A Tripartite Complex HIV-1 Tat-Cyclophilin A-Capsid Protein Enables Tat Encapsidation That Is Required for HIV-1 Infectivity.

HIV-1 Tat is a key viral protein that stimulates several steps of viral gene expression. Tat is especially required for the transcription of viral genes. Nevertheless, it is still not clear if and how Tat is incorporated into HIV-1 virions. Cyclophilin A (CypA) is a prolyl isomerase that binds to HIV-1 capsid protein (CA) and is thereby encapsidated at the level of 200 to 250 copies of CypA/virion. Here, we found that a Tat-CypA-CA tripartite complex assembles in HIV-1-infected cells and allows Tat encapsidation into HIV virions (1 Tat/1 CypA). Biochemical and biophysical studies showed that high-affinity interactions drive the assembly of the Tat-CypA-CA complex that could be purified by size exclusion chromatography. We prepared different types of viruses devoid of transcriptionally active Tat. They showed a 5- to 10 fold decrease in HIV infectivity, and conversely, encapsidating Tat into ΔTat viruses greatly enhanced infectivity. The absence of encapsidated Tat decreased the efficiency of reverse transcription by ~50% and transcription by more than 90%. We thus identified a Tat-CypA-CA complex that enables Tat encapsidation and showed that encapsidated Tat is required to initiate robust viral transcription and thus viral production at the beginning of cell infection, before neosynthesized Tat becomes available. IMPORTANCE The viral transactivating protein Tat has been shown to stimulate several steps of HIV gene expression. It was found to facilitate reverse transcription. Moreover, Tat is strictly required for the transcription of viral genes. Although the presence of Tat within HIV virions would undoubtedly favor these steps and therefore enable the incoming virus to boost initial viral production, whether and how Tat is present within virions has been a matter a debate. We here described and characterized a tripartite complex between Tat, HIV capsid protein, and the cellular chaperone cyclophilin A that enables efficient and specific Tat encapsidation within HIV virions. We further showed that Tat encapsidation is required for the virus to efficiently initiate infection and viral production. This effect is mainly due to the transcriptional activity of Tat.

Development of LM-41 and AF-2112, two flufenamic acid-derived TEAD inhibitors obtained through the replacement of the trifluoromethyl group by aryl rings.

The Hippo pathway regulates organ size and tissue homeostasis by controlling cell proliferation and apoptosis. The YAP-TEAD transcription factor, the downstream effector of the Hippo pathway, regulates the expression of genes such as CTGF, Cyr61, Axl and NF2. Aberrant Hippo activity has been identified in multiple types of cancers. Flufenamic acid (FA) was reported to bind in a liphophilic TEAD palmitic acid (PA) pocket, leading to reduction of the expression of Axl and NF2. Here, we show that the replacement of the trifluoromethyl moiety in FA by aromatic groups, directly connected to the scaffold or separated by a linker, leads to compounds with better affinity to TEAD. Co-crystallization studies show that these compounds bind similarly to FA, but deeper within the PA pocket. Our studies identified LM-41 and AF-2112 as two TEAD binders that strongly reduce the expression of CTGF, Cyr61, Axl and NF2. LM-41 gave the strongest reduction of migration of human MDA-MB-231 breast cancer cells.

Cyclophilin A Isomerisation of Septin 2 Mediates Abscission during Cytokinesis.

The isomerase activity of Cyclophilin A is important for midbody abscission during cell division, however, to date, midbody substrates remain unknown. In this study, we report that the GTP-binding protein Septin 2 interacts with Cyclophilin A. We highlight a dynamic series of Septin 2 phenotypes at the midbody, previously undescribed in human cells. Furthermore, Cyclophilin A depletion or loss of isomerase activity is sufficient to induce phenotypic Septin 2 defects at the midbody. Structural and molecular analysis reveals that Septin 2 proline 259 is important for interaction with Cyclophilin A. Moreover, an isomerisation-deficient EGFP-Septin 2 proline 259 mutant displays defective midbody localisation and undergoes impaired abscission, which is consistent with data from cells with loss of Cyclophilin A expression or activity. Collectively, these data reveal Septin 2 as a novel interacting partner and isomerase substrate of Cyclophilin A at the midbody that is required for abscission during cytokinesis in cancer cells.

Small-Molecule Cyclophilin Inhibitors Potently Reduce Platelet Procoagulant Activity.

Procoagulant platelets are associated with an increased risk for thrombosis. Procoagulant platelet formation is mediated via Cyclophilin D (CypD) mediated opening of the mitochondrial permeability transition pore. Inhibiting CypD activity could therefore be an interesting approach to limiting thrombosis. In this study, we investigated the potential of two novel, non-immunosuppressive, non-peptidic small-molecule cyclophilin inhibitors (SMCypIs) to limit thrombosis in vitro, in comparison with the cyclophilin inhibitor and immunosuppressant Cyclosporin A (CsA). Both cyclophilin inhibitors significantly decreased procoagulant platelet formation upon dual-agonist stimulation, shown by a decreased phosphatidylserine (PS) exposure, as well as a reduction in the loss of mitochondrial membrane potential. Furthermore, the SMCypIs potently reduced procoagulant platelet-dependent clotting time, as well as fibrin formation under flow, comparable to CsA. No effect was observed on agonist-induced platelet activation measured by P-selectin expression, as well as CypA-mediated integrin αIIbß3 activation. Importantly, whereas CsA increased Adenosine 5'-diphosphate (ADP)-induced platelet aggregation, this was unaffected in the presence of the SMCypIs. We here demonstrate specific cyclophilin inhibition does not affect normal platelet function, while a clear reduction in procoagulant platelets is observed. Reducing platelet procoagulant activity by inhibiting cyclophilins with SMCypIs forms a promising strategy to limit thrombosis.

A Histone Deacetylase (HDAC) Inhibitor with Pleiotropic In Vitro Anti-Toxoplasma and Anti-Plasmodium Activities Controls Acute and Chronic Toxoplasma Infection in Mice.

Toxoplasmosis is a highly prevalent human disease, and virulent strains of this parasite emerge from wild biotopes. Here, we report on the potential of a histone deacetylase (HDAC) inhibitor we previously synthesized, named JF363, to act in vitro against a large panel of Toxoplasma strains, as well as against the liver and blood stages of Plasmodium parasites, the causative agents of malaria. In vivo administration of the drug significantly increases the survival of mice during the acute phase of infection by T. gondii, thus delaying its spreading. We further provide evidence of the compound's efficiency in controlling the formation of cysts in the brain of T. gondii-infected mice. A convincing docking of the JF363 compound in the active site of the five annotated ME49 T. gondii HDACs was performed by extensive sequence-structure comparison modeling. The resulting complexes show a similar mode of binding in the five paralogous structures and a quite similar prediction of affinities in the micromolar range. Altogether, these results pave the way for further development of this compound to treat acute and chronic toxoplasmosis. It also shows promise for the future development of anti-Plasmodium therapeutic interventions.

A Phenyl-Pyrrolidine Derivative Reveals a Dual Inhibition Mechanism of Myocardial Mitochondrial Permeability Transition Pore, Which Is Limited by Its Myocardial Distribution.

Mitochondrial permeability transition pore (mPTP) opening is a key event in cell death during myocardial ischemia reperfusion. Inhibition of its modulator cyclophilin D (CypD) by cyclosporine A (CsA) reduces ischemia-reperfusion injury. The use of cyclosporine A in this indication is debated; however, targeting mPTP remains a major goal to achieve. We investigated the protective effects of a new original small-molecule cyclophilin inhibitor C31, which was specifically designed to target CypD. CypD peptidylprolyl cis-trans isomerase (PPIase) activity was assessed by the standard chemotrypsin-coupled assay. The effects of C31 on mPTP opening were investigated in isolated mouse cardiac mitochondria by measuring mitochondrial swelling and calcium retention capacity (CRC) in rat H9C2 cardiomyoblasts and in adult mouse cardiomyocytes by fluorescence microscopy in isolated perfused mouse hearts and ex vivo after drug infusion in mice. C31 potently inhibited CypD PPIase activity and mitochondrial swelling. C31 was more effective at increasing mitochondrial CRC than CsA and was still able to increase CRC in Ppif -/- (CypD-inactivated) cardiac mitochondria. C31 delayed both mPTP opening and cell death in cardiomyocytes subjected to hypoxia reoxygenation. However, high concentrations of both drugs were necessary to reduce mPTP opening in isolated perfused hearts, and neither CsA nor C31 inhibited mPTP opening in heart after in vivo infusion, underlying the importance of myocardial drug distribution for cardioprotection. C31 is an original inhibitor of mPTP opening involving both CypD-dependent and -independent mechanisms. It constitutes a promising new cytoprotective agent. Optimization of its pharmacokinetic properties is now required prior to its use against cardiac ischemia-reperfusion injury. SIGNIFICANCE STATEMENT: This study demonstrates that the new cyclophilin inhibitor C31 potently inhibits cardiac mitochondrial permeability transition pore (mPTP) opening in vitro and ex vivo. The dual mechanism of action of C31 allows the prevention of mPTP opening beyond cyclophilin D inhibition. Further development of the compound might bring promising drug candidates for cardioprotection. However, the lack of effect of both C31 and cyclosporine A after systemic administration demonstrates the difficulties of targeting myocardial mitochondria in vivo and should be taken into account in cardioprotective strategies.

Small-Molecule Inhibitors of Cyclophilins Block Opening of the Mitochondrial Permeability Transition Pore and Protect Mice From Hepatic Ischemia/Reperfusion Injury.

BACKGROUND & AIMS: Hepatic ischemia/reperfusion injury is a complication of liver surgery that involves mitochondrial dysfunction resulting from mitochondrial permeability transition pore (mPTP) opening. Cyclophilin D (PPIF or CypD) is a peptidyl-prolyl cis-trans isomerase that regulates mPTP opening in the inner mitochondrial membrane. We investigated whether and how recently created small-molecule inhibitors of CypD prevent opening of the mPTP in hepatocytes and the resulting effects in cell models and livers of mice undergoing ischemia/reperfusion injury. METHODS: We measured the activity of 9 small-molecule inhibitors of cyclophilins in an assay of CypD activity. The effects of the small-molecule CypD inhibitors or vehicle on mPTP opening were assessed by measuring mitochondrial swelling and calcium retention in isolated liver mitochondria from C57BL/6J (wild-type) and Ppif-/- (CypD knockout) mice and in primary mouse and human hepatocytes by fluorescence microscopy. We induced ischemia/reperfusion injury in livers of mice given a small-molecule CypD inhibitor or vehicle before and during reperfusion and collected samples of blood and liver for histologic analysis. RESULTS: The compounds inhibited peptidyl-prolyl isomerase activity (half maximal inhibitory concentration values, 0.2-16.2 µmol/L) and, as a result, calcium-induced mitochondrial swelling, by preventing mPTP opening (half maximal inhibitory concentration values, 1.4-132 µmol/L) in a concentration-dependent manner. The most potent inhibitor (C31) bound CypD with high affinity and inhibited swelling in mitochondria from livers of wild-type and Ppif-/- mice (indicating an additional, CypD-independent effect on mPTP opening) and in primary human and mouse hepatocytes. Administration of C31 in mice with ischemia/reperfusion injury before and during reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage compared with vehicle. CONCLUSIONS: Recently created small-molecule inhibitors of CypD reduced calcium-induced swelling in mitochondria from mouse and human liver tissues. Administration of these compounds to mice during ischemia/reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage. These compounds might be developed to protect patients from ischemia/reperfusion injury after liver surgery or for other hepatic or nonhepatic disorders related to abnormal mPTP opening.

A Tiny Change Makes a Big Difference in the Anti-Parasitic Activities of an HDAC Inhibitor.

We previously synthesized an hydroxamate derivative (N-hydroxy-4-[2-(3- methoxyphenyl)acetamido]benzamide) named 363 with potent anti-Toxoplasma gondii activity and histone deacetylase inhibitor (HDACi) effects. Here we show that 1-N-hydroxy-4-N- [(2-methoxyphenyl)methyl]benzene-1,4-dicarboxamide, a 363 isomer, does not have antiparasitic potency and has a 13-fold decrease in HDACi activity. The in silico modeling of T. gondii HDACs of the type II strain discloses identity varying from 25% to 62% on more than 250 residues for S8EP32_TOXG and A0A125YPH4_TOXGM. We observed a high conservation degree with the human HDAC2 (53% and 64% identity, respectively) and a moderate one with the human HDAC8 (30-40%). Two other TgHDACs, S8F6L4_TOXGM and S8GEI3_TOXGM, were identified as displaying a higher similarity with some bacterial orthologs (~35%) than with the human enzymes (~25%). The docking in parallel of the two compounds on the models generated allowed us to gain insights on the docking of these hydroxamate derivatives that guide their specificity and potency against T. gondii histone deacetylase. This information would constitute the rationale from which more specific derivatives can be synthetized.

Characterization of the Anti-Hepatitis C Virus Activity of New Nonpeptidic Small-Molecule Cyclophilin Inhibitors with the Potential for Broad Anti-Flaviviridae Activity.

Although members of the Flaviviridae display high incidence, morbidity, and mortality rates, the development of specific antiviral drugs for each virus is unlikely. Cyclophilins, a family of host peptidyl-prolyl cis-trans isomerases (PPIases), play a pivotal role in the life cycles of many viruses and therefore represent an attractive target for broad-spectrum antiviral development. We report here the pangenotypic anti-hepatitis C virus (HCV) activity of a small-molecule cyclophilin inhibitor (SMCypI). Mechanistic and modeling studies revealed that the SMCypI bound to cyclophilin A in competition with cyclosporine (CsA), inhibited its PPIase activity, and disrupted the CypA-nonstructural protein 5A (NS5A) interaction. Resistance selection showed that the lead SMCypI hardly selected amino acid substitutions conferring low-level or no resistance in vitro Interestingly, the SMCypI selected D320E and Y321H substitutions, located in domain II of the NS5A protein. These substitutions were previously associated with low-level resistance to cyclophilin inhibitors such as alisporivir. Finally, the SMCypI inhibited the replication of other members of the Flaviviridae family with higher 50% effective concentrations (EC50s) than for HCV. Thus, because of its chemical plasticity and simplicity of synthesis, our new family of SMCypIs represents a promising new class of drugs with the potential for broad-spectrum anti-Flaviviridae activity as well as an invaluable tool to explore the role of cyclophilins in viral life cycles.

Aminoglycoside binding and catalysis specificity of aminoglycoside 2″-phosphotransferase IVa: A thermodynamic, structural and kinetic study.

BACKGROUND: Aminoglycoside O-phosphotransferases make up a large class of bacterial enzymes that is widely distributed among pathogens and confer a high resistance to several clinically used aminoglycoside antibiotics. Aminoglycoside 2″-phosphotransferase IVa, APH(2″)-IVa, is an important member of this class, but there is little information on the thermodynamics of aminoglycoside binding and on the nature of its rate-limiting step. METHODS: We used isothermal titration calorimetry, electrostatic potential calculations, molecular dynamics simulations and X-ray crystallography to study the interactions between the enzyme and different aminoglycosides. We determined the rate-limiting step of the reaction by the means of transient kinetic measurements. RESULTS: For the first time, Kd values were determined directly for APH(2″)-IVa and different aminoglycosides. The affinity of the enzyme seems to anti-correlate with the molecular weight of the ligand, suggesting a limited degree of freedom in the binding site. The main interactions are electrostatic bonds between the positively charged amino groups of aminoglycosides and Glu or Asp residues of APH. In spite of the significantly different ratio Kd/Km, there is no large difference in the transient kinetics obtained with the different aminoglycosides. We show that a product release step is rate-limiting for the overall reaction. CONCLUSIONS: APH(2″)-IVa has a higher affinity for aminoglycosides carrying an amino group in 2' and 6', but tighter bindings do not correlate with higher catalytic efficiencies. As with APH(3')-IIIa, an intermediate containing product is preponderant during the steady state. GENERAL SIGNIFICANCE: This intermediate may constitute a good target for future drug design.

New imidazoquinoxaline derivatives: Synthesis, biological evaluation on melanoma, effect on tubulin polymerization and structure-activity relationships.

Microtubules are considered as important targets of anticancer therapy. EAPB0503 and its structural imidazo[1,2-a]quinoxaline derivatives are major microtubule-interfering agents with potent anticancer activity. In this study, the synthesis of several new derivatives of EAPB0503 is described, and the anticancer efficacy of 13 novel derivatives on A375 human melanoma cell line is reported. All new compounds show significant antiproliferative activity with IC50 in the range of 0.077-122µM against human melanoma cell line (A375). Direct inhibition of tubulin polymerization assay in vitro is also assessed. Results show that compounds 6b, 6e, 6g, and EAPB0503 highly inhibit tubulin polymerization with percentages of inhibition of 99%, 98%, 90%, and 84% respectively. Structure-activity relationship studies within the series are also discussed in line with molecular docking studies into the colchicine-binding site of tubulin.

Vpr expression abolishes the capacity of HIV-1 infected cells to repair uracilated DNA.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein binds to the cellular uracil-DNA glycosylase UNG2 and induces its degradation through the assembly with the DDB1-CUL4 ubiquitin ligase complex. This interaction counteracts the antiviral activity exerted by UNG2 on HIV-1 gene transcription, as previously reported by us. In this work, we show that Vpr expression in the context of HIV-1 infection markedly decreases UNG2 expression in transformed or primary CD4(+) T lymphocytes. We demonstrate for the first time that Vpr-UNG2 interaction significantly impairs the uracil excision activity of infected cells. The loss of uracil excision activity coincides with a significant accumulation of uracilated bases in the genome of infected cells without changes in cell division. Although UNG2 expression and uracil-DNA glycosylase activity are recovered after the peak of retroviral replication, the mutagenic effect of transient DNA uracilation in cycling cells should be taken into account. Therefore, the possible consequences of Vpr-mediated temporary depletion of endogenous nuclear UNG2 and subsequent alteration of the genomic integrity of infected cells need to be evaluated in the physiopathogenesis of HIV infection.

Inhibition of RNA binding to hepatitis C virus RNA-dependent RNA polymerase: a new mechanism for antiviral intervention.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) is a key target for antiviral intervention. The goal of this study was to identify the binding site and unravel the molecular mechanism by which natural flavonoids efficiently inhibit HCV RdRp. Screening identified the flavonol quercetagetin as the most potent inhibitor of HCV RdRp activity. Quercetagetin was found to inhibit RdRp through inhibition of RNA binding to the viral polymerase, a yet unknown antiviral mechanism. X-ray crystallographic structure analysis of the RdRp-quercetagetin complex identified quercetagetin's binding site at the entrance of the RNA template tunnel, confirming its original mode of action. This antiviral mechanism was associated with a high barrier to resistance in both site-directed mutagenesis and long-term selection experiments. In conclusion, we identified a new mechanism for non-nucleoside inhibition of HCV RdRp through inhibition of RNA binding to the enzyme, a mechanism associated with broad genotypic activity and a high barrier to resistance. Our results open the way to new antiviral approaches for HCV and other viruses that use an RdRp based on RNA binding inhibition, that could prove to be useful in human, animal or plant viral infections.

Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity.

In vertebrates, smooth muscle cells (SMCs) can reversibly switch between contractile and proliferative phenotypes. This involves various molecular mechanisms to reactivate developmental signaling pathways and induce cell dedifferentiation. The protein RBPMS2 regulates early development and plasticity of digestive SMCs by inhibiting the bone morphogenetic protein pathway through its interaction with NOGGIN mRNA. RBPMS2 contains only one RNA recognition motif (RRM) while this motif is often repeated in tandem or associated with other functional domains in RRM-containing proteins. Herein, we show using an extensive combination of structure/function analyses that RBPMS2 homodimerizes through a particular sequence motif (D-x-K-x-R-E-L-Y-L-L-F: residues 39-51) located in its RRM domain. We also show that this specific motif is conserved among its homologs and paralogs in vertebrates and in its insect and worm orthologs (CPO and MEC-8, respectively) suggesting a conserved molecular mechanism of action. Inhibition of the dimerization process through targeting a conserved leucine inside of this motif abolishes the capacity of RBPMS2 to interact with the translational elongation eEF2 protein, to upregulate NOGGIN mRNA in vivo and to drive SMC dedifferentiation. Our study demonstrates that RBPMS2 possesses an RRM domain harboring both RNA-binding and protein-binding properties and that the newly identified RRM-homodimerization motif is crucial for the function of RBPMS2 at the cell and tissue levels.

Two crystal structures of the FK506-binding domain of Plasmodium falciparum FKBP35 in complex with rapamycin at high resolution.

Antimalarial chemotherapy continues to be challenging in view of the emergence of drug resistance, especially artemisinin resistance in Southeast Asia. It is critical that novel antimalarial drugs are identified that inhibit new targets with unexplored mechanisms of action. It has been demonstrated that the immunosuppressive drug rapamycin, which is currently in clinical use to prevent organ-transplant rejection, has antimalarial effects. The Plasmodium falciparum target protein is PfFKBP35, a unique immunophilin FK506-binding protein (FKBP). This protein family binds rapamycin, FK506 and other immunosuppressive and non-immunosuppressive macrolactones. Here, two crystallographic structures of rapamycin in complex with the FK506-binding domain of PfFKBP35 at high resolution, in both its oxidized and reduced forms, are reported. In comparison with the human FKBP12-rapamycin complex reported previously, the structures reveal differences in the ß4-ß6 segment that lines the rapamycin binding site. Structural differences between the Plasmodium protein and human hFKBP12 include the replacement of Cys106 and Ser109 by His87 and Ile90, respectively. The proximity of Cys106 to the bound rapamycin molecule (4-5 Å) suggests possible routes for the rational design of analogues of rapamycin with specific antiparasitic activity. Comparison of the structures with the PfFKBD-FK506 complex shows that both drugs interact with the same binding-site residues. These two new structures highlight the structural differences and the specific interactions that must be kept in consideration for the rational design of rapamycin analogues with antimalarial activity that specifically bind to PfFKBP35 without immunosuppressive effects.

Combining 'dry' co-crystallization and in situ diffraction to facilitate ligand screening by X-ray crystallography.

X-ray crystallography is an established technique for ligand screening in fragment-based drug-design projects, but the required manual handling steps - soaking crystals with ligand and the subsequent harvesting - are tedious and limit the throughput of the process. Here, an alternative approach is reported: crystallization plates are pre-coated with potential binders prior to protein crystallization and X-ray diffraction is performed directly 'in situ' (or in-plate). Its performance is demonstrated on distinct and relevant therapeutic targets currently being studied for ligand screening by X-ray crystallography using either a bending-magnet beamline or a rotating-anode generator. The possibility of using DMSO stock solutions of the ligands to be coated opens up a route to screening most chemical libraries.

Protein-ligand structure guided by backbone and side-chain proton chemical shift perturbations.

The fragment-based drug design approach consists of screening libraries of fragment-like ligands, to identify hits that typically bind the protein target with weak affinity (100 µM-5 mM). The determination of the protein-fragment complex 3D structure constitutes a crucial step for uncovering the key interactions responsible for the protein-ligand recognition, and for growing the initial fragment into potent active compounds. The vast majority of fragments are aromatic compounds that induce chemical shift perturbations (CSP) on protein NMR spectra. These experimental CSPs can be quantitatively used to guide the ligand docking, through the comparison between experimental CSPs and CSP back-calculation based on the ring current effect. Here we implemented the CSP back-calculation into the scoring function of the program PLANTS. We compare the results obtained with CSPs measured either on amide or aliphatic protons of the human peroxiredoxin 5. We show that the different kinds of protons lead to different results for resolving the 3D structures of protein-fragment complexes, with the best results obtained with the Hα protons.

Cancer selective cell death induction by a bivalent CK2 inhibitor targeting the ATP site and the allosteric αD pocket.

Although the involvement of protein kinase CK2 in cancer is well-documented, there is a need for selective CK2 inhibitors suitable for investigating CK2 specific roles in cancer-related biological pathways and further exploring its therapeutic potential. Here, we report the discovery of AB668, an outstanding selective inhibitor that binds CK2 through a bivalent mode, interacting both at the ATP site and an allosteric αD pocket unique to CK2. Using caspase activation assay, live-cell imaging, and transcriptomic analysis, we have compared the effects of this bivalent inhibitor to representative ATP-competitive inhibitors, CX-4945, and SGC-CK2-1. Our results show that in contrast to CX-4945 or SGC-CK2-1, AB668, by targeting the CK2 αD pocket, has a distinct mechanism of action regarding its anti-cancer activity, inducing apoptotic cell death in several cancer cell lines and stimulating distinct biological pathways in renal cell carcinoma.

e-Drug3D: 3D structure collections dedicated to drug repurposing and fragment-based drug design.

MOTIVATION: In the drug discovery field, new uses for old drugs, selective optimization of side activities and fragment-based drug design (FBDD) have proved to be successful alternatives to high-throughput screening. e-Drug3D is a database of 3D chemical structures of drugs that provides several collections of ready-to-screen SD files of drugs and commercial drug fragments. They are natural inputs in studies dedicated to drug repurposing and FBDD. AVAILABILITY: e-Drug3D collections are freely available at http://chemoinfo.ipmc.cnrs.fr/e-drug3d.html either for download or for direct in silico web-based screenings.

Linkers in fragment-based drug design: an overview of the literature.

INTRODUCTION: In fragment-based drug design, fragment linking is a popular strategy where two fragments binding to different sub-pockets of a target are linked together. This attractive method remains challenging especially due to the design of ideal linkers. AREAS COVERED: The authors review the types of linkers and chemical reactions commonly used to the synthesis of linkers, including those utilized in protein-templated fragment self-assembly, where fragments are directly linked in the presence of the protein. Finally, they detail computational workflows and software including generative models that have been developed for fragment linking. EXPERT OPINION: The authors believe that fragment linking offers key advantages for compound design, particularly for the design of bivalent inhibitors linking two distinct pockets of the same or different subunits. On the other hand, more studies are needed to increase the potential of protein-templated approaches in FBDD. Important computational tools such as structure-based de novo software are emerging to select suitable linkers. Fragment linking will undoubtedly benefit from developments in computational approaches and machine learning models.