Affinity selection mass spectrometry speeding drug discovery.

Affinity selection mass spectrometry (AS-MS) has gained momentum in drug discovery. This review summarizes how this technology has slowly risen as a new paradigm in hit identification and its potential synergy with DNA encoded library technology. It presents an overview of the recent results on challenging targets and perspectives on new areas of research, such as RNA targeting with small molecules. The versatility of the approach is illustrated and strategic drivers discussed in terms of the experience of a small-medium CRO and a big pharma organization.

Development of Macrocyclic PRMT5-Adaptor Protein Interaction Inhibitors.

The PRMT5-MEP50 methyltransferase is a major target for anticancer drug discovery, and modulators of its interactions with different regulatory proteins are in high demand because they modulate PRMT5 substrate selectivity. We describe a strategy for the development of a PRMT5/adaptor protein PPI inhibitor, which includes the design and synthesis of macrocyclic peptides based on the motif for the interaction of PRMT5 with its adaptor protein RioK1. After the initial exploration of different macrocycle sizes and cyclization linkages, analysis of a peptide library identified hot spots for the variation of the amino acid structure. The incorporation of nonproteinogenic amino acids into the macrocyclic peptide led to a potent cyclic PRMT5 binding peptide (Ki = 66 nM), which selectively inhibits the interaction of PRMT5 with the adaptor proteins RioK1 and pICln (IC50 = 654 nM) but not with the alternative adaptor protein MEP50. The inhibitor is a promising tool for further biological investigation of this intriguing protein interface.

Preclinical Evaluation of a Novel Small Molecule Inhibitor of LIM Kinases (LIMK) CEL_Amide in Philadelphia-Chromosome Positive (BCR::ABL+) Acute Lymphoblastic Leukemia (ALL).

Ph+ (BCR::ABL+) B-ALL was considered to be high risk, but recent advances in BCR::ABL-targeting TKIs has shown improved outcomes in combination with backbone chemotherapy. Nevertheless, new treatment strategies are needed, including approaches without chemotherapy for elderly patients. LIMK1/2 acts downstream from various signaling pathways, which modifies cytoskeleton dynamics via phosphorylation of cofilin. Upstream of LIMK1/2, ROCK is constitutively activated by BCR::ABL, and upon activation, ROCK leads to the phosphorylation of LIMK1/2, resulting in the inactivation of cofilin by its phosphorylation and subsequently abrogating its apoptosis-promoting activity. Here, we demonstrate the anti-leukemic effects of a novel LIMK1/2 inhibitor (LIMKi) CEL_Amide in vitro and in vivo for BCR::ABL-driven B-ALL. The IC50 value of CEL_Amide was ≤1000 nM in BCR::ABL+ TOM-1 and BV-173 cells and induced dose-dependent apoptosis and cell cycle arrest in these cell lines. LIMK1/2 were expressed in BCR::ABL+ cell lines and patient cells and LIMKi treatment decreased LIMK1 protein expression, whereas LIMK2 expression was unaffected. As expected, CEL_Amide exposure caused specific activating downstream dephosphorylation of cofilin in cell lines and primary cells. Combination experiments with CEL_Amide and BCR::ABL TKIs imatinib, dasatinib, nilotinib, and ponatinib were synergistic for the treatment of both TOM-1 and BV-173 cells. CDKN2Ako/BCR::ABL1+ B-ALL cells were transplanted in mice, which were treated with combinations of CEL_Amide and nilotinib or ponatinib, which significantly prolonged their survival. Altogether, the LIMKi CEL_Amide yields activity in Ph+ ALL models when combined with BCR::ABL-targeting TKIs, showing promising synergy that warrants further investigation.

Characterization of Signalling Pathways That Link Apoptosis and Autophagy to Cell Death Induced by Estrone Analogues Which Reversibly Depolymerize Microtubules.

The search for novel anti-cancer compounds which can circumvent chemotherapeutic drug resistance and limit systemic toxicity remains a priority. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)15-tetraene-3-ol-17one (ESE-15-one) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) are sulphamoylated 2-methoxyestradiol (2-ME) analogues designed by our research team. Although their cytotoxicity has been demonstrated in vitro, the temporal and mechanistic responses of the initiated intracellular events are yet to be determined. In order to do so, assays investigating the compounds' effects on microtubules, cell cycle progression, signalling cascades, autophagy and apoptosis were conducted using HeLa cervical- and MDA-MB-231 metastatic breast cancer cells. Both compounds reversibly disrupted microtubule dynamics as an early event by binding to the microtubule colchicine site, which blocked progression through the cell cycle at the G1/S- and G2/M transitions. This was supported by increased pRB and p27Kip1 phosphorylation. Induction of apoptosis with time-dependent signalling involving the p-JNK, Erk1/2 and Akt/mTOR pathways and loss of mitochondrial membrane potential was demonstrated. Inhibition of autophagy attenuated the apoptotic response. In conclusion, the 2-ME analogues induced a time-dependent cross-talk between cell cycle checkpoints, apoptotic signalling and autophagic processes, with an increased reactive oxygen species formation and perturbated microtubule functioning appearing to connect the processes. Subtle differences in the responses were observed between the two compounds and the different cell lines.

Exploring new targets and chemical space with affinity selection-mass spectrometry.

Affinity selection-mass spectrometry (AS-MS) is a high-throughput screening (HTS) technique for drug discovery that enables rapid screening of large collections of compounds to identify ligands for a specific biomolecular target. AS-MS is a binding assay that is insensitive to the functional effects a ligand might have, which is important because it lets us identify novel ligands irrespective of their binding site. This approach is gaining popularity, notably due to its role in the emergence of useful agents for targeted protein degradation. This Perspective highlights the use of AS-MS techniques to explore broad chemical space and identify small-molecule ligands for biological targets that have proven challenging to address with other screening paradigms. We present chemical structures of reported AS-MS hits to illustrate the potential of this screening approach to deliver high-quality hits for further optimization. AS-MS has, thus, evolved from being an infrequent alternative to traditional HTS or DNA-encoded library strategies to now firmly establishing itself as a HTS approach for drug discovery.

Synergy of FLT3 inhibitors and the small molecule inhibitor of LIM kinase1/2 CEL_Amide in FLT3-ITD mutated Acute Myeloblastic Leukemia (AML) cells.

Patients with FLT3-ITD mutated (FLT3-ITD+) Acute Myeloid Leukemia (AML), have frequently relapsed or refractory disease and FLT3-ITD+ inhibitors have limited efficacy. Rho kinases (ROCK) are constitutively activated by FLT3-ITD+ in AML via PI3 kinase and Rho GTPase. Upon activation by ROCK, LIM kinases (LIMK) inactivate cofilin by phosphorylation which affects cytoskeleton dynamics, cell growth and apoptosis. LIMK inhibition leads to cofilin activation via dephosphorylation and activated cofilin localizes to mitochondria inducing apoptosis. Thus, we investigated the therapeutic potential of the LIMK1/2 inhibitor CEL_Amide (LIMKi) in FLT3-ITD+ AML. Expression of LIMK1/2 in FLT3-ITD+ cell lines MOLM-13 and MV-4-11 cells could be detected by RT-qPCR and at the protein level. IC50 after LIMKi monotherapy was 440 nM in MOLM-13 cells and 420 nM in MV4-11 cells. Treatment with LIMKi decreased LIMK1 protein levels and repression of inactivating phosphorylation of cofilin in FLT3-ITD+ cells. Combination experiments with LIMKi and FLT3 inhibitors including midostaurin, crenolanib and gilteritinib were synergistic for treatment of MOLM-13 cells while combinations with quizartinib were additive. Combinations of LIMKi and the hypomethylating agent azacitidine or the ROCK inhibitor fasudil were additive. In NOD-SCID mice engrafted with MOLM13-LUC cells, the FLT3 inhibitor midostaurin and LIMKi delayed MOLM13-LUC engraftment as detected by in vivo bioluminescence imaging and the LIMKi and midostaurin combination prolonged significantly survival of leukemic mice. LIMK1/2 inhibition by the small molecule CEL_Amide seems to have promising activity in combination with FLT3 inhibitors in vitro as well as in vivo and may constitute a novel treatment strategy for FLT3-ITD+ AML.

Two Antagonistic Microtubule Targeting Drugs Act Synergistically to Kill Cancer Cells.

Paclitaxel is a microtubule stabilizing agent and a successful drug for cancer chemotherapy inducing, however, adverse effects. To reduce the effective dose of paclitaxel, we searched for pharmaceutics which could potentiate its therapeutic effect. We screened a chemical library and selected Carba1, a carbazole, which exerts synergistic cytotoxic effects on tumor cells grown in vitro, when co-administrated with a low dose of paclitaxel. Carba1 targets the colchicine binding-site of tubulin and is a microtubule-destabilizing agent. Catastrophe induction by Carba1 promotes paclitaxel binding to microtubule ends, providing a mechanistic explanation of the observed synergy. The synergistic effect of Carba1 with paclitaxel on tumor cell viability was also observed in vivo in xenografted mice. Thus, a new mechanism favoring paclitaxel binding to dynamic microtubules can be transposed to in vivo mouse cancer treatments, paving the way for new therapeutic strategies combining low doses of microtubule targeting agents with opposite mechanisms of action.

Discovery of holoenzyme-disrupting chemicals as substrate-selective CK2 inhibitors.

CK2 is a constitutively active protein kinase overexpressed in numerous malignancies. Interaction between CK2α and CK2ß subunits is essential for substrate selectivity. The CK2α/CK2ß interface has been previously targeted by peptides to achieve functional effects; however, no small molecules modulators were identified due to pocket flexibility and open shape. Here we generated numerous plausible conformations of the interface using the fumigation modeling protocol, and virtually screened a compound library to discover compound 1 that suppressed CK2α/CK2ß interaction in vitro and inhibited CK2 in a substrate-selective manner. Orthogonal SPR, crystallography, and NMR experiments demonstrated that 4 and 6, improved analogs of 1, bind to CK2α as predicted. Both inhibitors alter CK2 activity in cells through inhibition of CK2 holoenzyme formation. Treatment with 6 suppressed MDA-MB231 triple negative breast cancer cell growth and induced apoptosis. Altogether, our findings exemplify an innovative computational-experimental approach and identify novel non-peptidic inhibitors of CK2 subunit interface disclosing substrate-selective functional effects.

A quantitative proteomic analysis of cofilin phosphorylation in myeloid cells and its modulation using the LIM kinase inhibitor Pyr1.

LIM kinases are located at a strategic crossroad, downstream of several signaling pathways and upstream of effectors such as microtubules and the actin cytoskeleton. Cofilin is the only LIM kinases substrate that is well described to date, and its phosphorylation on serine 3 by LIM kinases controls cofilin actin-severing activity. Consequently, LIM kinases inhibition leads to actin cytoskeleton disorganization and blockade of cell motility, which makes this strategy attractive in anticancer treatments. LIMK has also been reported to be involved in pathways that are deregulated in hematologic malignancies, with little information regarding cofilin phosphorylation status. We have used proteomic approaches to investigate quantitatively and in detail the phosphorylation status of cofilin in myeloid tumor cell lines of murine and human origin. Our results show that under standard conditions, only a small fraction (10 to 30% depending on the cell line) of cofilin is phosphorylated (including serine 3 phosphorylation). In addition, after a pharmacological inhibition of LIM kinases, a residual cofilin phosphorylation is observed on serine 3. Interestingly, this 2D gel based proteomic study identified new phosphorylation sites on cofilin, such as threonine 63, tyrosine 82 and serine 108.

Specific Targeting of Plant and Apicomplexa Parasite Tubulin through Differential Screening Using In Silico and Assay-Based Approaches.

Dinitroanilines are chemical compounds with high selectivity for plant cell α-tubulin in which they promote microtubule depolymerization. They target α-tubulin regions that have diverged over evolution and show no effect on non-photosynthetic eukaryotes. Hence, they have been used as herbicides over decades. Interestingly, dinitroanilines proved active on microtubules of eukaryotes deriving from photosynthetic ancestors such as Toxoplasma gondii and Plasmodium falciparum, which are responsible for toxoplasmosis and malaria, respectively. By combining differential in silico screening of virtual chemical libraries on Arabidopsis thaliana and mammal tubulin structural models together with cell-based screening of chemical libraries, we have identified dinitroaniline related and non-related compounds. They inhibit plant, but not mammalian tubulin assembly in vitro, and accordingly arrest A. thaliana development. In addition, these compounds exhibit a moderate cytotoxic activity towards T. gondii and P. falciparum. These results highlight the potential of novel herbicidal scaffolds in the design of urgently needed anti-parasitic drugs.

LIM kinases: cofilin and beyond.

LIM kinases are common downstream effectors of several signalization pathways and function as a signaling node that controls cytoskeleton dynamics through the phosphorylation of the cofilin family proteins. These last 10 years, several reports indicate that the functions of LIM kinases are more extended than initially described and, specifically, that LIM kinases also control microtubule dynamics, independently of their regulation of actin microfilament. In this review we analyze the data supporting these conclusions and the possible mechanisms that could be involved in the control of microtubules by LIM kinases. The demonstration that LIM kinases also control microtubule dynamics has pointed to new therapeutic opportunities. Consistently, several new LIM kinase inhibitors have been recently developed. We provide a comprehensive comparison of these inhibitors, of their chemical structure, their specificity, their cellular effects as well as their effects in animal models of various diseases including cancer.

LIM Kinase Inhibitor Pyr1 Reduces the Growth and Metastatic Load of Breast Cancers.

LIM kinases (LIMK) are emerging targets for cancer therapy, and they function as network hubs to coordinate actin and microtubule dynamics. When LIMKs are inhibited, actin microfilaments are disorganized and microtubules are stabilized. Owing to their stabilizing effect on microtubules, LIMK inhibitors may provide a therapeutic strategy to treat taxane-resistant cancers. In this study, we investigated the effect of LIMK inhibition on breast tumor development and on paclitaxel-resistant tumors, using a novel selective LIMK inhibitor termed Pyr1. Treatment of breast cancer cells, including paclitaxel-resistant cells, blocked their invasion and proliferation in vitro and their growth in vivo in tumor xenograft assays. The tumor-invasive properties of Pyr1 were investigated in vivo by intravital microscopy of tumor xenografts. A striking change of cell morphology was observed with a rounded phenotype arising in a subpopulation of cells, while other cells remained elongated. Notably, although Pyr1 decreased the motility of elongated cells, it increased the motility of rounded cells in the tumor. Pyr1 administration prevented the growth of metastasis but not their spread. Overall, our results provided a preclinical proof of concept concerning how a small-molecule inhibitor of LIMK may offer a strategy to treat taxane-resistant breast tumors and metastases. Cancer Res; 76(12); 3541-52. ©2016 AACR.

Novel synthetic pharmacophores inducing a stabilization of cellular microtubules.

Microtubule drugs have been widely used in cancer chemotherapies. Although microtubules are subject to regulation by signal transduction mechanisms, their pharmacological modulation has so far relied on compounds that bind to the tubulin subunit. Using a cell-based assay designed to probe the microtubule polymerization status, we identified two pharmacophores, CM09 and CM10, as cell-permeable microtubule stabilizing agents. These synthetic compounds do not affect the assembly state of purified microtubules in vitro but they profoundly suppress microtubule dynamics in vivo. Moreover, they exert cytotoxic effects on several cancer cell lines including multidrug resistant cell lines. Therefore, these classes of compounds represent novel attractive leads for cancer chemotherapy.

Novel in silico-designed estradiol analogues are cytotoxic to a multidrug-resistant cell line at nanomolar concentrations.

PURPOSE: 2-Methoxyestradiol (2ME) is a promising anti-cancer agent that disrupts the integrity and dynamics of the spindle network. In order to overcome the pharmacokinetic constraints of this compound, a panel of sulphamoylated estradiol analogues were in silico-designed by our laboratory. In this study, we analysed the potential of each analogue to induce cell death on a panel of cancer cell lines. Moreover, the mechanism of action of the most effective compounds was determined. METHODS: Cytotoxicity screening of the compounds and intermediates was performed on five different cancer cell lines to determine IG50 values. An in vitro tubulin polymerization assay was done to determine the effect of the drugs on tubulin polymerization while their intracellular effects on the microtubule network were assessed by immunofluorescence microscopy. RESULTS: IG50 calculations showed that the sulphamoylated analogues induce cytotoxicity at nanomolar concentrations in all cell lines, including the P-glycoprotein pump overexpressing multidrug-resistant uterine sarcoma cell line. The non-sulphamoylated compounds were only cytotoxic at micromolar ranges, if at all. The sulphamoylated compounds inhibited pure tubulin polymerization in a dose-dependent manner and induced microtubule destruction in cells after 24-h exposure. CONCLUSION: Results revealed that the novel sulphamoylated 2ME derivatives have potential as anti-cancer drugs, possibly even against chemoresistant cancer cells. These compounds disrupt the intracellular microtubule integrity which leads to mitotic block of the cells.

[Phenotypic screens or one stone to kill two birds: discover the target and its pharmacological regulator]. / Les criblages phénotypiques ou comment faire d'une pierre deux coups: découvrir la cible et la molécule pharmacologique capable de la réguler.

Phenotypic screens, in which chemical libraries are assayed on cells with the aim to isolate compounds that interfere with a given cell function, are a risky but powerful strategy to discover, in the same approach, new therapeutic targets and the compounds able to regulate them. With a strong experience of nearly 10 years in the field, we present the advantages of such an approach, the possible troubles and technical solutions. We also present in this paper a french network which has been recently built and that gather all the competencies needed for screening approaches.

Sulphamoylated 2-methoxyestradiol analogues induce apoptosis in adenocarcinoma cell lines.

2-Methoxyestradiol (2ME2) is a naturally occurring estradiol metabolite which possesses antiproliferative, antiangiogenic and antitumor properties. However, due to its limited biological accessibility, synthetic analogues have been synthesized and tested in attempt to develop drugs with improved oral bioavailability and efficacy. The aim of this study was to evaluate the antiproliferative effects of three novel in silico-designed sulphamoylated 2ME2 analogues on the HeLa cervical adenocarcinoma cell line and estrogen receptor-negative breast adenocarcinoma MDA-MB-231 cells. A dose-dependent study (0.1-25 µM) was conducted with an exposure time of 24 hours. Results obtained from crystal violet staining indicated that 0.5 µM of all 3 compounds reduced the number of cells to 50%. Lactate dehydrogenase assay was used to assess cytotoxicity, while the mitotracker mitochondrial assay and caspase-6 and -8 activity assays were used to investigate the possible occurrence of apoptosis. Tubulin polymerization assays were conducted to evaluate the influence of these sulphamoylated 2ME2 analogues on tubulin dynamics. Double immunofluorescence microscopy using labeled antibodies specific to tyrosinate and detyrosinated tubulin was conducted to assess the effect of the 2ME2 analogues on tubulin dynamics. An insignificant increase in the level of lactate dehydrogenase release was observed in the compounds-treated cells. These sulphamoylated compounds caused a reduction in mitochondrial membrane potential, cytochrome c release and caspase 3 activation indicating apoptosis induction by means of the intrinsic pathway in HeLa and MDA-MB-231 cells. Microtubule depolymerization was observed after exposure to these three sulphamoylated analogues.

Azaindole derivatives are inhibitors of microtubule dynamics, with anti-cancer and anti-angiogenic activities.

BACKGROUND AND PURPOSE: Drugs targeting microtubules are commonly used for cancer treatment. However, the potency of microtubule inhibitors used clinically is limited by the emergence of resistance. We thus designed a strategy to find new cell-permeable microtubule-targeting agents. EXPERIMENTAL APPROACH: Using a cell-based assay designed to probe for microtubule polymerization status, we screened a chemical library and identified two azaindole derivatives, CM01 and CM02, as cell-permeable microtubule-depolymerizing agents. The mechanism of the anti-tumour effects of these two compounds was further investigated both in vivo and in vitro. KEY RESULTS: CM01 and CM02 induced G2/M cell cycle arrest and exerted potent cytostatic effects on several cancer cell lines including multidrug-resistant (MDR) cell lines. In vitro experiments revealed that the azaindole derivatives inhibited tubulin polymerization and competed with colchicines for this effect, strongly indicating that tubulin is the cellular target of these azaindole derivatives. In vivo experiments, using a chicken chorioallantoic xenograft tumour assay, established that these compounds exert a potent anti-tumour effect. Furthermore, an assay probing the growth of vessels out of endothelial cell spheroids showed that CM01 and CM02 exert anti-angiogenic activities. CONCLUSIONS AND IMPLICATIONS: CM01 and CM02 are reversible microtubule-depolymerizing agents that exert potent cytostatic effects on human cancer cells of diverse origins, including MDR cells. They were also shown to inhibit angiogenesis and tumour growth in chorioallantoic breast cancer xenografts. Hence, these azaindole derivatives are attractive candidates for further preclinical investigations.

Pharmacological inhibition of LIM kinase stabilizes microtubules and inhibits neoplastic growth.

The emergence of tumor resistance to conventional microtubule-targeting drugs restricts their clinical use. Using a cell-based assay that recognizes microtubule polymerization status to screen for chemicals that interact with regulators of microtubule dynamics, we identified Pyr1, a cell permeable inhibitor of LIM kinase, which is the enzyme that phosphorylates and inactivates the actin-depolymerizing factor cofilin. Pyr1 reversibly stabilized microtubules, blocked actin microfilament dynamics, inhibited cell motility in vitro and showed anticancer properties in vivo, in the absence of major side effects. Pyr1 inhibition of LIM kinase caused a microtubule-stabilizing effect, which was independent of any direct effects on the actin cytoskeleton. In addition, Pyr1 retained its activity in multidrug-resistant cancer cells that were resistant to conventional microtubule-targeting agents. Our findings suggest that LIM kinase functions as a signaling node that controls both actin and microtubule dynamics. LIM kinase may therefore represent a targetable enzyme for cancer treatment.

Antitumoral activity of allosteric inhibitors of protein kinase CK2.

INTRODUCTION: Due to its physiological role into promoting cell survival and its dysregulation in most cancer cells, protein kinase CK2 is a relevant physiopathological target for development of chemical inhibitors. We report the discovery of azonaphthalene derivatives, as a new family of highly specific CK2 inhibitors. First, we demonstrated that CK2 inhibition (IC50= 0.4 µM) was highly specific, reversible and non ATP-competitive. Small Angle X-ray Scattering experiments showed that this inhibition was due to large conformational change of CK2α upon binding of these inhibitors. We showed that several compounds of the family were cell-potent CK2 inhibitors promoting cell cycle arrest of human glioblastoma U373 cells. Finally, in vitro and in vivo assays showed that these compounds could decrease U373 cell tumor mass by 83 % emphasizing their efficacy against these apoptosis-resistant tumors. In contrast, Azonaphthalene derivatives inactive on CK2 activity showed no effect in colony formation and tumor regression assays. These findings illustrate the emergence of nonclassical CK2 inhibitors and provide exciting opportunities for the development of novel allosteric CK2 inhibitors. BACKGROUND: CK2 is an emerging therapeutic target and ATP-competitive inhibitors have been identified. CK2 is endowed with specific structural features providing alternative strategies for inhibition. RESULTS: Azonaphthalene compounds are allosteric CK2 inhibitors showing antitumor activity. CONCLUSION: CK2 may be targeted allosterically. SIGNIFICANCE: These inhibitors provide a foundation for a new paradigm for specific CK2 inhibition.

Structure-function analysis of the beta regulatory subunit of protein kinase CK2 by targeting embryonic stem cell.

Programs that govern stem cell maintenance and pluripotency are dependent on extracellular factors and of intrinsic cell modulators. Embryonic stem (ES) cells with a specific depletion of the gene encoding the regulatory subunit of protein kinase CK2 (CK2ß) revealed a viability defect. However, analysis of CK2ß functions along the neural lineage established CK2ß as a positive regulator for neural stem/progenitor cell (NSC) proliferation and multipotency. By using an in vitro genetic conditional approach, we demonstrate in this work that specific domains of CK2ß involved in the regulatory function towards CK2 catalytic subunits are crucial structural determinants for ES cell homeostasis.