Fragment-based drug design is an established routine approach in both experimental and computational spheres. Growing fragment hits into viable ligands has increasingly shifted into the spotlight. FastGrow is an application based on a shape search algorithm that addresses this challenge at high speeds of a few milliseconds per fragment. It further features a pharmacophoric interaction description, ensemble flexibility, as well as geometry optimization to become a fully fledged structure-based modeling tool. All features were evaluated in detail on a previously reported collection of fragment growing scenarios extracted from crystallographic data. FastGrow was also shown to perform competitively versus established docking software. A case study on the DYRK1A kinase, using recently reported new chemotypes, illustrates FastGrow's features in practice and its ability to identify active fragments. FastGrow is freely available to the public as a web server at https://fastgrow.plus/ and is part of the SeeSAR 3D software package.

The accurate prediction of molecular properties, such as lipophilicity and aqueous solubility, are of great importance and pose challenges in several stages of the drug discovery pipeline. Machine learning methods, such as graph-based neural networks (GNNs), have shown exceptionally good performance in predicting these properties. In this work, we introduce a novel GNN architecture, called directed edge graph isomorphism network (D-GIN). It is composed of two distinct sub-architectures (D-MPNN, GIN) and achieves an improvement in accuracy over its sub-architectures employing various learning, and featurization strategies. We argue that combining models with different key aspects help make graph neural networks deeper and simultaneously increase their predictive power. Furthermore, we address current limitations in assessment of deep-learning models, namely, comparison of single training run performance metrics, and offer a more robust solution.

Since the first approval of a protein kinase inhibitor (PKI) by the Food and Drug Administration (FDA) in 2001, 55 new PKIs have reached the market, and many inhibitors are currently being evaluated in clinical trials. This is a clear indication that protein kinases still represent major drug targets for the pharmaceutical industry. In a previous work, we have introduced PKIDB, a publicly available database, gathering PKIs that have already been approved (Phase 4), as well as those currently in clinical trials (Phases 0 to 3). This database is updated frequently, and an analysis of the new data is presented here. In addition, we compared the set of PKIs present in PKIDB with the PKIs in early preclinical studies found in ChEMBL, the largest publicly available chemical database. For each dataset, the distribution of physicochemical descriptors related to drug-likeness is presented. From these results, updated guidelines to prioritize compounds for targeting protein kinases are proposed. The results of a principal component analysis (PCA) show that the PKIDB dataset is fully encompassed within all PKIs found in the public database. This observation is reinforced by a principal moments of inertia (PMI) analysis of all molecules. Interestingly, we notice that PKIs in clinical trials tend to explore new 3D chemical space. While a great majority of PKIs is located on the area of "flatland", we find few compounds exploring the 3D structural space. Finally, a scaffold diversity analysis of the two datasets, based on frequency counts was performed. The results give insight into the chemical space of PKIs, and can guide researchers to reach out new unexplored areas. PKIDB is freely accessible from the following website: http://www.icoa.fr/pkidb.

Here, we evaluate the performance of our range of ensemble simulation based binding free energy calculation protocols, called ESMACS (enhanced sampling of molecular dynamics with approximation of continuum solvent) for use in fragment based drug design scenarios. ESMACS is designed to generate reproducible binding affinity predictions from the widely used molecular mechanics Poisson-Boltzmann surface area (MMPBSA) approach. We study ligands designed to target two binding pockets in the lactate dehydogenase A target protein, which vary in size, charge and binding mode. When comparing to experimental results, we obtain excellent statistical rankings across this highly diverse set of ligands. In addition, we investigate three approaches to account for entropic contributions not captured by standard MMPBSA calculations: (1) normal mode analysis, (2) weighted solvent accessible surface area (WSAS) and (3) variational entropy.

Over the past two decades, the use of fragment-based lead generation has become a common, mature approach to identify tractable starting points in chemical space for the drug discovery process. This approach naturally involves the study of the binding properties of highly heterogeneous ligands. Such datasets challenge computational techniques to provide comparable binding free energy estimates from different binding modes. The performance of a range of statistically robust ensemble-based binding free energy calculation protocols, called ESMACS (enhanced sampling of molecular dynamics with approximation of continuum solvent), is evaluated. Ligands designed to target two binding pockets in the lactate dehydogenase, a target protein, which vary in size, charge, and binding mode, are studied. When compared to experimental results, excellent statistical rankings are obtained across this highly diverse set of ligands. In addition, three approaches to account for entropic contributions are investigated: 1) normal mode analysis, 2) weighted solvent accessible surface area (WSAS), and 3) variational entropy. Normal mode analysis and WSAS correlate strongly with each other-although the latter is computationally far cheaper-but do not improve rankings. Variational entropy corrects exaggerated discrimination of ligands bound in different pockets but creates three outliers which reduce the quality of the overall ranking.

We investigate the robustness of our ensemble molecular dynamics binding free energy protocols, known as ESMACS, to different choices of forcefield, starting structure and analysis. ESMACS is based on MMPBSA and we examinge the influence of multiple trajectories, explicit water molecules and estimates of the entropic contribution to the binding free energy.Simulation input and binding affinity calculation data:https://doi.org/10.5281/zenodo.1484050

We designed and synthesized a new series of fatty acid synthase (FASN) inhibitors with potential utility for the treatment of cancer. Extensive SAR studies led to highly active FASN inhibitors with good cellular activity and oral bioavailability, exemplified by compound 34. Compound 34 is a potent inhibitor of human FASN (IC50 = 28 nM) that effectively inhibits proliferation of A2780 ovarian cells (IC50= 13 nM) in lipid-reduced serum (LRS). This cellular activity can be rescued by addition of palmitate, consistent with an on-target effect. Compound 34 is also active in many other cell types, including PC3M (IC50 = 25 nM) and LnCaP-Vancouver prostate cells (IC50 = 66 nM), and is highly bioavailable (F 61%) with good exposure after oral administration. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with compound 34 results in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate, fully consistent with the desired target engagement. (C) 2018 Elsevier Ltd. All rights reserved.

The number of protein kinase inhibitors (PKIs) approved worldwide continues to grow steadily, with 39 drugs approved in the period between 2001 and January 2018. PKIs on the market have been the subject of many reviews, and structure-property relationships specific to this class of drugs have been inferred. However, the large number of PKIs under development is often overlooked. In this paper, we present PKIDB (Protein Kinase Inhibitor Database), a monthly-updated database gathering approved PKIs as well as PKIs currently in clinical trials. The database compiles currently 180 inhibitors ranging from phase 0 to 4 clinical trials along with annotations extracted from seven public resources. The distribution and property ranges of standard physicochemical properties are presented. They can be used as filters to better prioritize compound selection for future screening campaigns. Interestingly, more than one-third of the kinase inhibitors violate at least one Lipinski's rule. A Principal Component Analysis (PCA) reveals that Type-II inhibitors are mapped to a distinct chemical space as compared to orally administrated drugs as well as to other types of kinase inhibitors. Using a Principal Moment of Inertia (PMI) analysis, we show that PKIs under development tend to explore new shape territories as compared to approved PKIs. In order to facilitate the analysis of the protein space, the kinome tree has been annotated with all protein kinases being targeted by PKIs. Finally, we analyzed the pipeline of the pharmaceutical companies having PKIs on the market or still under development. We hope that this work will assist researchers in the kinase field in identifying and designing the next generation of kinase inhibitors for still untargeted kinases. The PKIDB database is freely accessible from a website at http://www.icoa.fr/pkidb and can be easily browsed through a user-friendly spreadsheet-like interface.

Protocols for the design of kinase-focused compound libraries are presented. Kinase-focused compound libraries can be differentiated based on the design goal. Depending on whether the library should be a discovery library specific for one particular kinase, a general discovery library for multiple distinct kinase projects, or even phenotypic screening, there exists today a variety of in silico methods to design candidate compound libraries. We address the following scenarios: 1) Datamining of SAR databases and kinase focused vendor catalogues; 2) Predictions and virtual screening; 3) Structure-based design of combinatorial kinase inhibitors; 4) Design of covalent kinase inhibitors; 5) Design of macrocyclic kinase inhibitors; and 6) Design of allosteric kinase inhibitors and activators.

Compound selectivity is an important issue when developing a new drug. In many instances, a lack of selectivity can translate to increased toxicity. Protein kinases are particularly concerned with this issue because they share high sequence and structural similarity. However, selectivity may be assessed early on using data generated from protein kinase profiling panels.

So far, 518 protein kinases have been identified in the human genome. They share a common mechanism of protein phosphorylation and are involved in many critical biological processes of eukaryotic cells. Deregulation of the kinase phosphorylation function induces severe illnesses such as cancer, diabetes, or inflammatory diseases. Many actors in the pharmaceutical domain have made significant efforts to design potent and selective protein kinase inhibitors as new potential drugs. Because the ATP binding site is highly conserved in the protein kinase family, the design of selective inhibitors remains a challenge and has negatively impacted the progression of drug candidates to late-stage clinical development. The work presented here adopts a 2.5D kinochemometrics (KCM) approach, derived from proteochemometrics (PCM), in which protein kinases are depicted by a novel 3D descriptor and the ligands by 2D fingerprints. We demonstrate in two examples that the protein descriptor successfully classified protein kinases based on their group membership and their Asp-Phe-Gly (DFG) conformation. We also compared the performance of our models with those obtained from a full 2D KCM model and QSAR models. In both cases, the internal validation of the models demonstrated good capabilities to distinguish "active" from "inactive" protein kinase ligand pairs. However, the, external validation performed on two independent data sets showed that the two statistical models tended to overestimate the number of "inactive" pairs.

Because of the success of imatinib, the first type-II kinase inhibitor approved by the FDA in 2001, sustained efforts have been made by the pharmaceutical industry to discover novel compounds stabilizing the inactive conformation of protein kinases. On the seven type-II inhibitors having reached the market, four were released in 2012, suggesting an acceleration of the research of such a class of compounds. Still, they represent less than a third of the protein kinase inhibitors available to patients today. The identification of key residues involved in the binding of this type of ligands in the kinase active site might ease the design of potent and selective type-II inhibitors. In order to identify those discriminant residues, we have developed a proteometric approach combining residue descriptors of protein kinase sequences and biological activities of various type-II kinase inhibitors. We applied Partial Least Squares (PLS) regression to identify 29 key residues that influence the binding of four type-II inhibitors to most proteins of the kinome. The gatekeeper residue was found to be the most relevant, confirming an essential role in ligand binding as well as in protein kinase conformational changes. Using the newly developed proteometric model, we predicted the propensity of each protein kinase to be inhibited by type-II ligands. The model was further validated using an external data set of protein/ligand activity pairs. Other residues present in the kinase domain, and more specifically in the binding site, have been highlighted by this approach, but their role in biological mechanisms is still unknown.

Abstract Fatty Acid Synthase (FASN) catalyzes the final step in palmitate (PA) synthesis, using acetyl-CoA, malonyl-CoA and NADPH. Most normal tissues express low levels of FASN and rely on uptake of fatty acids (FA) from the diet. It has been proposed that FASN overexpressing tumors including prostate and breast tumors depend on de novo FA synthesis, which is advantageous to tumors by providing lipids for membrane synthesis and increased growth factor receptor expression/signaling in lipid rafts. Overexpression of FASN leads to a higher amount of saturated lipids in membranes which can lead to resistance to cytotoxic chemotherapy. Lastly, NADPH consumption during PA synthesis keeps the redox balance in check. All of the above imply that FASN represents a potential therapeutic target for the treatment of multiple cancer types. At this AACR we report two novel chemical series (posters Connolly et al., Lu et al.). JNJ-53793220 and JNJ-54302833 potently and selectively (< 100nM) inhibit the FASN enzyme and proliferative activity in cells in lipid reduced medium (LRM). Using these, we investigated the underlying hypothesis that tumor cells do not utilize circulating FA and are dependent on de novo synthesis of FA. In a lipid reduced environment many cell lines, particularly of prostate, breast, ovarian or heme origin, proved to be sensitive to JNJ-53793220. However co-administration of PA dose-dependently reversed the anti-proliferative effects. Also androgen driven proliferation of LNCaP cells was potently blocked by JNJ-53793220 (EC50 30 nM), and decreased PSA levels. Both effects were partially rescued by the addition of PA. While the rescue of tumor cells by PA confirmed the on-target activity of the compounds, it also suggested that cancer cells are capable of using external FA. To extend these findings, we screened more than 400 cell lines in lipid containing medium (LCM) for their sensitivity to JNJ-53793220. In LCM sensitivity to FASN inhibition was lower than in LRM conditions. In most, but not all, cases the addition of PA reverted the antiproliferative effects of JNJ-53793220, although target engagement was not reduced in LCM conditions. The EC50 of 14C-acetate incorporation in lipids of ∼30 nM corresponded well with enzymatic and anti-proliferative effects in LRM (27 and 13 nM respectively). Furthermore, growth of pre-established LNCaP xenografts in vivo was not blocked significantly by JNJ-53793220, even though malonyl-CoA levels were increased as expected upon FASN inhibition in the tumor. While circulating lipids in vivo are likely culprits for the lack of efficacy, other factors may play a role as well. In a 3D culture model (poster Vidic et al.) the growth of LNCaP and PC346c spheroids was blocked by JNJ-54302833 (1µM), but growth of PC346c spheroids co-cultured with cancer associated fibroblasts was not inhibited. Taken together our data suggest that the outcome of FASN inhibition is influenced by the tumor environment. Citation Format: Karine A. Smans, Sabine De Breucker, Norbert Esser, Erwin Fraiponts, Ron Gilissen, Ralph Graeser, Boudewijn Janssen, Lieven Meerpoel, Danielle Peeters, Geert Van Hecke, Luc Van Nuffel, Yolanda Chong, Peter Vermeulen, Gilles Bignan, James Bischoff, Peter Connolly, Bruce Grasberger, Tianbao Lu, Donald Ludovici, Carsten Schubert, Michael Parker, Christophe Meyer, Suzana Vidic. Sensitivity of cell lines to Fatty Acid Synthase inhibitors depends on the lipid content in the cellular environment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 801. doi:10.1158/1538-7445.AM2014-801

Abstract The unfolded protein response (UPR) is a signal transduction pathway that coordinates cellular adaptation to microenvironmental stress and the accumulation of malfolded proteins in the endoplasmic reticulum (ER). In highly secretory malignancies, such as multiple myeloma (MM), characteristically there is both an extensively developed ER and a markedly elevated UPR to ensure a homeostatic balance between ER burden and ER capacity - thus making components of the UPR attractive targets for therapeutic intervention. One such target is PERK (PKR-like endoplasmic reticulum kinase), a member of the eukaryotic initiation factor (eIF)2α kinase family, that is activated during the UPR to chronic ER stress. Here, we describe the identification and characterization of compound (1) - a novel, orally bioavailable, potent and highly selective small molecule inhibitor of PERK. (1) is a sub-nM inhibitor of PERK, with a >100-fold window against other kinases (including other eIF2α kinases) - targeting only 6 out of a 400 kinase panel with sub-μM IC50's. It inhibits phosphorylation of eIF2α at 10 nM (IC50) in HEK293 cells (incubated with the ER stressor tunicamycin) and proves to be selectively anti-proliferative in an ER-stressed epithelial cancer model (A549 cells with tunicamycin) at nM concentrations, but to a lesser extent in the absence of ER stress. Furthermore, in the absence of an exogenous ER stressor, (1) induced excessive ER stress (eg, as evidenced by induction of the pro-apoptotic CHOP gene), and decreased cell viability selectively in MM cell lines and certain B-cell lymphoma lines at low nM concentrations, but not in normal or malignant epithelial cells. However, despite a sustained impact on eIF2α phosphorylation, interestingly compound (1) elicited a biphasic concentration dependent induction of ER stress (and consequent inhibition of cell viability) - with ER stress induction maximal at inhibitor concentrations corresponding to circa 50-75 % inhibition of PERK, returning progressively to baseline with concentrations rising to > IC90. Whole-genome expression profiling revealed that all significant changes seen at 10-100 nM of (1), returned to baseline levels at 1000 nM, suggesting a compensatory mechanism very proximal to phosphorylation of eIF2α. In summary, the highly selective PERK inhibitor, (1), validated the unique sensitivity of subsets of multiple myeloma and B-cell lymphoma to disruption of the UPR, but unexpectedly revealed a compensatory mechanism associated with potent PERK inhibition. Citation Format: Ian Stansfield, Yannick Ligny, Yvan Simonnet, Christophe Demestre, Nathalie Amblard, Christophe Meyer, Tamara Geerts, Jeroen Van de Ven, Ilse Van den Wyngaert, Peter Vermeulen, Inge Beerden, Danielle Peeters, Johnny Liebregts, Kurt Van Baelen, Cedric Simillion, Boudewijn Janssen, Tinne Verhulst, Norbert Esser, James Bischoff, Lieven Meerpoel, Matthias Versele. Characterization of a novel, orally bioavailable, potent and highly selective small molecule inhibitor of PERK: A tool to probe the biphasic concentration-dependent induction of ER stress in models of multiple myeloma and B-cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3228. doi:10.1158/1538-7445.AM2014-3228

Abstract Fatty Acid Synthase (FASN) is a multi-domain protein that carries out de novo fatty acid (palmitate) synthesis from acetate and malonate in mammalian cells. FASN is up-regulated in cancer cells, providing fatty acid building blocks for rapid cell growth and cell division. Increased FASN expression is correlated with disease progression and poor prognosis in many cancers including prostate, breast, ovary, colon, and lung. FASN has been demonstrated to play an important role in carcinogenesis by protecting cells from apoptosis. Herein we report a new series of potent, selective and orally bioavailable FASN inhibitors. Recent publications disclose several FASN inhibitor chemotypes that share a common pharmacophore, wherein an aromatic group and an acylated cyclic amine are attached to a central scaffold. We postulated that a spirocyclic imidazolinone core would be an acceptable and drug-like scaffold, inspired by the precedent of irbesartan, an approved antihypertensive drug in which a spirocyclopentyl-imidazolinone core replaces the substituted imidazole ring of losartan, an older approved agent from the same drug class. This hypothesis led to a new spirocyclic imidazolinone based FASN inhibitors. Extensive SAR efforts resulted in FASN inhibitors with potent enzyme and cell activity, selectivity, and oral bioavailability exemplified by JNJ-54302833. JNJ-54302833 is a potent inhibitor of human FASN (IC50 = 28 nM) and also potently inhibits proliferation of A2780 ovarian cells (IC50 = 13 nM) in lipid-reduced medium. This cellular activity can be rescued by addition of palmitate, demonstrating on-target effects. JNJ-54302833 is also potent in many other cells, including PC3M (IC50 = 25 nM) and LnCaP-Vancouver prostate cells (IC50 = 66 nM), and is highly bioavailable (F 61%) with good exposures. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with JNJ-54302833 resulted in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate. This novel series potently inhibits the FASN KR domain (IC50 = 54 nM for JNJ-54302833); specific binding to KR was confirmed by crystal structures.In summary, we have designed and discovered a new series of FASN inhibitors that are potent both in enzyme and in cell proliferation assays, are highly bioavailable, and bind to KR domain. Additionally, palmitate rescue of lipid-reduced cellular activity suggests selectivity and pharmacodynamics studies confirm target engagement. Citation Format: Tianbao Lu, Richard Alexander, Gilles Bignan, James Bischoff, Peter Connolly, Max Cummings, Sabine De Breucker, Norbert Esser, Erwin Fraiponts, Ron Gilissen, Bruce Grasberger, Boudewijn Janssens, Donald Ludovici, Lieven Meerpoel, Christophe Meyer, Michael Parker, Danielle Peeters, Carsten Schubert, Karine Smans, Luc Van Nuffel, Peter Vermeulen. Design and synthesis of a series highly potent and bioavailable FASN KR domain inhibitors for cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4747. doi:10.1158/1538-7445.AM2014-4747

Abstract De novo synthesis of fatty acids in mammalian cells is catalyzed by Fatty Acid Synthase (FASN), a complex dimeric protein with seven catalytic domains that processes acetyl-CoA and malonyl-CoA into palmitic acid. Palmitate and other fatty acids are essential for normal physiological processes like energy storage and production and are key intermediates in the biosynthesis of hormones and other essential biomolecules. The role of FASN in cancer has been well documented over the past decade. While FASN is expressed at low levels in most normal tissue (except liver and adipose tissue), it is highly expressed in many tumors including prostate, breast, and colon. FASN overexpression and activity is correlated with poor prognosis and disease progression. Numerous publications describing anti-cancer effects of small molecule FASN inhibitors have appeared in the scientific and patent literature. Recently, several groups reported anticancer and antiviral activities of N-benzoyl arylpiperidine FASN inhibitors. In this work we describe the discovery of novel N-benzoyl arylpiperidine and arylazetidine compounds that potently inhibit FASN enzymatic activity in vitro and antiproliferative activity in FASN-sensitive cell lines. We conducted focused structure-activity (SAR) studies exploring the benzoyl group and the aryl substituent on the piperidine or azetidine heterocycle to optimize potency, improve properties and oral bioavailability, and achieve in vivo FASN-driven pharmacodynamic (PD) activity. X-ray crystal structures of these molecules confirm a unique binding mode within the KR subdomain of FASN. Using structural information and molecular modeling, we were able to rationalize SAR trends and design FASN inhibitors with excellent in vitro potency and cellular activity. These efforts resulted in the discovery of compounds exemplified by JNJ-54380482, a potent FASN inhibitor (IC50 = 26 nM) that inhibits proliferation of A2780 ovarian cells in lipid-reduced medium (LRM, IC50 = 8.9 nM). Engagement of FASN in cellular assays is confirmed by rescue of proliferation upon addition of palmitate. JNJ-54380482 is orally bioavailable in mice (F% = 51) and exhibits excellent plasma exposure when dosed at 10 mg/kg (Cmax = 2.6 µM, T1/2 = 5 h). In a PD study using H460 lung xenograft-bearing mice, oral treatment with 30 mg/kg of JNJ-54380482 results in 36-fold higher tumor levels of malonyl-CoA vs vehicle. In summary, we synthesized several hundred compounds having the N-benzoyl arylpiperidine and arylazetidine chemotypes. Utilizing structural information from X-ray co-crystals to understand SAR and guide the design of more potent molecules, we identified >100 analogs with highly potent FASN enzymatic (IC50 ≤ 50 nM) and antiproliferative activities in A2780 cells in LRM (IC50 ≤ 100 nM). Key examples from these series have good in vitro properties, are orally bioavailable, and demonstrate FASN target engagement in a mouse PD model. Citation Format: Peter J. Connolly, Gilles Bignan, James Bischoff, Sabine De Breucker, Norbert Esser, Erwin Fraiponts, Ron Gilissen, Bruce Grasberger, Boudewijn Janssens, Tianbao Lu, Donald Ludovici, Lieven Meerpoel, Christophe Meyer, Michael Parker, Danielle Peeters, Carsten Schubert, Karine Smans, Luc Van Nuffel, Peter Vermeulen. Structure-activity relationships of novel N-benzoyl arylpiperidine and arylazetidine FASN inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2535. doi:10.1158/1538-7445.AM2014-2535

Abstract Plasma cell malignancies, such as multiple myeloma, are characterized by an extensively developed endoplasmic reticulum (ER) to accommodate the high secretion rate of immunoglobulins. An adaptive stress response mechanism, termed the unfolded protein response (UPR), is markedly elevated in multiple myeloma cells to ensure a homeostatic balance between ER burden and ER capacity. One branch of the UPR consists of the activation of PERK under chronic ER stress: PERK phosphorylates and impairs the function of the translation initiation factor, eIF2α, thereby downregulating global protein synthesis, and reducing the secretory burden on the ER. To test the therapeutic potential of PERK inhibitors in multiple myeloma, we identified two chemically diverse PERK inhibitors: both are sub-nM inhibitors of PERK, and have a >100-fold window against other kinases (including other eIF2α kinases). These compounds inhibit phosphorylation of eIF2α at 10-20nM (IC50) in HEK293 cells, incubated with the ER stressor tunicamycin. Both PERK inhibitors were selectively anti-proliferative in an ER-stressed epithelial cancer model (A549 cells with tunicamycin) at nM concentrations, but not in the absence of ER stress. Furthermore, in the absence of an exogenous ER stressor, both PERK inhibitors induced ER stress (eg, as evidenced by induction of the pro-apoptotic CHOP gene) selectively in multiple myeloma cell lines (and certain B-cell lymphoma lines) at low nM concentrations but not in normal or malignant epithelial cells (induction of CHOP at 10 μM). The magnitude of this induction was comparable to well-established ER stressors, such as bortezomib or tunicamycin, and correlated closely with reduced proliferation in malignant B-cell lines. The potent induction of CHOP occurred both in vitro and upon oral dosing of mice, with a xenografted multiple myeloma tumour (JIM-1); however, in both instances, the induction of ER stress was maximal at a dose corresponding to approximately 50-75% inhibition of PERK, whereas, remarkably, at dose levels corresponding to more complete PERK inhibition (as evidenced by inhibition of P-eIF2α), CHOP induction (and ER-stress induction in general as determined by a genome-wide expression profile) was reduced, eventually down to baseline levels. Consistently, both PERK inhibitors resulted in a biphasic concentration-dependent reduction of proliferation of certain myeloma and B-cell lymphoma lines. Hence, PERK inhibition triggers a biphasic induction of ER stress and inhibition of cell proliferation, selectively in B-cell malignancies. The possible underlying mechanism as well as the therapeutic implications of these findings will be discussed. Citation Format: Matthias Versele, Tamara Geerts, Jeroen Van De Ven, Ilse Van den Wyngaert, Peter Vermeulen, Inge Beerden, Danielle Peeters, Johnny Liebregts, Kurt Van Baelen, Cedric Simillion, Boudewijn Janssen, Tinne Verhulst, Norbert Esser, Christophe Meyer, Ian Stansfield, James Bischoff. Selective PERK kinase inhibition triggers a biphasic concentration-dependent induction of ER stress in multiple myeloma and B-cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2926. doi:10.1158/1538-7445.AM2013-2926

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis and resistance to current anti-TB drugs call for the discovery and development of new effective anti-TB drugs. TMC207 is the lead candidate of a novel class of antimycobacterial agents, the diarylquinolines, which specifically inhibit mycobacterial ATP synthase and displays high activity against both drug-susceptible and multidrug-resistant strains of Mycobacterium tuberculosis. This article covers both synthesis pathways as well as qualitative and quantitative analyses of the structure-activity relationships of the diarylquinoline series on Mycobacterium smegmatis activity.