Chemical Biology Publications 2017

Fernández-Montalván AE, Berger M, Kuropka B, Koo SJ, Badock V, Weiske J, Puetter V, Holton SJ, Stöckigt D, Ter Laak A et al. 2017. Isoform-Selective ATAD2 Chemical Probe with Novel Chemical Structure and Unusual Mode of Action. ACS Chem Biol, 12 (11), pp. 2730-2736. | Citations: 14 (Web of Science Lite) | Show Abstract | Read more

ATAD2 (ANCCA) is an epigenetic regulator and transcriptional cofactor, whose overexpression has been linked to the progress of various cancer types. Here, we report a DNA-encoded library screen leading to the discovery of BAY-850, a potent and isoform selective inhibitor that specifically induces ATAD2 bromodomain dimerization and prevents interactions with acetylated histones in vitro, as well as with chromatin in cells. These features qualify BAY-850 as a chemical probe to explore ATAD2 biology.

Frejno M, Zenezini Chiozzi R, Wilhelm M, Koch H, Zheng R, Klaeger S, Ruprecht B, Meng C, Kramer K, Jarzab A et al. 2017. Pharmacoproteomic characterisation of human colon and rectal cancer. Mol Syst Biol, 13 (11), pp. 951. | Citations: 2 (Scopus) | Show Abstract | Read more

Most molecular cancer therapies act on protein targets but data on the proteome status of patients and cellular models for proteome-guided pre-clinical drug sensitivity studies are only beginning to emerge. Here, we profiled the proteomes of 65 colorectal cancer (CRC) cell lines to a depth of > 10,000 proteins using mass spectrometry. Integration with proteomes of 90 CRC patients and matched transcriptomics data defined integrated CRC subtypes, highlighting cell lines representative of each tumour subtype. Modelling the responses of 52 CRC cell lines to 577 drugs as a function of proteome profiles enabled predicting drug sensitivity for cell lines and patients. Among many novel associations, MERTK was identified as a predictive marker for resistance towards MEK1/2 inhibitors and immunohistochemistry of 1,074 CRC tumours confirmed MERTK as a prognostic survival marker. We provide the proteomic and pharmacological data as a resource to the community to, for example, facilitate the design of innovative prospective clinical trials.

Hadzijusufovic E, Albrecht-Schgoer K, Huber K, Hoermann G, Grebien F, Eisenwort G, Schgoer W, Herndlhofer S, Kaun C, Theurl M et al. 2017. Nilotinib-induced vasculopathy: identification of vascular endothelial cells as a primary target site. Leukemia, 31 (11), pp. 2388-2397. | Citations: 14 (Web of Science Lite) | Show Abstract | Read more

The BCR/ABL1 inhibitor Nilotinib is increasingly used to treat patients with chronic myeloid leukemia (CML). Although otherwise well-tolerated, Nilotinib has been associated with the occurrence of progressive arterial occlusive disease (AOD). Our objective was to determine the exact frequency of AOD and examine in vitro and in vivo effects of Nilotinib and Imatinib on endothelial cells to explain AOD-development. In contrast to Imatinib, Nilotinib was found to upregulate pro-atherogenic adhesion-proteins (ICAM-1, E-selectin, VCAM-1) on human endothelial cells. Nilotinib also suppressed endothelial cell proliferation, migration and tube-formation and bound to a distinct set of target-kinases, relevant to angiogenesis and atherosclerosis, including angiopoietin receptor-1 TEK, ABL-2, JAK1 and MAP-kinases. Nilotinib and siRNA against ABL-2 also suppressed KDR expression. In addition, Nilotinib augmented atherosclerosis in ApoE-/- mice and blocked reperfusion and angiogenesis in a hindlimb-ischemia model of arterial occlusion, whereas Imatinib showed no comparable effects. Clinically overt AOD-events were found to accumulate over time in Nilotinib-treated patients. After a median observation-time of 2.0 years, the AOD-frequency was higher in these patients (29.4%) compared to risk factor- and age-matched controls (<5%). Together, Nilotinib exerts direct pro-atherogenic and anti-angiogenic effects on vascular endothelial cells, which may contribute to development of AOD in patients with CML.

Schuller M, Riedel K, Gibbs-Seymour I, Uth K, Sieg C, Gehring AP, Ahel I, Bracher F, Kessler BM, Elkins JM, Knapp S. 2017. Discovery of a Selective Allosteric Inhibitor Targeting Macrodomain 2 of Polyadenosine-Diphosphate-Ribose Polymerase 14. ACS Chem Biol, 12 (11), pp. 2866-2874. | Citations: 4 (European Pubmed Central) | Show Abstract | Read more

Macrodomains are conserved protein interaction modules that can be found in all domains of life including in certain viruses. Macrodomains mediate recognition of sequence motifs harboring adenosine diphosphate ribose (ADPR) modifications, thereby regulating a variety of cellular processes. Due to their role in cancer or viral pathogenesis, macrodomains have emerged as potential therapeutic targets, but the unavailability of small molecule inhibitors has hampered target validation studies so far. Here, we describe an efficient screening strategy for identification of small molecule inhibitors that displace ADPR from macrodomains. We report the discovery and characterization of a macrodomain inhibitor, GeA-69, selectively targeting macrodomain 2 (MD2) of PARP14 with low micromolar affinity. Co-crystallization of a GeA-69 analogue with PARP14 MD2 revealed an allosteric binding mechanism explaining its selectivity over other human macrodomains. We show that GeA-69 engages PARP14 MD2 in intact cells and prevents its localization to sites of DNA damage.

Aldeghi M, Bodkin MJ, Knapp S, Biggin PC. 2017. Statistical Analysis on the Performance of Molecular Mechanics Poisson-Boltzmann Surface Area versus Absolute Binding Free Energy Calculations: Bromodomains as a Case Study. J Chem Inf Model, 57 (9), pp. 2203-2221. | Citations: 15 (Scopus) | Show Abstract | Read more

Binding free energy calculations that make use of alchemical pathways are becoming increasingly feasible thanks to advances in hardware and algorithms. Although relative binding free energy (RBFE) calculations are starting to find widespread use, absolute binding free energy (ABFE) calculations are still being explored mainly in academic settings due to the high computational requirements and still uncertain predictive value. However, in some drug design scenarios, RBFE calculations are not applicable and ABFE calculations could provide an alternative. Computationally cheaper end-point calculations in implicit solvent, such as molecular mechanics Poisson-Boltzmann surface area (MMPBSA) calculations, could too be used if one is primarily interested in a relative ranking of affinities. Here, we compare MMPBSA calculations to previously performed absolute alchemical free energy calculations in their ability to correlate with experimental binding free energies for three sets of bromodomain-inhibitor pairs. Different MMPBSA approaches have been considered, including a standard single-trajectory protocol, a protocol that includes a binding entropy estimate, and protocols that take into account the ligand hydration shell. Despite the improvements observed with the latter two MMPBSA approaches, ABFE calculations were found to be overall superior in obtaining correlation with experimental affinities for the test cases considered. A difference in weighted average Pearson ([Formula: see text]) and Spearman ([Formula: see text]) correlations of 0.25 and 0.31 was observed when using a standard single-trajectory MMPBSA setup ([Formula: see text] = 0.64 and [Formula: see text] = 0.66 for ABFE; [Formula: see text] = 0.39 and [Formula: see text] = 0.35 for MMPBSA). The best performing MMPBSA protocols returned weighted average Pearson and Spearman correlations that were about 0.1 inferior to ABFE calculations: [Formula: see text] = 0.55 and [Formula: see text] = 0.56 when including an entropy estimate, and [Formula: see text] = 0.53 and [Formula: see text] = 0.55 when including explicit water molecules. Overall, the study suggests that ABFE calculations are indeed the more accurate approach, yet there is also value in MMPBSA calculations considering the lower compute requirements, and if agreement to experimental affinities in absolute terms is not of interest. Moreover, for the specific protein-ligand systems considered in this study, we find that including an explicit ligand hydration shell or a binding entropy estimate in the MMPBSA calculations resulted in significant performance improvements at a negligible computational cost.

Meier JC, Tallant C, Fedorov O, Witwicka H, Hwang S-Y, van Stiphout RG, Lambert J-P, Rogers C, Yapp C, Gerstenberger BS et al. 2017. Selective Targeting of Bromodomains of the Bromodomain-PHD Fingers Family Impairs Osteoclast Differentiation. ACS Chem Biol, 12 (10), pp. 2619-2630. | Citations: 3 (Web of Science Lite) | Show Abstract | Read more

Histone acetyltransferases of the MYST family are recruited to chromatin by BRPF scaffolding proteins. We explored functional consequences and the therapeutic potential of inhibitors targeting acetyl-lysine dependent protein interaction domains (bromodomains) present in BRPF1-3 in bone maintenance. We report three potent and selective inhibitors: one (PFI-4) with high selectivity for the BRPF1B isoform and two pan-BRPF bromodomain inhibitors (OF-1, NI-57). The developed inhibitors displaced BRPF bromodomains from chromatin and did not inhibit cell growth and proliferation. Intriguingly, the inhibitors impaired RANKL-induced differentiation of primary murine bone marrow cells and human primary monocytes into bone resorbing osteoclasts by specifically repressing transcriptional programs required for osteoclastogenesis. The data suggest a key role of BRPF in regulating gene expression during osteoclastogenesis, and the excellent druggability of these bromodomains may lead to new treatment strategies for patients suffering from bone loss or osteolytic malignant bone lesions.

Dalvit C, Knapp S. 2017. 19 F NMR isotropic chemical shift for efficient screening of fluorinated fragments which are racemates and/or display multiple conformers. Magn Reson Chem, 55 (12), pp. 1091-1095. | Citations: 2 (Scopus) | Show Abstract | Read more

Fluorine ligand-based NMR spectroscopy is now an established method for performing binding screening against a macromolecular target. Typically, the transverse relaxation rate of the fluorine signals is monitored in the absence and presence of the target. However, useful structural information can sometimes be obtained from the analysis of the fluorine isotropic chemical shift. This is particularly relevant for molecules that are racemates and/or display multiple conformers. The large difference in fluorine isotropic chemical shift between free and bound state deriving mainly from the breaking and/or making of intramolecular and/or intermolecular hydrogen bonds allows the detection of very weak affinity ligands. According to our experimental results, racemates should always be included in the generation of the fluorinated fragment libraries. The selection or the availability of only one of the enantiomers for the fluorinated screening library could result in missing relevant chemical scaffold motifs.

Igoe N, Bayle ED, Tallant C, Fedorov O, Meier JC, Savitsky P, Rogers C, Morias Y, Scholze S, Boyd H et al. 2017. Design of a Chemical Probe for the Bromodomain and Plant Homeodomain Finger-Containing (BRPF) Family of Proteins. J Med Chem, 60 (16), pp. 6998-7011. | Citations: 9 (Web of Science Lite) | Show Abstract | Read more

The bromodomain and plant homeodomain finger-containing (BRPF) family are scaffolding proteins important for the recruitment of histone acetyltransferases of the MYST family to chromatin. Here, we describe NI-57 (16) as new pan-BRPF chemical probe of the bromodomain (BRD) of the BRPFs. Inhibitor 16 preferentially bound the BRD of BRPF1 and BRPF2 over BRPF3, whereas binding to BRD9 was weaker. Compound 16 has excellent selectivity over nonclass IV BRD proteins. Target engagement of BRPF1B and BRPF2 with 16 was demonstrated in nanoBRET and FRAP assays. The binding of 16 to BRPF1B was rationalized through an X-ray cocrystal structure determination, which showed a flipped binding orientation when compared to previous structures. We report studies that show 16 has functional activity in cellular assays by modulation of the phenotype at low micromolar concentrations in both cancer and inflammatory models. Pharmacokinetic data for 16 was generated in mouse with single dose administration showing favorable oral bioavailability.

Ferguson FM, Doctor ZM, Chaikuad A, Sim T, Kim ND, Knapp S, Gray NS. 2017. Characterization of a highly selective inhibitor of the Aurora kinases. Bioorg Med Chem Lett, 27 (18), pp. 4405-4408. | Citations: 1 (European Pubmed Central) | Show Abstract | Read more

Aurora kinases play an essential role in mitosis and cell cycle regulation. In recent years Aurora kinases have proved popular cancer targets and many inhibitors have been developed. The majority of these clinical candidates are multi-targeted, rendering them inappropriate as tools for studying Aurora kinase mediated signaling. Here we report discovery of a highly selective inhibitor of Aurora kinases A, B and C, with potent cellular activity and minimal off-target activity (PLK4). The X-ray co-crystal structure of Aurora A in complex with compound 2 is reported, and provides insights into the structural determinants of ligand binding and selectivity.

Drewry DH, Wells CI, Andrews DM, Angell R, Al-Ali H, Axtman AD, Capuzzi SJ, Elkins JM, Ettmayer P, Frederiksen M et al. 2017. Progress towards a public chemogenomic set for protein kinases and a call for contributions. PLoS One, 12 (8), pp. e0181585. | Citations: 20 (Scopus) | Show Abstract | Read more

Protein kinases are highly tractable targets for drug discovery. However, the biological function and therapeutic potential of the majority of the 500+ human protein kinases remains unknown. We have developed physical and virtual collections of small molecule inhibitors, which we call chemogenomic sets, that are designed to inhibit the catalytic function of almost half the human protein kinases. In this manuscript we share our progress towards generation of a comprehensive kinase chemogenomic set (KCGS), release kinome profiling data of a large inhibitor set (Published Kinase Inhibitor Set 2 (PKIS2)), and outline a process through which the community can openly collaborate to create a KCGS that probes the full complement of human protein kinases.

Melo-Hanchuk TD, Slepicka PF, Meirelles GV, Basei FL, Lovato DV, Granato DC, Pauletti BA, Domingues RR, Leme AFP, Pelegrini AL et al. 2017. NEK1 kinase domain structure and its dynamic protein interactome after exposure to Cisplatin. Sci Rep, 7 (1), pp. 5445. | Citations: 1 (European Pubmed Central) | Show Abstract | Read more

NEK family kinases are serine/threonine kinases that have been functionally implicated in the regulation of the disjunction of the centrosome, the assembly of the mitotic spindle, the function of the primary cilium and the DNA damage response. NEK1 shows pleiotropic functions and has been found to be mutated in cancer cells, ciliopathies such as the polycystic kidney disease, as well as in the genetic diseases short-rib thoracic dysplasia, Mohr-syndrome and amyotrophic lateral sclerosis. NEK1 is essential for the ionizing radiation DNA damage response and priming of the ATR kinase and of Rad54 through phosphorylation. Here we report on the structure of the kinase domain of human NEK1 in its apo- and ATP-mimetic inhibitor bound forms. The inhibitor bound structure may allow the design of NEK specific chemo-sensitizing agents to act in conjunction with chemo- or radiation therapy of cancer cells. Furthermore, we characterized the dynamic protein interactome of NEK1 after DNA damage challenge with cisplatin. Our data suggest that NEK1 and its interaction partners trigger the DNA damage pathways responsible for correcting DNA crosslinks.

Verçoza BRF, Godinho JLP, de Macedo-Silva ST, Huber K, Bracher F, de Souza W, Rodrigues JCF. 2017. KH-TFMDI, a novel sirtuin inhibitor, alters the cytoskeleton and mitochondrial metabolism promoting cell death in Leishmania amazonensis. Apoptosis, 22 (9), pp. 1169-1188. | Citations: 6 (Scopus) | Show Abstract | Read more

Treatment of leishmaniasis involves the use of antimonials, miltefosine, amphotericin B or pentamidine. However, the side effects of these drugs and the reports of drug-resistant parasites demonstrate the need for new treatments that are safer and more efficacious. Histone deacetylase inhibitors are a new class of compounds with potential to treat leishmaniasis. Herein, we evaluated the effects of KH-TFMDI, a novel histone deacetylase inhibitor, on Leishmania amazonensis promastigotes and intracellular amastigotes. The IC50 values of this compound for promastigotes and intracellular amastigotes were 1.976 and 1.148 μM, respectively, after 72 h of treatment. Microscopic analyses revealed that promastigotes became elongated and thinner in response to KH-TFMDI, indicating changes in cytoskeleton organization. Immunofluorescence microscopy, western blotting and flow cytometry using an anti-acetylated tubulin antibody revealed an increase in the expression of acetylated tubulin. Furthermore, transmission electron microscopy revealed several ultrastructural changes, such as (a) mitochondrial swelling, followed by the formation of many vesicles inside the matrix; (b) presence of lipid bodies randomly distributed through the cytoplasm; (c) abnormal chromatin condensation; and (d) formation of blebs on the plasma membrane. Physiological studies for mitochondrial function, flow cytometry with propidium iodide and TUNEL assay confirmed the alterations in the mitochondrial metabolism, cell cycle, and DNA fragmentation, respectively, which could result to cell death by mechanisms related to apoptosis-like. All these together indicate that histone deacetylases are promising targets for the development of new drugs to treat Leishmania, and KH-TFMDI is a promising drug candidate that should be tested in vivo.

Chaikuad A, Filippakopoulos P, Marcsisin SR, Picaud S, Schröder M, Sekine S, Ichijo H, Engen JR, Takeda K, Knapp S. 2017. Structures of PGAM5 Provide Insight into Active Site Plasticity and Multimeric Assembly. Structure, 25 (7), pp. 1089-1099.e3. | Citations: 4 (Scopus) | Show Abstract | Read more

PGAM5 is a mitochondrial membrane protein that functions as an atypical Ser/Thr phosphatase and is a regulator of oxidative stress response, necroptosis, and autophagy. Here we present several crystal structures of PGAM5 including the activating N-terminal regulatory sequences, providing a model for structural plasticity, dimerization of the catalytic domain, and the assembly into an enzymatically active dodecameric form. Oligomeric states observed in structures were supported by hydrogen exchange mass spectrometry, size-exclusion chromatography, and analytical ultracentrifugation experiments in solution. We report that the catalytically important N-terminal WDPNWD motif acts as a structural integrator assembling PGAM5 into a dodecamer, allosterically activating the phosphatase by promoting an ordering of the catalytic loop. Additionally the observed active site plasticity enabled visualization of essential conformational rearrangements of catalytic elements. The comprehensive biophysical characterization offers detailed structural models of this key mitochondrial phosphatase that has been associated with the development of diverse diseases.

Wang B-D, Ceniccola K, Hwang S, Andrawis R, Horvath A, Freedman JA, Olender J, Knapp S, Ching T, Garmire L et al. 2017. Alternative splicing promotes tumour aggressiveness and drug resistance in African American prostate cancer. Nat Commun, 8 pp. 15921. | Citations: 13 (Web of Science Lite) | Show Abstract | Read more

Clinical challenges exist in reducing prostate cancer (PCa) disparities. The RNA splicing landscape of PCa across racial populations has not been fully explored as a potential molecular mechanism contributing to race-related tumour aggressiveness. Here, we identify novel genome-wide, race-specific RNA splicing events as critical drivers of PCa aggressiveness and therapeutic resistance in African American (AA) men. AA-enriched splice variants of PIK3CD, FGFR3, TSC2 and RASGRP2 contribute to greater oncogenic potential compared with corresponding European American (EA)-expressing variants. Ectopic overexpression of the newly cloned AA-enriched variant, PIK3CD-S, in EA PCa cell lines enhances AKT/mTOR signalling and increases proliferative and invasive capacity in vitro and confers resistance to selective PI3Kδ inhibitor, CAL-101 (idelalisib), in mouse xenograft models. High PIK3CD-S expression in PCa specimens associates with poor survival. These results highlight the potential of RNA splice variants to serve as novel biomarkers and molecular targets for developmental therapeutics in aggressive PCa.

Radic-Sarikas B, Halasz M, Huber KVM, Winter GE, Tsafou KP, Papamarkou T, Brunak S, Kolch W, Superti-Furga G. 2017. Lapatinib potentiates cytotoxicity of  YM155 in neuroblastoma via inhibition of the ABCB1 efflux transporter. Sci Rep, 7 (1), pp. 3091. | Citations: 3 (Web of Science Lite) | Show Abstract | Read more

Adverse side effects of cancer agents are of great concern in the context of childhood tumors where they can reduce the quality of life in young patients and cause life-long adverse effects. Synergistic drug combinations can lessen potential toxic side effects through lower dosing and simultaneously help to overcome drug resistance. Neuroblastoma is the most common cancer in infancy and extremely heterogeneous in clinical presentation and features. Applying a systematic pairwise drug combination screen we observed a highly potent synergy in neuroblastoma cells between the EGFR kinase inhibitor lapatinib and the anticancer compound YM155 that is preserved across several neuroblastoma variants. Mechanistically, the synergy was based on a lapatinib induced inhibition of the multidrug-resistance efflux transporter ABCB1, which is frequently expressed in resistant neuroblastoma cells, which allowed prolonged and elevated cytotoxicity of YM155. In addition, the drug combination (i.e. lapatinib plus YM155) decreased neuroblastoma tumor size in an in vivo model.

Urbanucci A, Barfeld SJ, Kytölä V, Itkonen HM, Coleman IM, Vodák D, Sjöblom L, Sheng X, Tolonen T, Minner S et al. 2017. Androgen Receptor Deregulation Drives Bromodomain-Mediated Chromatin Alterations in Prostate Cancer. Cell Rep, 19 (10), pp. 2045-2059. | Citations: 17 (Scopus) | Show Abstract | Read more

Global changes in chromatin accessibility may drive cancer progression by reprogramming transcription factor (TF) binding. In addition, histone acetylation readers such as bromodomain-containing protein 4 (BRD4) have been shown to associate with these TFs and contribute to aggressive cancers including prostate cancer (PC). Here, we show that chromatin accessibility defines castration-resistant prostate cancer (CRPC). We show that the deregulation of androgen receptor (AR) expression is a driver of chromatin relaxation and that AR/androgen-regulated bromodomain-containing proteins (BRDs) mediate this effect. We also report that BRDs are overexpressed in CRPCs and that ATAD2 and BRD2 have prognostic value. Finally, we developed gene stratification signature (BROMO-10) for bromodomain response and PC prognostication, to inform current and future trials with drugs targeting these processes. Our findings provide a compelling rational for combination therapy targeting bromodomains in selected patients in which BRD-mediated TF binding is enhanced or modified as cancer progresses.

Vladimer GI, Snijder B, Krall N, Bigenzahn JW, Huber KVM, Lardeau C-H, Sanjiv K, Ringler A, Berglund UW, Sabler M et al. 2017. Global survey of the immunomodulatory potential of common drugs. Nat Chem Biol, 13 (6), pp. 681-690. | Citations: 10 (Web of Science Lite) | Show Abstract | Read more

Small-molecule drugs may complement antibody-based therapies in an immune-oncology setting, yet systematic methods for the identification and characterization of the immunomodulatory properties of these entities are lacking. We surveyed the immumomodulatory potential of 1,402 small chemical molecules, as defined by their ability to alter the cell-cell interactions among peripheral mononuclear leukocytes ex vivo, using automated microscopy and population-wide single-cell image analysis. Unexpectedly, ∼10% of the agents tested affected these cell-cell interactions differentially. The results accurately recapitulated known immunomodulatory drug classes and revealed several clinically approved drugs that unexpectedly harbor the ability to modulate the immune system, which could potentially contribute to their physiological mechanism of action. For instance, the kinase inhibitor crizotinib promoted T cell interactions with monocytes, as well as with cancer cells, through inhibition of the receptor tyrosine kinase MSTR1 and subsequent upregulation of the expression of major histocompatibility complex molecules. The approach offers an attractive platform for the personalized identification and characterization of immunomodulatory therapeutics.

Lines KE, Stevenson M, Filippakopoulos P, Müller S, Lockstone HE, Wright B, Grozinsky-Glasberg S, Grossman AB, Knapp S, Buck D et al. 2017. Epigenetic pathway inhibitors represent potential drugs for treating pancreatic and bronchial neuroendocrine tumors. Oncogenesis, 6 (5), pp. e332. | Citations: 10 (Web of Science Lite) | Show Abstract | Read more

Cancer is associated with alterations in epigenetic mechanisms such as histone modifications and methylation of DNA, and inhibitors targeting epigenetic mechanisms represent a novel class of anti-cancer drugs. Neuroendocrine tumors (NETs) of the pancreas (PNETs) and bronchus (BNETs), which may have 5-year survivals of <50% and as low as 5%, respectively, represent targets for such drugs, as >40% of PNETs and ~35% of BNETs have mutations of the multiple endocrine neoplasia type 1 (MEN1) gene, which encodes menin that modifies histones by interacting with histone methyltransferases. We assessed 9 inhibitors of epigenetic pathways, for their effects on proliferation, by CellTiter Blue assay, and apoptosis, by CaspaseGlo assay, using 1 PNET and 2 BNET cell lines. Two inhibitors, referred to as (+)-JQ1 (JQ1) and PFI-1, targeting the bromo and extra terminal (BET) protein family which bind acetylated histone residues, were most effective in decreasing proliferation (by 40-85%, P<0.001) and increasing apoptosis (by 2-3.6 fold, P<0.001) in all 3 NET cell lines. The anti-proliferative effects of JQ1 and PFI-1 remained present for at least 48 hours after removal of the compound. JQ1, but not PFI-1, had cell cycle effects, assessed by propidium iodide staining and flow cytometry, resulting in increased and decreased proportions of NET cells in G1, and S and G2 phases, respectively. RNA Sequencing analysis revealed that these JQ1 effects were associated with increased histone 2B expression, and likely mediated through altered activity of bromodomain-containing (Brd) proteins. Assessment of JQ1 in vivo, using a pancreatic beta cell-specific conditional Men1 knockout mouse model that develops PNETs, revealed that JQ1 significantly reduced proliferation (by ~50%, P<0.0005), assessed by bromodeoxyuridine incorporation, and increased apoptosis (by ~3 fold, P<0.0005), assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling, of PNETs. Thus, our studies demonstrate that BET protein inhibitors may provide new treatments for NETs.

Zimmermann G, Rieder U, Bajic D, Vanetti S, Chaikuad A, Knapp S, Scheuermann J, Mattarella M, Neri D. 2017. A Specific and Covalent JNK-1 Ligand Selected from an Encoded Self-Assembling Chemical Library. Chemistry, 23 (34), pp. 8152-8155. | Citations: 10 (Scopus) | Show Abstract | Read more

We describe the construction of a DNA-encoded chemical library comprising 148 135 members, generated through the self-assembly of two sub-libraries, containing 265 and 559 members, respectively. The library was designed to contain building blocks potentially capable of forming covalent interactions with target proteins. Selections performed with JNK1, a kinase containing a conserved cysteine residue close to the ATP binding site, revealed the preferential enrichment of a 2-phenoxynicotinic acid moiety (building block A82) and a 4-(3,4-difluorophenyl)-4-oxobut-2-enoic acid moiety (building block B272). When the two compounds were joined by a short PEG linker, the resulting bidentate binder (A82-L-B272) was able to covalently modify JNK1 in the presence of a large molar excess of glutathione (0.5 mm), used to simulate intracellular reducing conditions. By contrast, derivatives of the individual building blocks were not able to covalently modify JNK1 in the same experimental conditions. The A82-L-B272 ligand was selective over related kinases (BTK and GAK), which also contain targetable cysteine residues in the vicinity of the active site.

Kashima R, Redmond PL, Ghatpande P, Roy S, Kornberg TB, Hanke T, Knapp S, Lagna G, Hata A. 2017. Hyperactive locomotion in a Drosophila model is a functional readout for the synaptic abnormalities underlying fragile X syndrome. Sci Signal, 10 (477), pp. eaai8133-eaai8133. | Citations: 11 (Scopus) | Show Abstract | Read more

Fragile X syndrome (FXS) is the most common cause of heritable intellectual disability and autism and affects ~1 in 4000 males and 1 in 8000 females. The discovery of effective treatments for FXS has been hampered by the lack of effective animal models and phenotypic readouts for drug screening. FXS ensues from the epigenetic silencing or loss-of-function mutation of the fragile X mental retardation 1 (FMR1) gene, which encodes an RNA binding protein that associates with and represses the translation of target mRNAs. We previously found that the activation of LIM kinase 1 (LIMK1) downstream of augmented synthesis of bone morphogenetic protein (BMP) type 2 receptor (BMPR2) promotes aberrant synaptic development in mouse and Drosophila models of FXS and that these molecular and cellular markers were correlated in patients with FXS. We report that larval locomotion is augmented in a Drosophila FXS model. Genetic or pharmacological intervention on the BMPR2-LIMK pathway ameliorated the synaptic abnormality and locomotion phenotypes of FXS larvae, as well as hyperactivity in an FXS mouse model. Our study demonstrates that (i) the BMPR2-LIMK pathway is a promising therapeutic target for FXS and (ii) the locomotion phenotype of FXS larvae is a quantitative functional readout for the neuromorphological phenotype associated with FXS and is amenable to the screening novel FXS therapeutics.

Bouché L, Christ CD, Siegel S, Fernández-Montalván AE, Holton SJ, Fedorov O, Ter Laak A, Sugawara T, Stöckigt D, Tallant C et al. 2017. Benzoisoquinolinediones as Potent and Selective Inhibitors of BRPF2 and TAF1/TAF1L Bromodomains. J Med Chem, 60 (9), pp. 4002-4022. | Citations: 10 (Scopus) | Show Abstract | Read more

Bromodomains (BD) are readers of lysine acetylation marks present in numerous proteins associated with chromatin. Here we describe a dual inhibitor of the bromodomain and PHD finger (BRPF) family member BRPF2 and the TATA box binding protein-associated factors TAF1 and TAF1L. These proteins are found in large chromatin complexes and play important roles in transcription regulation. The substituted benzoisoquinolinedione series was identified by high-throughput screening, and subsequent structure-activity relationship optimization allowed generation of low nanomolar BRPF2 BD inhibitors with strong selectivity against BRPF1 and BRPF3 BDs. In addition, a strong inhibition of TAF1/TAF1L BD2 was measured for most derivatives. The best compound of the series was BAY-299, which is a very potent, dual inhibitor with an IC50 of 67 nM for BRPF2 BD, 8 nM for TAF1 BD2, and 106 nM for TAF1L BD2. Importantly, no activity was measured for BRD4 BDs. Furthermore, cellular activity was evidenced using a BRPF2- or TAF1-histone H3.3 or H4 interaction assay.

Bataille CJR, Brennan MB, Byrne S, Davies SG, Durbin M, Fedorov O, Huber KVM, Jones AM, Knapp S, Liu G et al. 2017. Thiazolidine derivatives as potent and selective inhibitors of the PIM kinase family. Bioorg Med Chem, 25 (9), pp. 2657-2665. | Citations: 7 (Web of Science Lite) | Show Abstract | Read more

The PIM family of serine/threonine kinases have become an attractive target for anti-cancer drug development, particularly for certain hematological malignancies. Here, we describe the discovery of a series of inhibitors of the PIM kinase family using a high throughput screening strategy. Through a combination of molecular modeling and optimization studies, the intrinsic potencies and molecular properties of this series of compounds was significantly improved. An excellent pan-PIM isoform inhibition profile was observed across the series, while optimized examples show good selectivity over other kinases. Two PIM-expressing leukemic cancer cell lines, MV4-11 and K562, were employed to evaluate the in vitro anti-proliferative effects of selected inhibitors. Encouraging activities were observed for many examples, with the best example (44) giving an IC50 of 0.75μM against the K562 cell line. These data provide a promising starting point for further development of this series as a new cancer therapy through PIM kinase inhibition.

Batson J, Toop HD, Redondo C, Babaei-Jadidi R, Chaikuad A, Wearmouth SF, Gibbons B, Allen C, Tallant C, Zhang J et al. 2017. Development of Potent, Selective SRPK1 Inhibitors as Potential Topical Therapeutics for Neovascular Eye Disease. ACS Chem Biol, 12 (3), pp. 825-832. | Citations: 17 (Scopus) | Show Abstract | Read more

Serine/arginine-protein kinase 1 (SRPK1) regulates alternative splicing of VEGF-A to pro-angiogenic isoforms and SRPK1 inhibition can restore the balance of pro/antiangiogenic isoforms to normal physiological levels. The lack of potency and selectivity of available compounds has limited development of SRPK1 inhibitors, with the control of alternative splicing by splicing factor-specific kinases yet to be translated. We present here compounds that occupy a binding pocket created by the unique helical insert of SRPK1, and trigger a backbone flip in the hinge region, that results in potent (<10 nM) and selective inhibition of SRPK1 kinase activity. Treatment with these inhibitors inhibited SRPK1 activity and phosphorylation of serine/arginine splicing factor 1 (SRSF1), resulting in alternative splicing of VEGF-A from pro-angiogenic to antiangiogenic isoforms. This property resulted in potent inhibition of blood vessel growth in models of choroidal angiogenesis in vivo. This work identifies tool compounds for splice isoform selective targeting of pro-angiogenic VEGF, which may lead to new therapeutic strategies for a diversity of diseases where dysfunctional splicing drives disease development.

Huber KVM. 2017. Target engagement: Shining a light. Nat Chem Biol, 13 (2), pp. 133-134. | Citations: 2 (European Pubmed Central) | Read more

Moustakim M, Clark PGK, Trulli L, Fuentes de Arriba AL, Ehebauer MT, Chaikuad A, Murphy EJ, Mendez-Johnson J, Daniels D, Hou C-FD et al. 2017. Discovery of a PCAF Bromodomain Chemical Probe. Angew Chem Int Ed Engl, 56 (3), pp. 827-831. | Citations: 19 (Scopus) | Show Abstract | Read more

The p300/CBP-associated factor (PCAF) and related GCN5 bromodomain-containing lysine acetyl transferases are members of subfamily I of the bromodomain phylogenetic tree. Iterative cycles of rational inhibitor design and biophysical characterization led to the discovery of the triazolopthalazine-based L-45 (dubbed L-Moses) as the first potent, selective, and cell-active PCAF bromodomain (Brd) inhibitor. Synthesis from readily available (1R,2S)-(-)-norephedrine furnished L-45 in enantiopure form. L-45 was shown to disrupt PCAF-Brd histone H3.3 interaction in cells using a nanoBRET assay, and a co-crystal structure of L-45 with the homologous Brd PfGCN5 from Plasmodium falciparum rationalizes the high selectivity for PCAF and GCN5 bromodomains. Compound L-45 shows no observable cytotoxicity in peripheral blood mononuclear cells (PBMC), good cell-permeability, and metabolic stability in human and mouse liver microsomes, supporting its potential for in vivo use.

Igoe N, Bayle ED, Fedorov O, Tallant C, Savitsky P, Rogers C, Owen DR, Deb G, Somervaille TCP, Andrews DM et al. 2017. Design of a Biased Potent Small Molecule Inhibitor of the Bromodomain and PHD Finger-Containing (BRPF) Proteins Suitable for Cellular and in Vivo Studies. J Med Chem, 60 (2), pp. 668-680. | Citations: 15 (Scopus) | Show Abstract | Read more

The BRPF (bromodomain and PHD finger-containing) family are scaffolding proteins important for the recruitment of histone acetyltransferases of the MYST family to chromatin. Evaluation of the BRPF family as a potential drug target is at an early stage although there is an emerging understanding of a role in acute myeloid leukemia (AML). We report the optimization of fragment hit 5b to 13-d as a biased, potent inhibitor of the BRD of the BRPFs with excellent selectivity over nonclass IV BRD proteins. Evaluation of 13-d in a panel of cancer cell lines showed a selective inhibition of proliferation of a subset of AML lines. Pharmacokinetic studies established that 13-d had properties compatible with oral dosing in mouse models of disease (Fpo 49%). We propose that NI-42 (13-d) is a new chemical probe for the BRPFs suitable for cellular and in vivo studies to explore the fundamental biology of these proteins.

Radic-Sarikas B, Tsafou KP, Emdal KB, Papamarkou T, Huber KVM, Mutz C, Toretsky JA, Bennett KL, Olsen JV, Brunak S et al. 2017. Combinatorial Drug Screening Identifies Ewing Sarcoma-specific Sensitivities. Mol Cancer Ther, 16 (1), pp. 88-101. | Citations: 6 (Scopus) | Show Abstract | Read more

Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pediatric tumor types, we identified Ewing sarcoma-specific interactions of a diverse set of targeted agents including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1 We generated a molecular target profile of PKC412, a multikinase inhibitor with strong synergistic propensity in Ewing sarcoma, revealing its targets in critical Ewing sarcoma signaling routes. Using a multilevel experimental approach including quantitative phosphoproteomics, we analyzed the molecular rationale behind the disease-specific synergistic effect of simultaneous application of PKC412 and IGF1R inhibitors. The mechanism of the drug synergy between these inhibitors is different from the sum of the mechanisms of the single agents. The combination effectively inhibited pathway crosstalk and averted feedback loop repression, in EWS-FLI1-dependent manner. Mol Cancer Ther; 16(1); 88-101. ©2016 AACR.

Aldeghi M, Heifetz A, Bodkin MJ, Knapp S, Biggin PC. 2017. Predictions of Ligand Selectivity from Absolute Binding Free Energy Calculations. J Am Chem Soc, 139 (2), pp. 946-957. | Citations: 33 (Scopus) | Show Abstract | Read more

Binding selectivity is a requirement for the development of a safe drug, and it is a critical property for chemical probes used in preclinical target validation. Engineering selectivity adds considerable complexity to the rational design of new drugs, as it involves the optimization of multiple binding affinities. Computationally, the prediction of binding selectivity is a challenge, and generally applicable methodologies are still not available to the computational and medicinal chemistry communities. Absolute binding free energy calculations based on alchemical pathways provide a rigorous framework for affinity predictions and could thus offer a general approach to the problem. We evaluated the performance of free energy calculations based on molecular dynamics for the prediction of selectivity by estimating the affinity profile of three bromodomain inhibitors across multiple bromodomain families, and by comparing the results to isothermal titration calorimetry data. Two case studies were considered. In the first one, the affinities of two similar ligands for seven bromodomains were calculated and returned excellent agreement with experiment (mean unsigned error of 0.81 kcal/mol and Pearson correlation of 0.75). In this test case, we also show how the preferred binding orientation of a ligand for different proteins can be estimated via free energy calculations. In the second case, the affinities of a broad-spectrum inhibitor for 22 bromodomains were calculated and returned a more modest accuracy (mean unsigned error of 1.76 kcal/mol and Pearson correlation of 0.48); however, the reparametrization of a sulfonamide moiety improved the agreement with experiment.

Walter A, Chaikuad A, Loaëc N, Preu L, Knapp S, Meijer L, Kunick C, Koch O. 2017. Identification of CLK1 Inhibitors by a Fragment-linking Based Virtual Screening. Mol Inform, 36 (4), pp. 1600123-1600123. | Citations: 1 (Web of Science Lite) | Show Abstract | Read more

Alternative splicing plays an important role in the regulation of protein biosynthesis. CDC2-like kinases (CLKs) phosphorylate splicing factors rendering them a potential target for treating diseases caused by splicing dysregulation. As selective and potent inhibitors of CLK1 are still lacking, a fragment-linking based virtual screening campaign was successfully applied to identify new inhibitors showing activity on CLK1. These inhibitors exhibit a novel 2,4-substituted 1,3-thiazole scaffold that is suitable for further modification. A subsequently performed docking and protein structure based analysis revealed first hints for inhibitors showing preferred binding activity for CLK1 and DYRK2 over other splicing kinases.

Nagarajan S, Bedi U, Budida A, Hamdan FH, Mishra VK, Najafova Z, Xie W, Alawi M, Indenbirken D, Knapp S et al. 2017. BRD4 promotes p63 and GRHL3 expression downstream of FOXO in mammary epithelial cells. Nucleic Acids Res, 45 (6), pp. 3130-3145. | Citations: 6 (Scopus) | Show Abstract | Read more

Bromodomain-containing protein 4 (BRD4) is a member of the bromo- and extraterminal (BET) domain-containing family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelial-specific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXO transcription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression.

Olzscha H, Fedorov O, Kessler BM, Knapp S, La Thangue NB. 2017. CBP/p300 Bromodomains Regulate Amyloid-like Protein Aggregation upon Aberrant Lysine Acetylation. Cell Chem Biol, 24 (1), pp. 9-23. | Citations: 9 (Scopus) | Show Abstract | Read more

Lysine acetylation is becoming increasingly recognized as a general biological principle in cellular homeostasis, and is subject to abnormal control in different human pathologies. Here, we describe a global effect on amyloid-like protein aggregation in human cells that results from aberrant lysine acetylation. Bromodomain reader proteins are involved in the aggregation process and, using chemical biology and gene silencing, we establish that p300/CBP bromodomains are necessary for aggregation to occur. Moreover, protein aggregation disturbs proteostasis by impairing the ubiquitin proteasome system (UPS) and protein translation, resulting in decreased cell viability. p300/CBP bromodomain inhibitors impede aggregation, which coincides with enhanced UPS function and increased cell viability. Aggregation of a pathologically relevant form of huntingtin protein is similarly affected by p300/CBP inhibition. Our results have implications for understanding the molecular basis of protein aggregation, and highlight the possibility of treating amyloid-like pathologies and related protein folding diseases with bromodomain inhibitor-based strategies.

Li J, Casteels T, Frogne T, Ingvorsen C, Honoré C, Courtney M, Huber KVM, Schmitner N, Kimmel RA, Romanov RA et al. 2017. Artemisinins Target GABAA Receptor Signaling and Impair α Cell Identity. Cell, 168 (1-2), pp. 86-100.e15. | Citations: 102 (Scopus) | Show Abstract | Read more

Type 1 diabetes is characterized by the destruction of pancreatic β cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional β-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic β cell mass from α cells.

Ma Y, Wang L, Neitzel LR, Loganathan SN, Tang N, Qin L, Crispi EE, Guo Y, Knapp S, Beauchamp RD et al. 2017. The MAPK Pathway Regulates Intrinsic Resistance to BET Inhibitors in Colorectal Cancer. Clin Cancer Res, 23 (8), pp. 2027-2037. | Citations: 10 (Web of Science Lite) | Show Abstract | Read more

Purpose: The bromodomain and extra-terminal domain (BET) family proteins are epigenetic readers for acetylated histone marks. Emerging BET bromodomain inhibitors have exhibited antineoplastic activities in a wide range of human cancers through suppression of oncogenic transcription factors, including MYC. However, the preclinical activities of BET inhibitors in advanced solid cancers are moderate at best. To improve BET-targeted therapy, we interrogated mechanisms mediating resistance to BET inhibitors in colorectal cancer.Experimental Design: Using a panel of molecularly defined colorectal cancer cell lines, we examined the impact of BET inhibition on cellular proliferation and survival as well as MYC activity. We further tested the ability of inhibitors targeting the RAF/MEK/ERK (MAPK) pathway to enhance MYC suppression and circumvent intrinsic resistance to BET inhibitors. Key findings were validated using genetic approaches.Results: BET inhibitors as monotherapy moderately reduced colorectal cancer cell proliferation and MYC expression. Blockade of the MAPK pathway synergistically sensitized colorectal cancer cells to BET inhibitors, leading to potent apoptosis and MYC downregulation in vitro and in vivo A combination of JQ1 and trametinib, but neither agent alone, induced significant regression of subcutaneous colorectal cancer xenografts.Conclusions: Our findings suggest that the MAPK pathway confers intrinsic resistance to BET inhibitors in colorectal cancer and propose an effective combination strategy for the treatment of colorectal cancer. Clin Cancer Res; 23(8); 2027-37. ©2016 AACR.

Najafova Z, Tirado-Magallanes R, Subramaniam M, Hossan T, Schmidt G, Nagarajan S, Baumgart SJ, Mishra VK, Bedi U, Hesse E et al. 2017. BRD4 localization to lineage-specific enhancers is associated with a distinct transcription factor repertoire. Nucleic Acids Res, 45 (1), pp. 127-141. | Citations: 19 (Scopus) | Show Abstract | Read more

Proper temporal epigenetic regulation of gene expression is essential for cell fate determination and tissue development. The Bromodomain-containing Protein-4 (BRD4) was previously shown to control the transcription of defined subsets of genes in various cell systems. In this study we examined the role of BRD4 in promoting lineage-specific gene expression and show that BRD4 is essential for osteoblast differentiation. Genome-wide analyses demonstrate that BRD4 is recruited to the transcriptional start site of differentiation-induced genes. Unexpectedly, while promoter-proximal BRD4 occupancy correlated with gene expression, genes which displayed moderate expression and promoter-proximal BRD4 occupancy were most highly regulated and sensitive to BRD4 inhibition. Therefore, we examined distal BRD4 occupancy and uncovered a specific co-localization of BRD4 with the transcription factors C/EBPb, TEAD1, FOSL2 and JUND at putative osteoblast-specific enhancers. These findings reveal the intricacies of lineage specification and provide new insight into the context-dependent functions of BRD4.

da Motta LL, Ledaki I, Purshouse K, Haider S, De Bastiani MA, Baban D, Morotti M, Steers G, Wigfield S, Bridges E et al. 2017. The BET inhibitor JQ1 selectively impairs tumour response to hypoxia and downregulates CA9 and angiogenesis in triple negative breast cancer. Oncogene, 36 (1), pp. 122-132. | Citations: 37 (Scopus) | Show Abstract | Read more

The availability of bromodomain and extra-terminal inhibitors (BETi) has enabled translational epigenetic studies in cancer. BET proteins regulate transcription by selectively recognizing acetylated lysine residues on chromatin. BETi compete with this process leading to both downregulation and upregulation of gene expression. Hypoxia enables progression of triple negative breast cancer (TNBC), the most aggressive form of breast cancer, partly by driving metabolic adaptation, angiogenesis and metastasis through upregulation of hypoxia-regulated genes (for example, carbonic anhydrase 9 (CA9) and vascular endothelial growth factor A (VEGF-A). Responses to hypoxia can be mediated epigenetically, thus we investigated whether BETi JQ1 could impair the TNBC response induced by hypoxia and exert anti-tumour effects. JQ1 significantly modulated 44% of hypoxia-induced genes, of which two-thirds were downregulated including CA9 and VEGF-A. JQ1 prevented HIF binding to the hypoxia response element in CA9 promoter, but did not alter HIF expression or activity, suggesting some HIF targets are BET-dependent. JQ1 reduced TNBC growth in vitro and in vivo and inhibited xenograft vascularization. These findings identify that BETi dually targets angiogenesis and the hypoxic response, an effective combination at reducing tumour growth in preclinical studies.

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