocapped trigonal prism. This structure was compared with the seven-coordinated Mn(II) complexes deposited in the Cambridge Structured Database (CSD). The redox potential of the Mn(III)/Mn(II) couple was determined by cyclic voltammetry.

Several new ortho bridged biphenyls mimicking the structure of (−)-rhazinilam were synthesized and evaluated as cytotoxic compounds and as inhibitors of microtubules disassembly. These included azadibenzo[a,c]cyclononene derivatives having an ester, urea or carbamate function present in the 9-membered ring linking the two phenyl moieties. The compound bearing a carbamate function instead of the amide group of rhazinilam interacts better with tubulin than (−) rhazinilam.

Most antiretroviral medical treatments were developed and tested principally on HIV-1 B nonrecombinant strain, which represents less than 10% of the worldwide HIV-1-infected population. HIV-1 circulating recombinant form CRF02_AG is prevalent in West Africa and is becoming more frequent in other countries. Previous studies suggested that the HIV-1 polymorphisms might be associated to variable susceptibility to antiretrovirals. This study is pointed to compare the susceptibility to integrase (IN) inhibitors of HIV-1 subtype CRF02_AG IN respectively to HIV-1 B. Structural models of B and CRF02_AG HIV-1 INs as unbound enzymes and in complex with the DNA substrate were built by homology modeling. IN inhibitors—raltegravir (RAL), elvitegravir (ELV) and L731,988—were docked onto the models, and their binding affinity for both HIV-1 B and CRF02_AG INs was compared. CRF02_AG INs were cloned and expressed from plasma of integrase strand transfer inhibitor (INSTI)-naïve infected patients. Ourin silicoandin vitrostudies showed that the sequence variations between the INs of CRF02_AG and B strains did not lead to any notable difference in the structural features of the enzyme and did not impact the susceptibility to the IN inhibitors. The binding modes and affinities of INSTI inhibitors to B and CRF02_AG INs were found to be similar. Although previous studies suggested that several naturally occurring variations of CRF02_AG IN might alter either IN/vDNA interactions or INSTIs binding, our study demonstrate that these variations do affect neither IN activity nor its susceptibility to INSTIs.

The type III receptor tyrosine kinase (RTK) KIT plays a crucial role in the transmission of cellular signals through phosphorylation events that are associated with a switching of the protein conformation between inactive and active states. D816V KIT mutation is associated with various pathologies including mastocytosis and cancers. D816V-mutated KIT is constitutively active, and resistant to treatment with the anti-cancer drug Imatinib. To elucidate the activating molecular mechanism of this mutation, we applied a multi-approach procedure combining molecular dynamics (MD) simulations, normal modes analysis (NMA) and binding site prediction. Multiple 50-ns MD simulations of wild-type KIT and its mutant D816V were recorded using the inactive auto-inhibited structure of the protein, characteristic of type III RTKs. Computed free energy differences enabled us to quantify the impact of D816V on protein stability in the inactive state. We evidenced a local structural alteration of the activation loop (A-loop) upon mutation, and a long-range structural re-organization of the juxta-membrane region (JMR) followed by a weakening of the interaction network with the kinase domain. A thorough normal mode analysis of several MD conformations led to a plausible molecular rationale to propose that JMR is able to depart its auto-inhibitory position more easily in the mutant than in wild-type KIT and is thus able to promote kinase mutant dimerization without the need for extra-cellular ligand binding. Pocket detection at the surface of NMA-displaced conformations finally revealed that detachment of JMR from the kinase domain in the mutant was sufficient to open an access to the catalytic and substrate binding sites., Author Summary Protein kinases are involved in a huge amount of cellular processes through phosphorylation, a crucial mechanism in cell signaling, and their misregulation often results in disease. The deactivation of protein tyrosine kinases (PTKs) or their oncogenic activation arises from mutations which affect the protein primary structure and the configuration of the enzymatic site apparently by stabilizing the activation loop (A-loop) extended conformation. Particularly, mutation D816V of receptor tyrosine kinase (RTK) KIT, found in patients with pediatric mastocytosis, acute leukemia or germ cell tumors, can be considered as the archetype of mutation inducing a displacement of the population equilibrium toward the active conformation. We present a comprehensive computational study of the activating mechanism(s) of this mutation. Our multi-approach in silico procedure evidenced a local alteration of the A-loop structure, and a long-range structural re-organization of the juxta-membrane region (JMR) followed by a weakening of the interaction network with the kinase domain. Our results provided a plausible conception of how the observed departure of JMR from kinase domain in the mutant promotes kinase mutant dimerization without requiring extra-cellular ligand binding. The pocket profiles we obtained suggested putative allosteric binding sites that could be targeted by ligands/modulators that trap the mutated enzyme.

While searching for new HIV integrase inhibitors we discovered that some ethyl malonate amides (EMA) are active against this enzyme. Surprisingly, the main function can only very rarely be found among the reported drug candidates. We synthesised a series of compounds in order to establish and analyse the structure–activity relationship. The similarity to the important classes of HIV integrase inhibitors as well as the synthetic availability of the different targets including this pharmacophore makes EMA compounds an interesting object of investigations.

Several new ortho bridged biphenyls mimicking the structure of (−)-rhazinilam were synthesized and evaluated as cytotoxic compounds and as inhibitors of microtubules disassembly. These included azadibenzo[a,c]cyclononene derivatives having an ester, urea or carbamate function present in the 9-membered ring linking the two phenyl moieties. The compound bearing a carbamate function instead of the amide group of rhazinilam interacts better with tubulin than (−) rhazinilam.

Strand-transfer inhibitors, of which raltegravir, elvitegravir and S/GSK1349572, is a new class of antiretrovirals that inhibit HIV integrase-catalyzed insertion of the HIV-1 genome into cell chromosomes. The results of clinical trials were very encouraging regarding their viral efficiency and tolerance. However resistance mutations were identified in patients failing to respond to treatment with these inhibitors, involving primary mutations as well as numerous secondary mutations. This review focuses on recent advanced computational studies that have highlighted the contribution of those residues subject to primary mutations and the role of conformational flexibility of the enzyme in binding to strand-transfer inhibitors.

Raltegravir (MK-0518) is clinically used against viruses resistant to other antiretroviral compounds. However, it has been reported that Raltegravir induces integrase (IN) resistance very soon after the administration of the drug. Some residues in the protein seem to be preferentially targeted: E92Q, N155H, and the double mutant G140S/Q148H. It was demonstrated that most of these mutations altered both 3'-processing and strand transfer activities of IN. In this report, we addressed for the first time by real time PCR the kinetics of the integrase mutants as well their resistance towards Raltegravir. Furthermore, we characterised the activities of the recombinant proteins. Our data highly suggest that the resistance towards Raltegravir is due to the mutation Q148H. This mutation results in a slower replication kinetic which is compensate by the G140S mutation. These data explain why only the double mutant G140S/Q148H is observed in clinical trials as one of the most prevalent profile of raltegravir resistance. Two patients receiving raltegravir as part as their regimen were followed. After a viral load decrease, these patients harbored virological failure and selection of raltegravir resistance mutations in the integrase gene (N155H for patient 1 and G140S + Q148H for patient 2). At failure, patients harbouring viruses with these raltegravir mutations showed HIV viral load rebound corresponding to baseline values.

Received 9 October 2008; returned 21 November 2008; revised 12 December 2008; accepted 8 January 2009 Objectives: Raltegravir is the first approved inhibitor of HIV-1 integrase (IN). In most patients, raltegravir failure is associated with mutations in the IN gene, through two different genetic pathways: 155 (N155H) or 148 (Q148K/R/H). The objective of this study was to characterize the dynamics of HIV-1 quasispecies variant populations in patients who failed to respond to raltegravir treatment. Patients and methods: Bulk genotyping and clonal analysis were performed during the follow-up of 10 patients who failed to respond to raltegravir treatment. Results: Treatment failed through the 155 pathway in six patients and through the 148 pathway in two patients; two further patients switched from the 155 to the 148 pathway. In the two patients switching from the 155 to the 148 pathway, clonal analysis showed that Q148R/H and N155H mutations were present on different strands, suggesting that these two pathways are independent. This was consistent with our finding that each genetic profile was associated with different secondary mutations. We observed a greater variability among quasispecies associated with the 155 pathway, and IC50 determinations showed that the fold resistance to raltegravir, relative to wild-type, was 10 for the N155H mutant and 50 for the G140S1Q148H mutant. Conclusions: Clonal analysis strongly suggests that the two main genetic pathways, 155 and 148, involved in the development of resistance to raltegravir are independent and exclusive. Moreover, the switch of the resistance profile from 155 to 148 may be related to the higher level of resistance to raltegravir conferred by the 148 pathway and also to the higher instability of the 155 pathway.

International audience; Virologic failure during treatment with raltegravir, the first effective drug targeting HIV integrase, is associated with two exclusive pathways involving either Q148H/R/K, G140S/A or N155H mutations. We carried out a detailed analysis of the molecular and structural effects of these mutations. We observed no topological change in the integrase core domain, with conservation of a newly identified V-shaped hairpin containing the Q148 residue, in particular. In contrast, the mutations greatly altered the specificity of DNA recognition by integrase. The native residues displayed a clear preference for adenine, whereas the mutant residues strongly favored pyrimidines. Raltegravir may bind to N155 and/or Q148 residues as an adenine bioisoster. This may account for the selected mutations impairing raltegravir binding while allowing alternative DNA recognition by integrase. This study opens up new opportunities for the design of integrase inhibitors active against raltegravir-resistant viruses. Copyright Cop. 2009 John Wiley and Sons, Ltd. Supporting information may be found in the online version of this article.

International audience; HIV-1 integrase is one of the three essential enzyme required for viral replication and has a great potential as a novel target for anti-HIV drugs. The sequence variability of the entire integrase (IN) was examined in HIV-1 subtype B and CRF02-AG antiretroviral na? infected patients for the presence of naturally occurring polymorphisms IN gene sequences and protein structures from both subtypes were compared. The phylogenetic analysis showed a total concordance between the 3 pol gene sequences for patients identified as subtype B whereas 3% of patients identified as CRF02-AG showed a mixture of subtypes. The analysis of IN aa sequences showed that 13 positions (K/R14, V/I31, L/I101, T/V112, T/A124, T/A125, G/N134, I/V135, K/T136, V/I201, T/S206, L/I234, and S/G283) differed between subtypes B and CRF02-AG. As observed in the 3D model of the preintegration complex, these differences may impact the functional property of IN. The fact that most variations were grouped suggests that some of them are linked together through compensatory mechanisms. This comparison allowed us to identify several variations of amino acids in HIV-1 IN subtype CRF02-AG that could have a putative impact on anti-integrase sensitivity. In particular, the region formed by Thr125, Thr124, Val31 contains at least one residue, T125, which variation has been involved in eliciting resistance to the naphtyridine carboxamide L870,810 IN inhibitor. In conclusion, virological response to anti-integrase should be studied carefully, according to the subtype, in clinical trials.

Catechol and beta-ketoenol are important pharmacophores of HIV-1 integrase (IN) inhibitors. We investigated their recognition of the divalent metals, Mg and Mn, and of hydrogen bond donors (HBD) and acceptors (HBA). We used data retrieved from the Cambridge Structural Database (CSD), applying a 3-D structure-based, in silico-driven approach. We found that both biophores were stabilized by intramolecular H-bonding (IHB), which was weak in catechols and very strong in beta-ketoenols. Catechols tended to recognize environmental HBD and HBA, demonstrating their ability to make use of both hydroxyl groups to form multiple, strong intermolecular H-bonds. In contrast, beta-ketoenols stabilized by strong IHB inefficiently formed intermolecular H-bonds. beta-Ketoenolate chelated both Mg and Mn ions much more efficiently than dioxolene, which was highly selective for Mn cations. The significant differences in the ability of these two pharmacophores to bind HBD and HBA and in their ability to chelate Mg and Mn have important consequences for competitive inhibitor binding and selectivity for metals and integrase DNA-binding sites.

Context : Germline mutations in genes involved in the hypoxia sensing pathway (VHL, PHD2/EGLN1, HIF2A/EPAS1) predispose patients to erythrocytosis associated with normal or high serum erythropoietin level. The more frequent mutation, VHL-R200W (R200W), has been identified in homozygous carriers with a congenital erythrocytosis named Chuvash polycythemia. Survival in the Chuvash patients was found to be reduced compared to control groups due to higher rates of arterial and venous thromboses, and to haemorrhagic events. Noteworthily, a characteristic of these patients and their parents, heterozygous for the mutation, is the total absence of tumor development as the opposite of heterozygous carriers of other VHL mutations. Indeed, VHL is a tumor suppressor gene and heterozygous carriers of VHLmutations have von Hippel-Lindau disease and are at high risk of multiple tumors (e.g. CNS hemangioblastomas, pheochromocytoma, renal cell carcinoma). The absence of tumor development in patients with congenital erythrocytosis remained unexplained. The R200W mutation is transmitted within a 340-kilobases haplotype inherited 14,000 to 62,000 years ago from a single founder event. The absence of tumor development in patients carrying the R200W mutation raised the possibility of the presence of a protective element within this core haplotype. Objective: The purpose of this study was to determine the mechanisms underlying the absence of tumor development in patients carrying the R200W mutation, healthy (heterozygous carriers) or with congenital erythrocytosis (homozygous). Methods: We report here one particular family carring a heterozygous R200W mutation associated, for the first time, with a typical von Hippel-Lindau disease (with pheochromocytoma, hemangioblastoma and renal cell carcinoma) (Olswang et al., 1998). We performed a comprehensive study with genotyping, phenotypic analysis, structural modeling, functional and transcriptomic studies of the R200W mutant in comparison to classical mutants involved in von Hippel-Lindau disease. For the first time, the use of inducible expression vectors encoding untagged VHL in the functional studies allowed the observation of very subtle effects. Results: We show that the R200W mutation alone is definitely not associated with the von Hippel Lindau disease because this particular family actually carries another pathogenic mutation, VHL-R161Q, on the same VHL allele, and the double mutated allele segregates with disease. Our genotyping showed that the doubled mutations lies on the Chuvash core haplotype transmitted with the R200W mutation. We can thus conclude that this haplotype does not contain any protective elements against cancer development and that the VHLmutations are, by themselves, predominantly responsible for manifestations of the von Hippel-Lindau disease. Functional studies demonstrate that the function of the R200W mutation is close to the wild type protein and the severity of disease manifestations observed in VHL syndrome is perfectly correlated with a gradient of pVHL dysfunction in hypoxia signaling pathways. Conclusion: We show that the R200W mutation associated with congenital erythrocytosis is at the bottom of an oncogenic gradient. These results validate for the first time in humans the recently proposed “continuum” model of tumor suppression, which differs from the classical Knudson’s two-hit model and implies that subtle gene dosage-sensitivity may underlie tumor predisposition. This model may be of major importance in our understanding of tumor risk in patients with erythrocytosis carrying germline mutations in other genes belonging the HIF pathway. Indeed, in rare cases, PHD2 and HIF2A pathogenic mutations also predispose patients to pheochromocytomas or paragangliomas development. Disclosures No relevant conflicts of interest to declare.

Novel (−)- and (+)-rhazinilam derivatives substituted on the D-ring (compounds 3 , 4 , 5 and 6 ) have been prepared from (+)-vincadifformine 7 and (−)-tabersonine and evaluated against the disassembly of microtubules into tubulin. Along with this study, a reproducible ‘one pot’ semisynthesis of (−)-rhazinilam 1 from (+)-1,2-didehydroaspidospermidine 2 was performed allowing the easy preparation of these new compounds.

Abstract 823 Background: Despite extensive knowledge of the molecular defects causing β-thalassemia major (TM), less is known about the mechanisms responsible of the ineffective erythropoiesis. This latter is characterized by accelerated erythroid differentiation, apoptosis and maturation arrest at the polychromatophilic stage. It explains, at least in part, the profound anemia observed in this disease. Although it has been proposed that both the precipitation of unmatched globin chains as well as the accumulation of unbound iron could lead to oxidative stress and subsequent hemolysis, the mechanism by which apoptosis and maturation arrest are induced remains unclear. Hypothesis: We have previously demonstrated that normal human erythroid cell maturation requires the transcription factor GATA-1 and an transient activation of caspase-3 at the basophilic stage (Zermati Y, J Exp Med 2001). Although GATA-1 is a capsase-3 target, we have shown that it was protected during erythroid maturation by the nucleus translocation of heat shock protein 70 (Hsp70) at the onset of caspase-3 activation (Ribeil JA, Nature 2007). Hsp70 is an universally highly conserved protein chaperone involved in the regulation of protein folding and in the prevention of damaging aggregates formation (Hart HU, Nature 2011). We hypothesized that, in addition to AHSP the cognate chaperone of α-globin chains, Hsp70 could also serve as a secondary chaperone in case of excess of free α-globin chains or aggregates to reduce their toxicity. As a result, this interaction could be detrimental by preventing the nuclear localization of Hsp70 leading to the degradation of GATA-1, maturation arrest and apoptosis in TM erythroid mature cells. Results: During erythroid maturation of CD34+ derived from TM peripheral blood (n=8) or bone marrow (n=3), we have first shown by confocal microscopy that, in contrast to CD34+ from healthy donor (n=10), Hsp70 nuclear translocation did not occur at the late stage of maturation. As a result, GATA-1 was poorly expressed, end stage erythroid maturation was arrested and apoptosis occurred. Hsp70/α-globin complexes were evidenced by confocal analysis in cytosol (specific co-localization), by immunoprecipitation of lysed differentiated erythroid TM cells in liquid culture, and by yeast two-hydrid experiments. In order to better characterize this interaction, we have demonstrated by molecular modeling, docking and molecular dynamic simulation (3D models of the human full-length Hsp701-701generated by homology modeling from crystallographic structures) that α-globin bounds to a highly electronegative cavity formed by NBD, SBD and LID (3 major domains of Hsp70). The Hsp70/α-globin complex is characterized by extensive protein-protein interactions stabilized by multiple hydrogen bonds that we have accurately characterized. Then, to demonstrate the pathological role of the Hsp70 cytosolic sequestration, by lentivirus transfection of TM CD34+ cells, we have expressed a nucleus-targeted mutant of Hsp70 and a wtHsp70 (overexpressing Hsp70). As expected, both lentivirus increased nuclear Hsp70 expression and rescued GATA-1 expression. They also increased TM erythroid cell maturation (more mature cells -acidophils and reticulocytes- than in control transfected cells -21.5% vs 11,8%, p Conclusion: Our data indicate that, cytosolic Hsp70 sequestration by α-globin chains prevents its nuclear localization and is a key mechanism of the ineffective erythropoiesis observed in β-TM patients. In order to increase nuclear Hsp70 translocation, developing drugs or small molecules that could increase Hsp70 expression or better disrupt the Hsp70/α-globin complex could be a novel approach of targeted therapies to improve erythropoiesis in TM. Disclosures: Leboulch: bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

International audience; BACKGROUND: Sarcomas are rare mesenchymal malignancies whose pathogenesis is poorly understood; both environmental and genetic risk factors could contribute to their aetiology.METHODS AND RESULTS: We performed whole-exome sequencing (WES) in a familial aggregation of three individuals affected with soft-tissue sarcoma (STS) without TP53 mutation (Li-Fraumeni-like, LFL) and found a shared pathogenic mutation in CDKN2A tumour suppressor gene. We searched for individuals with sarcoma among 474 melanoma-prone families with a CDKN2A-/+ genotype and for CDKN2A mutations in 190 TP53-negative LFL families where the index case was a sarcoma. Including the initial family, eight independent sarcoma cases carried a germline mutation in the CDKN2A/p16INK4A gene. In five out of seven formalin-fixed paraffin-embedded sarcomas, heterozygosity was lost at germline CDKN2A mutations sites demonstrating complete loss of function. As sarcomas are rare in CDKN2A/p16INK4A carriers, we searched in constitutional WES of nine carriers for potential modifying rare variants and identified three in platelet-derived growth factor receptor (PDGFRA) gene. Molecular modelling showed that two never-described variants could impact the PDGFRA extracellular domain structure.CONCLUSION: Germline mutations in CDKN2A/P16INK4A, a gene known to predispose to hereditary melanoma, pancreatic cancer and tobacco-related cancers, account also for a subset of hereditary sarcoma. In addition, we identified PDGFRA as a candidate modifier gene