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Arginase-1, which converts the amino acid L-arginine into L-ornithine and urea, is a promising new drug target for cancer immunotherapy, as it has a role in the regulation of T-cell immunity in the tumor microenvironment. To enable the discovery of small-molecule Arginase-1 inhibitors by high-throughput screening, we developed a novel homogeneous (mix-and-measure) fluorescence-based activity assay. The assay measures the conversion of L-arginine into L-ornithine by a decrease in fluorescent signal due to quenching of a fluorescent probe, Arginase Gold. This way, inhibition of Arginase-1 results in a gain of signal when compared with the uninhibited enzyme. Side-by-side profiling of reference inhibitors in the fluorescence-based assay and a colorimetric urea formation assay revealed similar potencies and the same potency rank order among the two assay formats. The fluorescence-based assay was successfully automated for high-throughput screening of a small-molecule library in 384-well format with a good Z'-factor and hit confirmation rate. Finally, we show that the assay can be used to study the binding kinetics of inhibitors.

The protein kinase threonine tyrosine kinase (TTK; also known as Mps1) is a critical component of the spindle assembly checkpoint and a promising drug target for the treatment of aggressive cancers, such as triple negative breast cancer. While the first TTK inhibitors have entered clinical trials, little is known about how the inhibition of TTK with small-molecule compounds affects cellular activity. We studied the selective TTK inhibitor NTRC 0066-0, which was developed in our own laboratory, together with 11 TTK inhibitors developed by other companies, including Mps-BAY2b, BAY 1161909, BAY 1217389 (Bayer), TC-Mps1-12 (Shionogi), and MPI-0479605 (Myrexis). Parallel testing shows that the cellular activity of these TTK inhibitors correlates with their binding affinity to TTK and, more strongly, with target residence time. TTK inhibitors are therefore an example where target residence time determines activity in in vitro cellular assays. X-ray structures and thermal stability experiments reveal that the most potent compounds induce a shift of the glycine-rich loop as a result of binding to the catalytic lysine at position 553. This "lysine trap" disrupts the catalytic machinery. Based on these insights, we developed TTK inhibitors, based on a (5,6-dihydro)pyrimido[4,5-e]indolizine scaffold, with longer target residence times, which further exploit an allosteric pocket surrounding Lys553. Their binding mode is new for kinase inhibitors and can be classified as hybrid Type I/Type III. These inhibitors have very potent anti-proliferative activity that rivals classic cytotoxic therapy. Our findings will open up new avenues for more applications for TTK inhibitors in cancer treatment.

The journey towards a detailed mechanistic understanding of the anticoagulant action of heparin has resulted in synthetic mimetics with improved pharmacodynamic profiles. Inspired by the ternary complex formation of heparin with antithrombin III and thrombin, the active pentasaccharide fondaparinux has been succeeded by several clinical candidates, such as SR123781, that have tailor-made factor Xa and thrombin inhibitory activities combined with less aspecific binding (e.g. binding to platelet factor 4 involved in thrombocytopenia). Novel compounds with both antithrombin III-mediated inhibition of factor Xa and direct thrombin inhibition are emerging. Org42675 is one such compound, balancing dual inhibition of factor Xa and thrombin in one anticoagulant drug, with excellent pharmacokinetic properties and strong inhibitory activity toward clot-bound thrombin.

The protein kinase TTK is a critical component of the spindle assembly checkpoint (SAC), which regulates the proper attachment of sister chromatids during mitosis. TTK is essential for normal progression of the cell cycle and is upregulated in various aggressive cancers such as triple negative breast cancer (TNBC). Inhibition of TTK leads to an overriding of the SAC, premature progression of the cell cycle and mitotic catastrophe. TTK inhibition therefore contrasts with e.g. Aurora and PLK1 inhibition, which block the cell cycle [1]. Various studies have shown the clinical utility of TTK inhibition, particularly in combination therapy with paclitaxel [1,2]. In vivo, TTK inhibitor monotherapy has shown partial tumor growth inhibition without weight loss [1,2]. We have developed a novel class of TTK inhibitors based on a pyrimido-indolizine scaffold. These bind selectively and with high affinity to the ATP pocket of TTK and potently inhibit the proliferation of a variety of cell lines [1]. Previously we published a representative of this class, NTRC 0066-0, which has an IC50 of 0.6 nM in a biochemical assay for TTK activity and which is more than 200 times selective over a panel of 276 kinases [1]. NTRC 0066-0 has an average IC50 of 98 nM in proliferation assays in 66 different cell lines (Oncolines™ panel) [3]. To further improve the potency of the pyrimido-indolizine series, we determined the X-ray structures of a dozen of class representatives in complex with TTK. The results were compared to 3D complexes of other chemical scaffolds such as BAY-1161909, Mps-Bay2b, MPI-0479605, NMS-P715 and Mps1-IN1. The pyrimido-indolizine series uses an aromatic moiety to trap the catalytic lysine in the active site, enforcing a catalytically incompetent conformation of TTK. Thermal melting and surface plasmon resonance experiments demonstrate that this leads to a strong stabilization of the kinase domain and a slow dissociation rate for the compounds, which is one of the key determinants for potent cellular activity. We took advantage of these structure-activity relationships to develop analogs of NTRC 0066-0 with increased residence time and cellular potency. One such analog, NTRC 1501-0, inhibits the proliferation of 66 cancer cell lines with an average IC50 of 18 nM, while retaining selectivity over the kinome. It has good pharmacokinetic properties and shows no cross-reactivity with drug safety targets in vitro. In a xenograft model of the human TNBC cell line MDA-MB-231, it completely inhibited tumor growth at a low oral dose, without effect on body weight, indicating good tolerability. These data show, for the first time, that TTK inhibition as monotherapy can achieve complete inhibition of tumor growth. [1] Maia et al. (2015) Annals of Oncology 26, 2180-2192; [2] Wengner et al (2016) Mol. Canc. Therap. 15, 583-592; [3] Uitdehaag et al. (2016) Mol. Canc. Therap., in the press. Citation Format: Joost C. Uitdehaag, Jos de Man, Marion Libouban, Nicole Willemsen-Seegers, Jan Gerard Sterrenburg, Joeri J. de Wit, Jeroen A.D de Roos, Martine B. Prinsen, Rogier C. Buijsman, Guido J. Zaman. NTRC 1501-0, a TTK kinase inhibitor selected for its long target residence time, completely inhibits tumor growth in the MDA-MB-231 xenograft model for triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4185. doi:10.1158/1538-7445.AM2017-4185

Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are two structurally different enzymes that have a different tissue distribution and physiological roles, but both catalyze the conversion of tryptophan to N-formylkynurenine (NFK). IDO1 has been clinically validated as a small-molecule drug target for cancer, while preclinical studies indicate that TDO may be a target for cancer immunotherapy and neurodegenerative disease. We have developed a high-throughput screening assay for IDO1 and TDO based on a novel chemical probe, NFK Green, that reacts specifically with NFK to form a green fluorescent molecule with an excitation wavelength of 400 nm and an emission wavelength of 510 nm. We provide the first side-by-side comparison of a number of published inhibitors of IDO1 and TDO and reveal that the preclinical IDO1 inhibitor Compound 5l shows significant cross-reactivity with TDO, while the relative selectivity of other published inhibitors was confirmed. The suitability for high-throughput screening of the assays was demonstrated by screening a library of 87,000 chemical substances in 384- or 1536-well format. Finally, we demonstrate that the assay can also be used to measure the capacity of cells to metabolize tryptophan and to measure the cellular potency of IDO1 and TDO inhibitors.

A synthesis is presented of the cyclic trimeric d-oligonucleotide 3′-isopropylphosphate I, comprising one formacetal and two (3′ → 5′)-internucleosidic phosphodiester bonds. The ester linkages connect d-guanosine with the 3′ and 5′ ends of thymidine and 5-hydroxymethyl-2′-deoxyuridine-3′-isopropylphosphate (HMDUpiPr), respectively. The 5′-end of the thymidine unit is anchored via the formacetal bond to the allylic hydroxyl group of HMDUpiPr. The cyclic arrangement of the three d-nucleosides in I mimics, as based on molecular modeling, the key structural features of the conformationally constrained T7pG8pT9p-domain of the thrombin-binding DNA aptamer d(G1G2T3T4G5G6 T 7 G 8 T 9 G10G11T12T13G14G15). Biological evaluation showed that compound I did not exhibit anti-thrombin activity.

Trisubstrate UGT transition state analogue 2 is readily accessible by nucleophilic ring-opening of 1,2-anhydroglucose precursor 5 with diethylmalonate anion followed by reduction of the ethyl ester moieties ( 6→7 ). Subsequent C 6 oxidation ( 8→9 ), NIS/ cat . TfOH-mediated introduction of the androsterylmethylene unit ( 12→15 ) and phosphitylation with 5′-uridine phosphoramidite 16 furnished, after oxidation and deprotection, target derivative 2 , the two individual diastereomers of which ( 2a and 2b ) were separated by HPLC. Trisubstrate analogues 2a,b show a different inhibition pattern for several UGT isoforms, indicating isoenzyme selectivity. Moreover, C 7″ -epimers 2a and 2b exert a different inhibitory effect on UGT2B15.

[purpose] An abnormal number of chromosomes, or ‘aneuploidy’, is a common feature of solid human tumors and a predictor of poor prognosis in breast, lung, brain and colorectal cancer. Aneuploidy is caused by malfunctioning of the Spindle Assembly Checkpoint (SAC), a surveillance mechanism that ensures the fidelity of chromosome segregation. The protein kinase TTK (commonly referred to as Mps1) is a component of the SAC. Inhibition of TTK gene expression by RNA interference and inhibition of TTK kinase activity by small molecule kinase inhibitors causes chromosome missegregation and cancer cell death. [experimental procedures] A novel class of compounds was identified that potently inhibits TTK enzyme activity and cancer cell line proliferation [1]. Its binding mode and that of reference inhibitors was characterized by protein crystallography. Binding kinetics and target residence time were determined by surface plasmon resonance using Biacore T200. Anti-proliferative activity was measured on a broad panel of cancer cell lines [2]. [results] The clinical candidate, NTRC 0066-0, inhibits TTK with subnanomolar potency (IC50) in a kinase enzyme assay and is more than 200 times selective over 276 kinases examined, including mitotic and cell cycle dependent kinases (CDKs). X-ray structures of the TTK kinase domain in complex with NTRC 0066-0 and analogs indicate that this class of compounds induces a large conformational shift in the glycine-rich loop, invoking an inactive kinase conformation. In surface plasmon resonance experiments, NTRC 0066-0 exhibited slow dissociation kinetics, resulting in a long target residence time. Parallel surface plasmon resonance experiments with mitotic kinases confirmed the exquisite selectivity of NTRC 0066-0 for TTK over Aurora and Polo-like kinases. NTRC 0066-0 potently inhibited the proliferation of a wide variety of human cancer cell lines with potencies in the same range as marketed cytotoxic agents. The crystal structure, binding kinetics and cellular potency of NTRC 0066-0 were compared to that of other TTK inhibitors such as Mps1-IN-2, AZD-3146, Mps-BAY2b and Bay 1161909 as well as analogs from the NTRC 0066-0 series. This suggest that the unique binding mode of NTRC 0066-0 results in long target residence time which contributes to its strong anti-tumor activity. In subsequent mouse xenograft models of human cancer cell lines, NTRC 0066-0 inhibited tumor growth as a single agent after oral administration at 20 mg per kg. [conclusions] NTRC 0066-0 is a novel TTK inhibitor with outstanding in vitro properties and potent anti-tumor activity in mouse xenograft models. Our data suggest that long target residence time corresponds with potent cellular activity for TTK inhibitors. References [1] Maia et al. (2015) Annals of Oncology 26, 2180-2192; [2] Uitdehaag et al. (2014) PLOS ONE 9(3) e92146. Citation Format: Jos de Man, Joost C.M. Uitdehaag, Nicole Willemsen-Seegers, Jan Gerard Sterrenburg, Joeri J.P. de Wit, Jeroen A.D.M. de Roos, Martine B.W. Prinsen, Rogier C. Buijsman, Guido J.R. Zaman. The unique binding mode of NTRC 0066-0, a novel inhibitor of the spindle assembly checkpoint kinase TTK (Mps1), leads to long target residence time and potent antitumor activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2646.

[reaction--see text] Ionic liquid 1-butyl-3-methylimidazoliumhexafluorophosphate ([bmim]PF(6)) is described as an effective medium for ring-closing metathesis (RCM) using Grubbs catalysts. When [bmim]PF(6) was used as solvent, the RCM showed high conversions and a broad substrate tolerance. [bmim]PF(6) and the ruthenium catalyst were recycled after extraction of the product in the organic phase for at least three cycles.

Background: Pediatric T-cell acute lymphoblastic leukemia patients frequently harbor mutations in IL7Ra or downstream molecules encoded by JAK1, JAK3, N-RAS, K-RAS, NF1, AKT, and PTEN. These mutated signaling molecules can contribute to leukemia by disturbing a multitude of cellular processes such as the cell cycle, epigenetics, apoptosis, or affecting other important signal transduction pathways. Aims: We aimed to determine the overall incidence of mutations in IL7Ra and downstream signaling components in a large cohort of pediatric T-ALL patients. In order to find better treatment options for patients with these mutations, we analyzed the effect of selected IL7Ra-pathway inhibitors-individually and in combinations-on downstream signaling and cytotoxicity in Ba/F3 cells expressing each of the mutations. Methods: We sequenced 146 pediatric T-ALL patient samples for mutations in the FERM, pseudokinase and kinase domains of the Janus kinase gene family (JAK1, JAK2, JAK3, TYK2) and hotspot regions of N-RAS and K-RAS. We adapted the IL3-dependent Ba/F3 cell line to express mutant or wild type genes upon induction by doxycycline and assessed cell viability and signaling in the absence of IL3. Various IL7Ra-pathway inhibitors were tested using this system, and the synergy of combined inhibitors was determined by comparing the dose-response curve of different ratios of IC50-based inhibitor concentrations to the curves for each of the single inhibitors. The Combination Index was calculated using Calcusyn™ software. Results: IL7Ra, JAK, RAS, AKT and PTEN mutations are present in approximately 45% of patients and occur in a predominantly mutually exclusive fashion, suggesting they share aberrant activation of similar downstream targets. We found JAK1, JAK3 and RAS mutations as previously reported, but also identified new JAK1 mutations including V427M, L624YPILKV, E668Q, P815S, and T901G. A novel three-dimensional model of JAK1 reveals that mutations in JAK molecules affect important amino acids that are involved in the interaction between the pseudokinase and kinase domains, facilitating constitutive kinase activity. In our doxycycline-inducible IL3-dependent Ba/F3 system, expression of mutant genes-in contrast to the wild type genes-transforms Ba/F3 cells by supporting IL3-independent growth through activation of the RAS-MEK-ERK and PI3K-AKT pathways. We used this system to test the sensitivity to pharmacological inhibitors; IL7Ra and JAK mutant Ba/F3 cells are sensitive to JAK inhibition, so JAK inhibitors such as ruxolitinib may offer therapeutic potential for IL7Ra, JAK1 or most JAK3 mutated T-ALL patients. The RAS and AKT mutants respond to RAS-MEK and PI3K-AKT-mTOR inhibition, respectively, but are-as expected-insensitive to JAK inhibition. Remarkably, IL7Ra and JAK mutants are relatively resistant to downstream RAS-MEK-ERK or PI3K-AKT-mTOR inhibition, indicating that inhibiting just one of these downstream pathways is insufficient. We provide evidence of (cross-)activation of the alternate pathway when one of these pathways is inhibited. Combined inhibition of MEK and PI3K/AKT synergistically prevents proliferation of the IL7Ra- and JAK-mutants by efficiently blocking both downstream signaling pathways. Furthermore, this combined inhibition is cytotoxic in two out of five tested primary T-ALL specimens. Summary/Conclusion: We show that the combined inhibition of MEK and PI3K/AKT leads to strong and synergistic cytotoxic effects in the IL7Ra and JAK mutants and efficiently blocks signaling downstream of both pathways. This inhibitor combination is effective in two out of five primary T-ALL samples. Therefore, the cytotoxic effects of synergistic MEK and PI3K/AKT inhibition should be further explored as a therapeutic option for (relapsed) ALL patients. Disclosures Buijsman: Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder. Zaman:Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder.

Background: Many pediatric T-cell acute lymphoblastic leukemia patients harbor mutations in IL7Ra or downstream molecules encoded by JAK1, JAK3, N-RAS, K-RAS, NF1, AKT, and PTEN. These mutated signaling molecules can contribute to leukemia by disturbing a multitude of cellular processes such as the cell cycle, epigenetics, apoptosis, or affecting other important signal transduction pathways. Aims: We aimed to determine the overall incidence of JAK family mutations in a large cohort of T-ALL patients. We also aimed to generate a 3D JAK1 model including known and newly identified JAK mutations in order to better understand how these contribute to JAK kinase activity and the transformation of cells. Methods: We screened 146 pediatric T-ALL patient samples for mutations in the FERM, pseudokinase and kinase domains of the Janus kinase gene family (JAK1-3, TYK2). To establish a 3D JAK1 model, we superimposed individual pseudokinase and kinase crystallographic structures on the homologous TYK2 pseudokinase-kinase structure. We visualized JAK mutations and their effects on the 3D structure. We modified the IL3-dependent Ba/F3 cell line to express JAK mutant or wild type genes upon induction by doxycycline. We tested these Ba/F3 derivative lines for transforming ability, signaling, and resistance to various inhibitors in the absence of IL3. Results: JAK1 or JAK3 mutations were found in 10 patients; no mutations were found in JAK2 or TYK2. We found JAK1 and JAK3 mutations as previously reported, but also identified amino acid substitutions as a result of novel JAK1 mutations including V427M, L624YPILKV, E668Q, P815S, and T901G. Our novel 3D model of JAK1 places most mutations in one of two crucial pseudokinase-kinase interaction sites, which can weaken the interaction and facilitate constitutive kinase activity. One interaction is between the hinge region of the pseudokinase domain and the loop in the kinase domain, which is supported by four salt bridges. Mutations in T-ALL disrupting these salt bridges include E668Q, R724H and its JAK3 equivalent R657Q, and T901G. The second interaction with the kinase domain is formed by a helical domain in the pseudokinase domain, located just upstream of the conserved F575-F636-V658 triad. This F-F-V triad is predicted to act as a structural switch that controls the catalytic activity of JAK kinases. Various mutations occur in the direct vicinity and can affect the function of this switch. V658F in T-ALL and its JAK2 equivalent V617F in polycythemia vera patients are mutations in this triad. The frequent JAK3 mutation M511I in T-ALL flanks the F513 residue (equivalent of JAK1 F575) and also affects the F-F-V triad. The L624YPILKV insertion mutation is located in a loop near the helical domain, which may also subtly compromise the F-F-V triad structural switch leading to derepression of the kinase domain. Expression of mutant JAK genes-in contrast to the wild type genes-transforms Ba/F3 cells by supporting IL3-independent growth, and by activating downstream RAS-MEK-ERK and PI3K-AKT pathways. This pathway activation as a result of ligand-independent mutant JAK kinase activity was confirmed by measuring phospho-proteins including p-MEK, p-ERK, p-AKT, p-mTOR, and p-p70S6K, and can be blocked by JAK inhibitors. Notably, JAK3 mutants signal significantly weaker than JAK1 mutants, possibly due to different dependence on (endogenous) receptors that normally mediate wild type JAK signaling. Summary/Conclusion: In a 3D model, we show that JAK mutations are located in critical interface regions between the pseudokinase and kinase domains, maintaining the kinase in an open, active confirmation. The inducible Ba/F3 model system confirms the transforming capacity of JAK mutations, reveals constitutive active downstream signaling, and is also suitable to test the effect of various inhibitors. The visualization of various JAK mutations in a 3D model and how these contribute to kinase activity provides insight in how mutant JAK could be inhibited, helping guide the development of new small molecule inhibitors of mutant JAKs. Disclosures Buijsman: Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder. Zaman:Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder.

The synthesis of ionic support 1 and its application in the preparation of a set of amides and sulfonamides is described. The potential of 1 is further exemplified by its use in a one-pot multistep ionic liquid phase assisted synthesis of tirofiban analogue 2.

The synthesis of three heparin analogues (i.e. compounds VI-VIII) having perphosphorylated thrombin binding domains (TBDs) is reported. These compounds were tested in vitro for their antithrombin III (ATIII)-mediated anti-Xa and antithrombin activities. Conjugates VI and VIII show a remarkable increase in antithrombin activity compared to the structurally related conjugates with persulfated TBDs (i.e. compounds IV and V), whereas compound VII displays a diminished activity.

The synthesis of a novel antithrombotic consisting of a heparin pentasaccharide conjugated to the active site inhibitor N-(2- naphtalenesulfonyl)-glycyl-( d )-4-aminophenyl-alanyl-piperidine (NAPAP) (i.e. compound I) is reported. This conjugate shows a unique pharmacological profile both in vitro and in vivo having direct anti-thrombin and ATIII-mediated anti-Xa activity. Furthermore, conjugate I has a prolonged in vivo half-life compared to NAPAP (1.5 h vs 9 min.).