Structure-Guided Discovery of Potent and Selective DYRK1A Inhibitors.

The kinase DYRK1A is an attractive target for drug discovery programs due to its implication in multiple diseases. Through a fragment screen, we identified a simple biaryl compound that is bound to the DYRK1A ATP site with very high efficiency, although with limited selectivity. Structure-guided optimization cycles enabled us to convert this fragment hit into potent and selective DYRK1A inhibitors. Exploiting the structural differences in DYRK1A and its close homologue DYRK2, we were able to fine-tune the selectivity of our inhibitors. Our best compounds potently inhibited DYRK1A in the cell culture and in vivo and demonstrated drug-like properties. The inhibition of DYRK1A in vivo translated into dose-dependent tumor growth inhibition in a model of ovarian carcinoma.

Discovery of S64315, a Potent and Selective Mcl-1 Inhibitor.

Myeloid cell leukemia 1 (Mcl-1) has emerged as an attractive target for cancer therapy. It is an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Here we report the discovery of our clinical candidate S64315, a selective small molecule inhibitor of Mcl-1. Starting from a fragment derived lead compound, we have conducted structure guided optimization that has led to a significant (3 log) improvement of target affinity as well as cellular potency. The presence of hindered rotation along a biaryl axis has conferred high selectivity to the compounds against other members of the Bcl-2 family. During optimization, we have also established predictive PD markers of Mcl-1 inhibition and achieved both efficient in vitro cell killing and tumor regression in Mcl-1 dependent cancer models. The preclinical candidate has drug-like properties that have enabled its development and entry into clinical trials.

S55746 is a novel orally active BCL-2 selective and potent inhibitor that impairs hematological tumor growth.

Escape from apoptosis is one of the major hallmarks of cancer cells. The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Thus, BCL-2 has become an attractive target for therapeutic strategy in cancer, as demonstrated by the recent approval of ABT-199 (Venclexta™) in relapsed or refractory Chronic Lymphocytic Leukemia with 17p deletion. Here, we describe a novel orally bioavailable BCL-2 selective and potent inhibitor called S55746 (also known as BCL201). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile demonstrates no significant binding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. Accordingly, S55746 has no cytotoxic activity on BCL-XL-dependent cells, such as platelets. In a panel of hematological cell lines, S55746 induces hallmarks of apoptosis including externalization of phosphatidylserine, caspase-3 activation and PARP cleavage. Ex vivo, S55746 induces apoptosis in the low nanomolar range in primary Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma patient samples. Finally, S55746 administered by oral route daily in mice demonstrated robust anti-tumor efficacy in two hematological xenograft models with no weight lost and no change in behavior. Taken together, these data demonstrate that S55746 is a novel, well-tolerated BH3-mimetic targeting selectively and potently the BCL-2 protein.

The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models.

Avoidance of apoptosis is critical for the development and sustained growth of tumours. The pro-survival protein myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers, but the development of small molecules targeting this protein that are amenable for clinical testing has been challenging. Here we describe S63845, a small molecule that specifically binds with high affinity to the BH3-binding groove of MCL1. Our mechanistic studies demonstrate that S63845 potently kills MCL1-dependent cancer cells, including multiple myeloma, leukaemia and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptotic pathway. In vivo, S63845 shows potent anti-tumour activity with an acceptable safety margin as a single agent in several cancers. Moreover, MCL1 inhibition, either alone or in combination with other anti-cancer drugs, proved effective against several solid cancer-derived cell lines. These results point towards MCL1 as a target for the treatment of a wide range of tumours.

S49076 is a novel kinase inhibitor of MET, AXL, and FGFR with strong preclinical activity alone and in association with bevacizumab.

Aberrant activity of the receptor tyrosine kinases MET, AXL, and FGFR1/2/3 has been associated with tumor progression in a wide variety of human malignancies, notably in instances of primary or acquired resistance to existing or emerging anticancer therapies. This study describes the preclinical characterization of S49076, a novel, potent inhibitor of MET, AXL/MER, and FGFR1/2/3. S49076 potently blocked cellular phosphorylation of MET, AXL, and FGFRs and inhibited downstream signaling in vitro and in vivo. In cell models, S49076 inhibited the proliferation of MET- and FGFR2-dependent gastric cancer cells, blocked MET-driven migration of lung carcinoma cells, and inhibited colony formation of hepatocarcinoma cells expressing FGFR1/2 and AXL. In tumor xenograft models, a good pharmacokinetic/pharmacodynamic relationship for MET and FGFR2 inhibition following oral administration of S49076 was established and correlated well with impact on tumor growth. MET, AXL, and the FGFRs have all been implicated in resistance to VEGF/VEGFR inhibitors such as bevacizumab. Accordingly, combination of S49076 with bevacizumab in colon carcinoma xenograft models led to near total inhibition of tumor growth. Moreover, S49076 alone caused tumor growth arrest in bevacizumab-resistant tumors. On the basis of these preclinical studies showing a favorable and novel pharmacologic profile of S49076, a phase I study is currently underway in patients with advanced solid tumors. Mol Cancer Ther; 12(9); 1749-62. ©2013 AACR.

Characterization of a large panel of patient-derived tumor xenografts representing the clinical heterogeneity of human colorectal cancer.

PURPOSE: Patient-derived xenograft models are considered to represent the heterogeneity of human cancers and advanced preclinical models. Our consortium joins efforts to extensively develop and characterize a new collection of patient-derived colorectal cancer (CRC) models. EXPERIMENTAL DESIGN: From the 85 unsupervised surgical colorectal samples collection, 54 tumors were successfully xenografted in immunodeficient mice and rats, representing 35 primary tumors, 5 peritoneal carcinoses and 14 metastases. Histologic and molecular characterization of patient tumors, first and late passages on mice includes the sequence of key genes involved in CRC (i.e., APC, KRAS, TP53), aCGH, and transcriptomic analysis. RESULTS: This comprehensive characterization shows that our collection recapitulates the clinical situation about the histopathology and molecular diversity of CRC. Moreover, patient tumors and corresponding models are clustering together allowing comparison studies between clinical and preclinical data. Hence, we conducted pharmacologic monotherapy studies with standard of care for CRC (5-fluorouracil, oxaliplatin, irinotecan, and cetuximab). Through this extensive in vivo analysis, we have shown the loss of human stroma cells after engraftment, observed a metastatic phenotype in some models, and finally compared the molecular profile with the drug sensitivity of each tumor model. Through an experimental cetuximab phase II trial, we confirmed the key role of KRAS mutation in cetuximab resistance. CONCLUSIONS: This new collection could bring benefit to evaluate novel targeted therapeutic strategies and to better understand the basis for sensitivity or resistance of tumors from individual patients.

Specific oncogenic activity of the Src-family tyrosine kinase c-Yes in colon carcinoma cells.

c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of ß-catenin at cell membranes and a reduction of expression of ß-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in ß-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src.

Human hair follicle pigmentary unit as a direct target for modulators of melanogenesis, as studied by [14C]-2-thiouracil incorporation.

The purpose of this study was to evaluate human hair follicle melanogenic activity using the [14C]-2-thiouracil, which was known to incorporate into nascent melanins. Results obtained on pigmented, grey and non-pigmented hair follicles demonstrated that [(14)C]-2-TU incorporation was restricted to the melanogenic compartment with a strong accumulation located around dermal papilla and within the fibre of pigmented hair follicles. Quantitative analysis of [(14)C]-2-TU incorporation showed a significant increase in pigmented hair follicles upon stimulation with 1 microm forskolin concomitant to an increase in tyrosinase levels. A strong significant decrease in [14C]-2-TU incorporation was noted, when hair follicles were incubated with the tyrosinase competitive inhibitor kojic acid (200 microm). Incubation with the MC1-R agonist alpha-MSH (0.2 microm) did not induce a significant stimulation of hair melanogenesis. The present model could thus represent a useful new tool to identify modulators of human hair pigmentation.

Expression of NAD+ dependent 15-hydroxyprostaglandin dehydrogenase and protection of prostaglandins in human hair follicle.

NAD(+) dependent 15-hydroxyprostaglandin dehydrogenate (15-PGDH) catalyses oxidation of 15(S)-hydroxyl group of prostaglandins and as a result inactivates their physiological potential. Positive effects of prostaglandins or prostaglandin analogues were reported on terminal hair, vellus hair or eyelash growth and a complex prostaglandin network was recently described in human hair follicle. In the present study, we showed that 15-PGDH was expressed in human hair follicle mainly in melanocytes and keratinocytes, which brought us to consider this enzyme as a possible target to sustain local prostaglandin production. Using a recombinant enzymatic strategy, specific 15-PGDH inhibitors were screened. We identified a thiazolidine dione derivative exhibiting efficacy on follicular outer root sheath keratinocytes, since it concomitantly decreased the production of deactivated 13,14 dihydro 15-ketoprostaglandin F(2alpha) and sustained prostaglandin F(2alpha)in vitro production. In the context of recent interest in prostaglandins and prostaglandin analogues as hair regrowth agents, we postulated that the use of selected 15-PGDH inhibitors could reinforce or prolong the effect of these physiological mediators on hair and skin.

TRP-2 specifically decreases WM35 cell sensitivity to oxidative stress.

TRP-2 (dopachrome tautomerase) is a melanogenic enzyme whose expression was recently reported to modulate melanocyte response to different cytotoxic events. Here we studied a possible role of TRP-2 in the oxidative stress response in the amelanotic WM35 melanoma cell line. Cell viability assays showed that TRP-2 overexpression in WM35 cells reduced their sensitivity to oxidative stress. Comet assays linked TRP-2 expression to DNA damage protection, and high-performance liquid chromotography-tandem mass spectrometry experiments showed an increase in intracellular glutathione in TRP-2-overexpressing cells. These effects were specifically reversed when TRP-2 was silenced by RNA interference. Nevertheless, these properties appeared to depend on a particular cell environment because expression of TRP-2 failed to rescue HEK epithelial cells exposed to similar treatments.

Prostanoid receptors in anagen human hair follicles.

Prostanoid pathway in hair follicle gained closer attention since trichogenic side-effects on hair growth has been observed concomitantly with prostaglandin F(2alpha) receptor (FP) agonist treatment of intraocular pressure. We thus investigated prostanoid receptor distribution in anagen hair follicle and different cell types from hair and skin. Using RT-PCR, Western blot and immunohistochemistry (IHC), we found that all receptors were present in hair follicle. This data shed new light on an underestimated complex network involved in hair growth control. Indeed most of these receptors showed a wide spectrum of expression in cultured cells and the whole hair follicle. Using IHC, we observed that expression of prostaglandin E(2) receptors (EP(2), EP(3), EP(4)), prostaglandin D(2) receptor (DP(2)), prostanoid thromboxane A(2) receptor (TP) and to a lesser extent EP(1) involved several hair follicle compartments. On the opposite, Prostaglandin I(2) receptor (IP) and DP(1) were more specifically expressed in hair cuticle layer and outer root sheath (ORS) basal layer, respectively. FP expression was essentially restricted to ORS companion layer and dermal papilla (DP). Although extracting a clear functional significance from this intricate network remains open challenge, FP labelling, i.e. could explain the biological effect of PGF(2alpha) on hair regrowth, by directly modulating DP function.

Prostaglandin metabolism in human hair follicle.

Prostaglandins regulate a wide number of physiological functions. Recently PGF(2alpha) analogue such as latanoprost was shown to have a real impact on hair regrowth. The aim of this study was to investigate and describe the expression profile in human hair follicle of prostaglandin metabolism key enzymes, i.e. carbonyl reductase-1 (CBR1), microsomal prostaglandin E synthase-1 (mPGES-1) and microsomal prostaglandin E synthase-2 (mPGES-2), cytosolic prostaglandin E synthase (cPGES), the aldoketoreductase AKR1C1 and the prostaglandin F synthase AKR1C3. Quantitative RT-PCR on plucked hair follicles revealed some sex-related differences, mPGES-2 and AKR1C3 expression levels being higher in women. Cell and hair follicle compartment specificity was investigated using Western blot, PGE(2) and PGF(2alpha) ELISA assays and immunohistochemistry. Most of the hair cell types were endowed with prostaglandin metabolism machinery and were thus able to produce PGE(2) and/or PGF(2alpha). The epithelial part of the hair bulb was identified by immunohistology and EIA assays as the main source of prostaglandin synthesis and interconversion. All these observations support the concept that prostaglandins might be involved in hair growth and differentiation control.

Comparative proteomic analysis of extracellular matrix proteins secreted by two types of skin fibroblasts.

The hair follicle dermal papilla is composed primarily of extracellular matrix (ECM) proteins secreted by resident fibroblasts. Dermal papilla is endowed with hair morphogenic properties, yet its composition is poorly characterized. In an attempt to understand its specificity better, we compared the protein composition of ECM secreted by cultured dermal papilla fibroblasts with that of dermal fibroblasts. ECM proteins are generally large, difficult to solubilize, and abundantly post-translationally modified. We thus implemented an original protocol for analyzing them: ECM samples were enzymatically digested directly in the culture flasks and analyzed by LC-MS/MS. Sequencing of proteolytic peptides by MS/MS yielded protein identification. The relative abundance of a given protein in dermal fibroblast versus dermal papilla samples was estimated by comparing proteolytic peptide intensities detected by MS. Using this approach, several matrix proteins were found to be present at markedly different levels in each ECM type; in particular, thrombospondin 1 and fibronectin appeared to be overrepresented in the dermal papilla fibroblast ECM. MS results were supported by Western blot and immunostaining experiments. In addition, peptide intensities were processed in two ways, which proved to favor either the quantification accuracy or the information precision at the sequence level.

Systematic identification of mitochondrial proteins by LC-MS/MS.

In eukaryotic cells, the mitochondrion is the key organelle for cellular respiration. Mitochondrial proteome analysis is difficult to perform by the classical proteomic approach involving two-dimensional gel electrophoresis (2DE), because this organelle contains a large number of membrane-associated and highly alkaline proteins usually requiring specific treatments to be successfully analyzed. Here, an alternative approach was evaluated and led to the rapid and sensitive identification of approximately 35% of the yeast mitochondrial proteins. It consists of an SDS-PAGE gel electrophoresis of the total mitochondrial protein in combination with the LC-MS/MS analysis of the digestion products of gel slices. The use of only 40 microg of mitochondrial protein enabled the identification of 179 different gene products divided into similar proportions of membrane and soluble proteins. The distribution of the identified proteins in terms of pI and hydrophobicity revealed that the present analytical strategy is largely unbiased. The identification of 28 proteins of previously unknown subcellular localization demonstrated the ability of SDS-PAGE-LC-MS/MS to rapidly supplement the knowledge of the mitochondrial proteome.