Olivier Michielin

SwissParam: A fast force field generation tool for small organic molecules

The drug discovery process has been deeply transformed recently by the use of computational ligand‐based or structure‐based methods, helping the lead compounds identification and optimization, and finally the delivery of new drug candidates more quickly and at lower cost. Structure‐based computational methods for drug discovery mainly involve ligand‐protein docking and rapid binding free energy estimation, both of which require force field parameterization for many drug candidates. Here, we present a fast force field generation tool, called SwissParam, able to generate, for arbitrary small organic molecule, topologies, and parameters based on the Merck molecular force field, but in a functional form that is compatible with the CHARMM force field. Output files can be used with CHARMM or GROMACS. The topologies and parameters generated by SwissParam are used by the docking software EADock2 and EADock DSS to describe the small molecules to be docked, whereas the protein is described by the CHARMM force field, and allow them to reach success rates ranging from 56 to 78%. We have also developed a rapid binding free energy estimation approach, using SwissParam for ligands and CHARMM22/27 for proteins, which requires only a short minimization to reproduce the experimental binding free energy of 214 ligand‐protein complexes involving 62 different proteins, with a standard error of 2.0 kcal mol−1, and a correlation coefficient of 0.74. Together, these results demonstrate the relevance of using SwissParam topologies and parameters to describe small organic molecules in computer‐aided drug design applications, together with a CHARMM22/27 description of the target protein. SwissParam is available free of charge for academic users at www.swissparam.ch.

SwissDock, a protein-small molecule docking web service based on EADock DSS

Most life science processes involve, at the atomic scale, recognition between two molecules. The prediction of such interactions at the molecular level, by so-called docking software, is a non-trivial task. Docking programs have a wide range of applications ranging from protein engineering to drug design. This article presents SwissDock, a web server dedicated to the docking of small molecules on target proteins. It is based on the EADock DSS engine, combined with setup scripts for curating common problems and for preparing both the target protein and the ligand input files. An efficient Ajax/HTML interface was designed and implemented so that scientists can easily submit dockings and retrieve the predicted complexes. For automated docking tasks, a programmatic SOAP interface has been set up and template programs can be downloaded in Perl, Python and PHP. The web site also provides an access to a database of manually curated complexes, based on the Ligand Protein Database. A wiki and a forum are available to the community to promote interactions between users. The SwissDock web site is available online at http://www.swissdock.ch. We believe it constitutes a step toward generalizing the use of docking tools beyond the traditional molecular modeling community.

Exome sequencing identifies recurrent somatic MAP2K1 and MAP2K2 mutations in melanoma

We performed exome sequencing to detect somatic mutations in protein-coding regions in seven melanoma cell lines and donor-matched germline cells. All melanoma samples had high numbers of somatic mutations, which showed the hallmark of UV-induced DNA repair. Such a hallmark was absent in tumor sample–specific mutations in two metastases derived from the same individual. Two melanomas with non-canonical BRAF mutations harbored gain-of-function MAP2K1 and MAP2K2 (MEK1 and MEK2, respectively) mutations, resulting in constitutive ERK phosphorylation and higher resistance to MEK inhibitors. Screening a larger cohort of individuals with melanoma revealed the presence of recurring somatic MAP2K1 and MAP2K2 mutations, which occurred at an overall frequency of 8%. Furthermore, missense and nonsense somatic mutations were frequently found in three candidate melanoma genes, FAT4, LRP1B and DSC1.

Inhibition of the shade avoidance response by formation of non‐DNA binding bHLH heterodimers

In shade‐intolerant plants such as Arabidopsis, a reduction in the red/far‐red (R/FR) ratio, indicative of competition from other plants, triggers a suite of responses known as the shade avoidance syndrome (SAS). The phytochrome photoreceptors measure the R/FR ratio and control the SAS. The phytochrome‐interacting factors 4 and 5 (PIF4 and PIF5) are stabilized in the shade and are required for a full SAS, whereas the related bHLH factor HFR1 (long hypocotyl in FR light) is transcriptionally induced by shade and inhibits this response. Here we show that HFR1 interacts with PIF4 and PIF5 and limits their capacity to induce the expression of shade marker genes and to promote elongation growth. HFR1 directly inhibits these PIFs by forming non‐DNA‐binding heterodimers with PIF4 and PIF5. Our data indicate that PIF4 and PIF5 promote SAS by directly binding to G‐boxes present in the promoter of shade marker genes, but their action is limited later in the shade when HFR1 accumulates and forms non‐DNA‐binding heterodimers. This negative feedback loop is important to limit the response of plants to shade.

The endocrine disruptor monoethyl-hexyl-phthalate is a selective peroxisome proliferator-activated receptor gamma modulator that promotes adipogenesis

The ability of pollutants to affect human health is a major concern, justified by the wide demonstration that reproductive functions are altered by endocrine disrupting chemicals. The definition of endocrine disruption is today extended to broader endocrine regulations, and includes activation of metabolic sensors, such as the peroxisome proliferator-activated receptors (PPARs). Toxicology approaches have demonstrated that phthalate plasticizers can directly influence PPAR activity. What is now missing is a detailed molecular understanding of the fundamental basis of endocrine disrupting chemical interference with PPAR signaling. We thus performed structural and functional analyses that demonstrate how monoethyl-hexyl-phthalate (MEHP) directly activates PPARγ and promotes adipogenesis, albeit to a lower extent than the full agonist rosiglitazone. Importantly, we demonstrate that MEHP induces a selective activation of different PPARγ target genes. Chromatin immunoprecipitation and fluorescence microscopy in living cells reveal that this selective activity correlates with the recruitment of a specific subset of PPARγ coregulators that includes Med1 and PGC-1α, but not p300 and SRC-1. These results highlight some key mechanisms in metabolic disruption but are also instrumental in the context of selective PPAR modulation, a promising field for new therapeutic development based on PPAR modulation.

Vemurafenib in patients with BRAFV600 mutation-positive melanoma with symptomatic brain metastases: Final results of an open-label pilot study

BACKGROUND & AIM Brain metastases are frequent in patients with metastatic melanoma, indicating poor prognosis. We investigated the BRAF kinase inhibitor vemurafenib in patients with advanced melanoma with symptomatic brain metastases. METHODS This open-label trial assessed vemurafenib (960mg twice a day) in patients with BRAF(V600) mutation-positive metastatic melanoma with non-resectable, previously treated brain metastases. The primary end-point was safety. Secondary end-points included best overall response rate, and progression-free and overall survival. RESULTS Twenty-four patients received vemurafenib for a median treatment duration of 3.8 (0.1-11.3) months. The majority of discontinuations were due to disease progression (n=22). Twenty-three of 24 patients reported at least one adverse event (AE). Grade 3 AEs were reported in four (17%; 95% confidence interval [CI], 4.7-37.4%) patients and included cutaneous squamous cell carcinoma in four patients. Median progression-free survival was 3.9 (95% CI, 3.0-5.5) months, and median survival was 5.3 (95% CI, 3.9-6.6) months. An overall partial response (PR) at both intracranial and extracranial sites was achieved in 10 of 24 (42%; 95% CI, 22.1-63.4) evaluable patients, with stable disease in nine (38%; 95% CI, 18.8-59.4) patients. Of 19 patients with measurable intracranial disease, seven (37%) achieved >30% intracranial tumour regression, and three (16%; 95% CI, 3.4-39.6%) achieved a confirmed PR. Other signs of improvement included reduced need for corticosteroids and enhanced performance status. CONCLUSIONS Vemurafenib can be safely used in patients with advanced symptomatic melanoma that has metastasised to the brain and can result in meaningful tumour regression.

Anti–CTLA-4 therapy broadens the melanoma-reactive CD8+ T cell response

Anti–CTLA-4 treatment increases the diversity of the melanoma-specific CD8 T cell response. Anti–CTLA-4 Therapy Expands T Cell Range An antibody to the immune inhibitory molecule CTLA-4, ipilimumab, can improve survival in patients with advanced melanoma. However, how anti–CTLA-4 works to improve the tumor immune response in humans remains unclear. Now, Kvistborg et al. show that although the magnitude of T cell responses was largely unaltered after therapy, the number of different T cell responses was significantly increased. Indeed, this increased breadth suggests that anti–CTLA-4 may work by increasing priming of T cells to tumor-related antigens rather than boosting preexisting immune responses. If so, other strategies that improve the range of T cells may have similar success battling cancer. Anti–CTLA-4 treatment improves the survival of patients with advanced-stage melanoma. However, although the anti–CTLA-4 antibody ipilimumab is now an approved treatment for patients with metastatic disease, it remains unknown by which mechanism it boosts tumor-specific T cell activity. In particular, it is unclear whether treatment amplifies previously induced T cell responses or whether it induces new tumor-specific T cell reactivities. Using a combination ultraviolet (UV)–induced peptide exchange and peptide–major histocompatibility complex (pMHC) combinatorial coding, we monitored immune reactivity against a panel of 145 melanoma-associated epitopes in a cohort of patients receiving anti–CTLA-4 treatment. Comparison of pre- and posttreatment T cell reactivities in peripheral blood mononuclear cell samples of 40 melanoma patients demonstrated that anti–CTLA-4 treatment induces a significant increase in the number of detectable melanoma-specific CD8 T cell responses (P = 0.0009). In striking contrast, the magnitude of both virus-specific and melanoma-specific T cell responses that were already detected before start of therapy remained unaltered by treatment (P = 0.74). The observation that anti–CTLA-4 treatment induces a significant number of newly detected T cell responses—but only infrequently boosts preexisting immune responses—provides strong evidence for anti–CTLA-4 therapy–enhanced T cell priming as a component of the clinical mode of action.

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

Ipilimumab-dependent cell-mediated cytotoxicity of regulatory T cells ex vivo by nonclassical monocytes in melanoma patients.

Significance mAbs directed to inhibitory immune receptors represent a very promising class of immunotherapeutics. This study suggests a potential mechanism of action of ipilimumab (a fully human anti–cytotoxic T lymphocyte–associated antigen 4), by which FcγRIIIA (CD16)-expressing nonclassical monocytes kill regulatory T cells ex vivo via antibody-dependent cell-mediated cytotoxicity. Notably, at baseline, responder patients display significantly higher peripheral frequencies of nonclassical monocytes and a selective enrichment in tumor-infiltrating CD68+CD16+ macrophages compared with nonresponder patients. If further confirmed, these findings may contribute to the generation of predictive biomarker panels, antibody design, and the development of rational combination therapies to promote antitumor immunity. Enhancing immune responses with immune-modulatory monoclonal antibodies directed to inhibitory immune receptors is a promising modality in cancer therapy. Clinical efficacy has been demonstrated with antibodies blocking inhibitory immune checkpoints such as cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) or PD-1/PD-L1. Treatment with ipilimumab, a fully human CTLA-4–specific mAb, showed durable clinical efficacy in metastatic melanoma; its mechanism of action is, however, only partially understood. This is a study of 29 patients with advanced cutaneous melanoma treated with ipilimumab. We analyzed peripheral blood mononuclear cells (PBMCs) and matched melanoma metastases from 15 patients responding and 14 not responding to ipilimumab by multicolor flow cytometry, antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and immunohistochemistry. PBMCs and matched tumor biopsies were collected 24 h before (i.e., baseline) and up to 4 wk after ipilimumab. Our findings show, to our knowledge for the first time, that ipilimumab can engage ex vivo FcγRIIIA (CD16)-expressing, nonclassical monocytes resulting in ADCC-mediated lysis of regulatory T cells (Tregs). In contrast, classical CD14++CD16− monocytes are unable to do so. Moreover, we show that patients responding to ipilimumab display significantly higher baseline peripheral frequencies of nonclassical monocytes compared with nonresponder patients. In the tumor microenvironment, responders have higher CD68+/CD163+ macrophage ratios at baseline and show decreased Treg infiltration after treatment. Together, our results suggest that anti–CTLA-4 therapy may target Tregs in vivo. Larger translational studies are, however, warranted to substantiate this mechanism of action of ipilimumab in patients.

Antigenicity and immunogenicity of Melan-A/MART-1 derived peptides as targets for tumor reactive CTL in human melanoma.

Summary: Some cancer patients mount spontaneous T‐ and B‐cell responses against their tumor cells. Autologous tumor reactive CD8 cytolytic T lymphocyte (CTL) and CD4 T‐cell clones as well as antibodies from these patients have been used for the identification of genes encoding the target antigens. This knowledge opened the way for new approaches to the immunotherapy of cancer. In this review, we describe the characterization of the structure‐function properties of the melanocyte/melanoma tumor antigen Melan‐A/MART‐1, the assessment of the T‐cell repertoire available against this antigen in healthy individuals, and the analysis of naturally acquired and/or vaccine‐induced CTL responses to this antigen in patients with metastatic melanoma.