24 research outputs found
Identification of allosteric inhibitors of the ecto-5'-nucleotidase (CD73) targeting the dimer interface.
The ecto-5'-nucleotidase CD73 plays an important role in the production of immune-suppressive adenosine in tumor micro-environment, and has become a validated drug target in oncology. Indeed, the anticancer immune response involves extracellular ATP to block cell proliferation through T-cell activation. However, in the tumor micro-environment, two extracellular membrane-bound enzymes (CD39 and CD73) are overexpressed and hydrolyze efficiently ATP into AMP then further into immune-suppressive adenosine. To circumvent the impact of CD73-generated adenosine, we applied an original bioinformatics approach to identify new allosteric inhibitors targeting the dimerization interface of CD73, which should impair the large dynamic motions required for its enzymatic function. Several hit compounds issued from virtual screening campaigns showed a potent inhibition of recombinant CD73 with inhibition constants in the low micromolar range and exhibited a non-competitive inhibition mode. The structure-activity relationships studies indicated that several amino acid residues (D366, H456, K471, Y484 and E543 for polar interactions and G453-454, I455, H456, L475, V542 and G544 for hydrophobic contacts) located at the dimerization interface are involved in the tight binding of hit compounds and likely contributed for their inhibitory activity. Overall, the gathered information will guide the upcoming lead optimization phase that may lead to potent and selective CD73 inhibitors, able to restore the anticancer immune response
Hydrophobic contacts involved in the binding of RR compounds.
<p>Docking poses obtained for the two structurally-related compounds, (A) <b>RR4</b> (yellow) and (B) <b>RR6</b> (cyan) highlighting the binding differences (thick sticks correspond to residues that are inversely involved). Binding mode of compound having a stretched structure as for <b>RR11</b> (C) and for <b>RR20</b> (D) depicted as blue and orange sticks, respectively. Comparison of the binding mode for the inhibitory compound <b>RR3</b> (E) and the activator <b>RR28</b> (F) assuming a common binding site for both. Residues making halogen bonds are depicted in yellow sticks. All residues contributing to hydrophobic contacts (either with backbone or sidechain atoms) are depicted in solvent accessible surface and in thin sticks (all compounds are not oriented identically).</p
Akt inhibition improves irinotecan treatment and prevents cell emergence by switching the senescence response to apoptosis
International audienceActivated in response to chemotherapy, senescence is a tumor suppressive mechanism that induces a permanent loss of proliferation. However, in response to treatment, it is not really known how cells can escape senescence and how irreversible or incomplete this pathway is. We have recently described that cells that escape senescence are more transformed than non-treated parental cells, they resist anoikis and rely on Mcl-1. In this study, we further characterize this emergence in response to irinotecan, a first line treatment used in colorectal cancer. Our results indicate that Akt was activated as a feedback pathway during the early step of senescence. The inhibition of the kinase prevented cell emergence and improved treatment efficacy, both in vitro and in vivo. This improvement was correlated with senescence inhibition, p21waf1 downregulation and a concomitant activation of apoptosis due to Noxa upregulation and Mcl-1 inactivation. The inactivation of Noxa prevented apoptosis and increased the number of emergent cells. Using either RNA interference or p21waf1-deficient cells, we further confirmed that an intact p53-p21-senescence pathway favored cell emergence and that its downregulation improved treatment efficacy through apoptosis induction. Therefore, although senescence is an efficient suppressive mechanism, it also generates more aggressive cells as a consequence of apoptosis inhibition. We therefore propose that senescence-inducing therapies should be used sequentially with drugs favoring cell death such as Akt inhibitors. This should reduce cell emergence and tumor relapse through a combined induction of senescence and apoptosis
Structure-based drug design including cavity selection and dynamics of the enzyme target.
<p>(A) Flowchart illustrating the global strategy for developing allosteric CD73 inhibitors. (B) Five cavities detected using Fpocket on the closed dimeric form of CD73 (4H2G) and shown in colored mesh representations. (C) Top view of superimposed structures of CD73 during the TMD simulation highlighting the large rotating motion of N-domains (centers of mass depicted as spheres in arc shape). (D) Volumes changes and mean local hydrophobic densities observed during TMD for the blue cavity from panel “B” located at the dimerization interface. (E) Target cavity (mesh representation) outside the substrate binding site (AMP and Zn ions are depicted in cyan sticks). (F) Illustration of the target binding site in complex with one hit compound (green sticks) obtained by docking (Glu543 residues are depicted as spheres).</p
Comparison of hit compounds by using conventional metrics used in drug design.
<p>Inhibition constants (<i>K</i><sub>i</sub>) are expressed as pK<sub>i</sub> (A) and ligand (B), ligand-lipophilicity (C), binding and surface (D) efficiencies correspond to LE, LLE, BEI and SEI, respectively. Note that for compounds exhibiting a mixed inhibition mode, two inhibition constants (“a” and “b”) were determined as for <b>RR2</b> and <b>RR16</b>.</p
Detailed analysis of the binding mode for best-ranked hit compounds.
<p>(A) Overlay of the docking poses from all selected hits at the dimerization interface. Compounds are depicted in sticks and CD73 as solvent accessible surface (yellow and pink for differentiating the two monomers). (B) Overlay of the three most active compounds, <b>RR3</b> (green) <b>RR6</b> (cyan) and <b>RR16</b> (purple). Main polar interactions involved in the binding of <b>RR3</b> (C), <b>RR6</b> (D) and <b>RR16</b> (E) viewed in the same orientation. (F) Binding pose of hit compound <b>RR11</b> (blue) holding an extended and dimeric structure.</p
Hydrophobic contacts involved in the binding of RR compounds.
<p>Docking poses obtained for the two structurally-related compounds, (A) <b>RR4</b> (yellow) and (B) <b>RR6</b> (cyan) highlighting the binding differences (thick sticks correspond to residues that are inversely involved). Binding mode of compound having a stretched structure as for <b>RR11</b> (C) and for <b>RR20</b> (D) depicted as blue and orange sticks, respectively. Comparison of the binding mode for the inhibitory compound <b>RR3</b> (E) and the activator <b>RR28</b> (F) assuming a common binding site for both. Residues making halogen bonds are depicted in yellow sticks. All residues contributing to hydrophobic contacts (either with backbone or sidechain atoms) are depicted in solvent accessible surface and in thin sticks (all compounds are not oriented identically).</p
Determination of the kinetics inhibition profiles for the most representative compounds.
<p>Secondary plots and double-reciprocal of steady state rate constants as a function of AMP concentration in the absence (circles) or with increasing concentrations of hit compounds (squares, triangles and stars). (A): <b>RR3</b> at 0, 0.4, 0.8 and 1.6 ÎĽM; (B): <b>RR4</b> at 0, 0.3, 0.6 and 1.2 ÎĽM; (C): <b>RR6</b> at 0, 0.25, 0.5 and 1.0 ÎĽM; (D): <b>RR20</b> at 0, 0.3, 0.6 and 1.2 ÎĽM.</p
The Host DHX9 DExH-Box Helicase Is Recruited to Chikungunya Virus Replication Complexes for Optimal Genomic RNA Translation
International audienceBeyond their role in cellular RNA metabolism, DExD/H-box RNA heli-cases are hijacked by various RNA viruses in order to assist replication of the viral genome. Here, we identify the DExH-box RNA helicase 9 (DHX9) as a binding partner of chikungunya virus (CHIKV) nsP3 mainly interacting with the C-terminal hypervari-able domain. We show that during early CHIKV infection, DHX9 is recruited to the plasma membrane, where it associates with replication complexes. At a later stage of infection, DHX9 is, however, degraded through a proteasome-dependent mechanism. Using silencing experiments, we demonstrate that while DHX9 negatively controls viral RNA synthesis, it is also required for optimal mature nonstructural protein translation. Altogether, this study identifies DHX9 as a novel cofactor for CHIKV repli-cation in human cells that differently regulates the various steps of CHIKV life cycle and may therefore mediate a switch in RNA usage from translation to replication during the earliest steps of CHIKV replication. IMPORTANCE The reemergence of chikungunya virus (CHIKV), an alphavirus that is transmitted to humans by Aedes mosquitoes, is a serious global health threat. In the absence of effective antiviral drugs, CHIKV infection has a significant impact on human health, with chronic arthritis being one of the most serious complications. The molecular understanding of host-virus interactions is a prerequisite to the development of targeted therapeutics capable to interrupt viral replication and transmission. Here, we identify the host cell DHX9 DExH-Box helicase as an essential cofactor for early CHIKV genome translation. We demonstrate that CHIKV nsP3 protein acts as a key factor for DHX9 recruitment to replication complexes. Finally, we establish that DHX9 behaves as a switch that regulates the progression of the viral cycle from translation to genome replication. This study might therefore have a significant impact on the development of antiviral strategies. KEYWORDS chikungunya virus, DHX9, RNA helicase, nsP3, viral replication T he chikungunya virus (CHIKV), a mosquito-borne alphavirus transmitted by Aedes mosquitoes, represents an ongoing challenge to medicine and public health. The clinical manifestation of CHIKV infection is an acute syndrome (high fever, rash, myalgia, and intense arthralgia) that coincides with high viremia. In the absence of targeted therapeutics the infection evolves into a chronic incapacitating arthralgia in the distal joints in more than half of the cases, with patients requiring long-term administration of anti-inflammatory and immunosuppressive treatment (for a review, see reference 1). Because CHIKV recently caused major outbreaks worldwide with a disastrous socioeconomic impact and because antiviral molecules are still lacking, there is an urgent need to identify the mechanisms of infection that might be targeted therapeutically. Citation Matkovic R, Bernard E, Fontanel S, Eldin P, Chazal N, Hassan Hersi D, Merits A, Péloponèse J-M, Briant L. 2019. The host DHX9 DExH-box helicase is recruited to chikungunya virus replication complexes for optimal genomic RNA translation. J Virol 93:e01764-18
Binding mode of hit compound RR28 linking both enzyme monomers.
<p>Residues are depicted in yellow or pink thin sticks according to the monomer they belong and <b>RR28</b> in thick pink sticks.</p