Discovery, Characterization, and Structure-Based Optimization of Small-Molecule In Vitro and In Vivo Probes for Human DNA Polymerase Theta.

Human DNA polymerase theta (Polθ), which is essential for microhomology-mediated DNA double strand break repair, has been proposed as an attractive target for the treatment of BRCA deficient and other DNA repair pathway defective cancers. As previously reported, we recently identified the first selective small molecule Polθ in vitro probe, 22 (ART558), which recapitulates the phenotype of Polθ loss, and in vivo probe, 43 (ART812), which is efficacious in a model of PARP inhibitor resistant TNBC in vivo. Here we describe the discovery, biochemical and biophysical characterization of these probes including small molecule ligand co-crystal structures with Polθ. The crystallographic data provides a basis for understanding the unique mechanism of inhibition of these compounds which is dependent on stabilization of a "closed" enzyme conformation. Additionally, the structural biology platform provided a basis for rational optimization based primarily on reduced ligand conformational flexibility.

Parallel Optimization of Potency and Pharmacokinetics Leading to the Discovery of a Pyrrole Carboxamide ERK5 Kinase Domain Inhibitor.

The nonclassical extracellular signal-related kinase 5 (ERK5) mitogen-activated protein kinase pathway has been implicated in increased cellular proliferation, migration, survival, and angiogenesis; hence, ERK5 inhibition may be an attractive approach for cancer treatment. However, the development of selective ERK5 inhibitors has been challenging. Previously, we described the development of a pyrrole carboxamide high-throughput screening hit into a selective, submicromolar inhibitor of ERK5 kinase activity. Improvement in the ERK5 potency was necessary for the identification of a tool ERK5 inhibitor for target validation studies. Herein, we describe the optimization of this series to identify nanomolar pyrrole carboxamide inhibitors of ERK5 incorporating a basic center, which suffered from poor oral bioavailability. Parallel optimization of potency and in vitro pharmacokinetic parameters led to the identification of a nonbasic pyrazole analogue with an optimal balance of ERK5 inhibition and oral exposure.

Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance.

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.

Identification of a novel orally bioavailable ERK5 inhibitor with selectivity over p38α and BRD4.

Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC50 values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (∼3-fold) against ERK5, but importantly attenuated inhibition of p38α. Systematic variation of the substituents on the aroyl group led to the selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC50 0.82 µM for ERK5; IC50 > 120 µM for p38α). The crystal structure (PDB 5O7I) of this compound in complex with ERK5 has been solved. This compound was orally bioavailable and inhibited bFGF-driven Matrigel plug angiogenesis and tumour xenograft growth. The selective ERK5 inhibitor described herein provides a lead for further development into a tool compound for more extensive studies seeking to examine the role of ERK5 signalling in cancer and other diseases.

A High-Throughput Screening Triage Workflow to Authenticate a Novel Series of PFKFB3 Inhibitors.

A high-throughput screen (HTS) of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) resulted in several series of compounds with the potential for further optimization. Informatics was used to identify active chemotypes with lead-like profiles and remove compounds that commonly occurred as actives in other HTS screens. The activities were confirmed with IC50 measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC50 values at 10 times the enzyme concentration were used to identify artifact compounds. Several series of compounds were rejected as they had both high slopes and poor ratios. A small number of compounds representing the different leading series were assessed using isothermal titration calorimetry, and the X-ray crystal structure of the complex with PFKFB3 was solved. The orthogonal assay technology and isothermal calorimetry were demonstrated to be unreliable in identifying false-positive compounds in this case. Presented here is the discovery of the dihydropyrrolopyrimidinone series of compounds as active and novel inhibitors of PFKFB3, shown by X-ray crystallography to bind to the adenosine triphosphate site. The crystal structures of this series also reveal it is possible to flip the binding mode of the compounds, and the alternative orientation can be driven by a sigma-hole interaction between an aromatic chlorine atom and a backbone carbonyl oxygen. These novel inhibitors will enable studies to explore the role of PFKFB3 in driving the glycolytic phenotype of tumors.

High-Throughput Screening and Hit Validation of Extracellular-Related Kinase 5 (ERK5) Inhibitors.

The extracellular-related kinase 5 (ERK5) is a promising target for cancer therapy. A high-throughput screen was developed for ERK5, based on the IMAP FP progressive binding system, and used to identify hits from a library of 57 617 compounds. Four distinct chemical series were evident within the screening hits. Resynthesis and reassay of the hits demonstrated that one series did not return active compounds, whereas three series returned active hits. Structure-activity studies demonstrated that the 4-benzoylpyrrole-2-carboxamide pharmacophore had excellent potential for further development. The minimum kinase binding pharmacophore was identified, and key examples demonstrated good selectivity for ERK5 over p38α kinase.

Structure-Based Design of Potent and Selective Inhibitors of the Metabolic Kinase PFKFB3.

A weak screening hit with suboptimal physicochemical properties was optimized against PFKFB3 kinase using critical structure-guided insights. The resulting compounds demonstrated high selectivity over related PFKFB isoforms and modulation of the target in a cellular context. A selected example demonstrated exposure in animals following oral dosing. Examples from this series may serve as useful probes to understand the emerging biology of this metabolic target.

Morpholylureas are a new class of potent and selective inhibitors of the type 5 17-ß-hydroxysteroid dehydrogenase (AKR1C3).

Inhibitors of the aldo-keto reductase enzyme AKR1C3 are of interest as potential drugs for leukemia and hormone-related cancers. A series of non-carboxylate morpholino(phenylpiperazin-1-yl)methanones were prepared by palladium-catalysed coupling of substituted phenyl or pyridyl bromides with the known morpholino(piperazin-1-yl)methanone, and shown to be potent (IC50∼100nM) and very isoform-selective inhibitors of AKR1C3. Lipophilic electron-withdrawing substituents on the phenyl ring were positive for activity, as was an H-bond acceptor on the other terminal ring, and the ketone moiety (as a urea) was essential. These structure-activity relationships are consistent with an X-ray structure of a representative compound bound in the AKR1C3 active site, which showed H-bonding between the carbonyl oxygen of the drug and Tyr55 and His117 in the 'oxyanion hole' of the enzyme, with the piperazine bridging unit providing the correct twist to allow the terminal benzene ring to occupy the lipophilic pocket and align with Phe311.

Synthesis and structure-activity relationships for 1-(4-(piperidin-1-ylsulfonyl)phenyl)pyrrolidin-2-ones as novel non-carboxylate inhibitors of the aldo-keto reductase enzyme AKR1C3.

High expression of the aldo-keto reductase enzyme AKR1C3 in the human prostate and breast has implicated it in the development and progression of leukemias and of prostate and breast cancers. Inhibitors are thus of interest as potential drugs. Most inhibitors of AKR1C3 are carboxylic acids, whose transport into cells is likely dominated by carrier-mediated processes. We describe here a series of (piperidinosulfonamidophenyl)pyrrolidin-2-ones as potent (<100 nM) and isoform-selective non-carboxylate inhibitors of AKR1C3. Structure-activity relationships identified the sulfonamide was critical, and a crystal structure showed the 2-pyrrolidinone does not interact directly with residues in the oxyanion hole. Variations in the position, co-planarity or electronic nature of the pyrrolidinone ring severely diminished activity, as did altering the size or polarity of the piperidino ring. There was a broad correlation between the enzyme potencies of the compounds and their effectiveness at inhibiting AKR1C3 activity in cells.

3-(3,4-Dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic Acids: highly potent and selective inhibitors of the type 5 17-ß-hydroxysteroid dehydrogenase AKR1C3.

A high-throughput screen identified 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic acid as a novel, highly potent (low nM), and isoform-selective (1500-fold) inhibitor of aldo-keto reductase AKR1C3: a target of interest in both breast and prostate cancer. Crystal structure studies showed that the carboxylate group occupies the oxyanion hole in the enzyme, while the sulfonamide provides the correct twist to allow the dihydroisoquinoline to bind in an adjacent hydrophobic pocket. SAR studies around this lead showed that the positioning of the carboxylate was critical, although it could be substituted by acid isosteres and amides. Small substituents on the dihydroisoquinoline gave improvements in potency. A set of "reverse sulfonamides" showed a 12-fold preference for the R stereoisomer. The compounds showed good cellular potency, as measured by inhibition of AKR1C3 metabolism of a known dinitrobenzamide substrate, with a broad rank order between enzymic and cellular activity, but amide analogues were more effective than predicted by the cellular assay.

Mechanism-based screen for G1/S checkpoint activators identifies a selective activator of EIF2AK3/PERK signalling.

Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes.

Structure-based design of potent and selective 2-(quinazolin-2-yl)phenol inhibitors of checkpoint kinase 2.

Structure-based design was applied to the optimization of a series of 2-(quinazolin-2-yl)phenols to generate potent and selective ATP-competitive inhibitors of the DNA damage response signaling enzyme checkpoint kinase 2 (CHK2). Structure-activity relationships for multiple substituent positions were optimized separately and in combination leading to the 2-(quinazolin-2-yl)phenol 46 (IC(50) 3 nM) with good selectivity for CHK2 against CHK1 and a wider panel of kinases and with promising in vitro ADMET properties. Off-target activity at hERG ion channels shown by the core scaffold was successfully reduced by the addition of peripheral polar substitution. In addition to showing mechanistic inhibition of CHK2 in HT29 human colon cancer cells, a concentration dependent radioprotective effect in mouse thymocytes was demonstrated for the potent inhibitor 46 (CCT241533).

Improved ATM kinase inhibitor KU-60019 radiosensitizes glioma cells, compromises insulin, AKT and ERK prosurvival signaling, and inhibits migration and invasion.

Ataxia telangiectasia (A-T) mutated (ATM) is critical for cell cycle checkpoints and DNA repair. Thus, specific small molecule inhibitors targeting ATM could perhaps be developed into efficient radiosensitizers. Recently, a specific inhibitor of the ATM kinase, KU-55933, was shown to radiosensitize human cancer cells. Herein, we report on an improved analogue of KU-55933 (KU-60019) with K(i) and IC(50) values half of those of KU-55933. KU-60019 is 10-fold more effective than KU-55933 at blocking radiation-induced phosphorylation of key ATM targets in human glioma cells. As expected, KU-60019 is a highly effective radiosensitizer of human glioma cells. A-T fibroblasts were not radiosensitized by KU-60019, strongly suggesting that the ATM kinase is specifically targeted. Furthermore, KU-60019 reduced basal S473 AKT phosphorylation, suggesting that the ATM kinase might regulate a protein phosphatase acting on AKT. In line with this finding, the effect of KU-60019 on AKT phosphorylation was countered by low levels of okadaic acid, a phosphatase inhibitor, and A-T cells were impaired in S473 AKT phosphorylation in response to radiation and insulin and unresponsive to KU-60019. We also show that KU-60019 inhibits glioma cell migration and invasion in vitro, suggesting that glioma growth and motility might be controlled by ATM via AKT. Inhibitors of MEK and AKT did not further radiosensitize cells treated with KU-60019, supporting the idea that KU-60019 interferes with prosurvival signaling separate from its radiosensitizing properties. Altogether, KU-60019 inhibits the DNA damage response, reduces AKT phosphorylation and prosurvival signaling, inhibits migration and invasion, and effectively radiosensitizes human glioma cells.

A high-content, cell-based screen identifies micropolyin, a new inhibitor of microtubule dynamics.

High-content cell-based screens provide a powerful tool to identify new chemicals that interfere with complex biological processes. Here, we describe the identification of a new inhibitor of microtubule dynamics (micropolyin) using a high-content screen. Integrated high-resolution imaging allowed for fast selection of hits and progression to target identification. Treatment of cells with micropolyin efficiently causes a pro-metaphase arrest, with abnormal spindle morphology and with the spindle assembly checkpoint activated. The arrest appears to result from interference of micropolyin with microtubule dynamics. We show in vitro that tubulin is indeed the target of micropolyin and that micropolyin inhibits microtubule polymerization. Our results demonstrate the power of high-content image- and cell-based screening approaches to identify potential new drug candidates. As our approach is unbiased, it should allow for discovery of new targets that may otherwise be overlooked.

Pyranone, thiopyranone, and pyridone inhibitors of phosphatidylinositol 3-kinase related kinases. Structure-activity relationships for DNA-dependent protein kinase inhibition, and identification of the first potent and selective inhibitor of the ataxia telangiectasia mutated kinase.

Structure-activity relationships have been investigated for inhibition of DNA-dependent protein kinase (DNA-PK) and ATM kinase by a series of pyran-2-ones, pyran-4-ones, thiopyran-4-ones, and pyridin-4-ones. A wide range of IC50 values were observed for pyranones and thiopyranones substituted at the 6-position, with the 3- and 5-positions proving intolerant to substitution. Related pyran-2-ones, pyran-4-ones, and thiopyran-4-ones showed similar IC50 values against DNA-PK, whereas the pyridin-4-one system proved, in general, ineffective at inhibiting DNA-PK. Extended libraries exploring the 6-position of 2-morpholino-pyran-4-ones and 2-morpholino-thiopyrano-4-ones identified the first highly potent and selective ATM inhibitor 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (151C; ATM; IC50=13 nM) and revealed constrained SARs for ATM inhibition compared with DNA-PK. One of the most potent DNA-PK inhibitors identified, 2-(4-methoxyphenyl)-6-(morpholin-4-yl)pyran-4-one (16; DNA-PK; IC50=220 nM) effectively sensitized HeLa cells to the topoisomerase II inhibitor etoposide in vitro.

Discovery of potent chromen-4-one inhibitors of the DNA-dependent protein kinase (DNA-PK) using a small-molecule library approach.

Structure-activity relationships for inhibition of DNA-dependent protein kinase (DNA-PK) have been defined for substituted chromen-4-ones. For the 2-amino-substituted benzo[h]chromen-4-ones, a morpholine substituent at this position was essential for activity. Small libraries of 6- and 7-alkoxy-substituted chromen-4-ones showed that a number of 7-alkoxy-substituted chromenones displayed improved activity. Focused libraries incorporating 6-, 7-, and 8-aryl and heteroaryl substituents were prepared. In these cases, 6- and 7-substitution was disfavored, whereas 8-substitution was largely tolerated. Surprisingly, two compounds, 2-N-morpholino-8-dibenzofuranyl-chromen-4-one (NU7427, 32{38}) and the 2-N-morpholino-8-dibenzothiophenyl-chromen-4-one (NU7441, 32{26}) were excellent inhibitors (IC50 vs DNA-PK = 40 and 13 nM, respectively). The ring-saturated analogue 2-N-morpholino-8-(6',7',8',9'-tetrahydrodibenzothiophene)chromen-4-one, 36, retained potent activity (IC50 vs DNA-PK = 23 nM). The dibenzothiophene 32{38} sensitized HeLa cells to ionizing radiation in vitro, with dose modification factors of 2.5 at 10% survival being observed at 0.5 microM. The cytotoxicity of the topoisomerase II inhibitor etoposide was also potentiated.

Selective benzopyranone and pyrimido[2,1-a]isoquinolin-4-one inhibitors of DNA-dependent protein kinase: synthesis, structure-activity studies, and radiosensitization of a human tumor cell line in vitro.

A diverse range of chromen-2-one, chromen-4-one and pyrimidoisoquinolin-4-one derivatives was synthesized and evaluated for inhibitory activity against the DNA repair enzyme DNA-dependent protein kinase (DNA-PK), with a view to elucidating structure-activity relationships for potency and kinase selectivity. DNA-PK inhibitory activity varied widely over the series of compounds evaluated (IC(50) values ranged from 0.19 to >10 microM), with excellent activity being observed for the 7,8-benzochromen-4-one and pyrimido[2,1-a]isoquinolin-4-one templates. By contrast, inhibitors based on the benzochromen-2-one (coumarin) or 2-aryl-7,8-benzochromen-4-one (flavone) scaffolds were less potent. Crucially, these studies revealed a very constrained structure-activity relationship at the 2-position of the benzopyranone and pyrimido[2,1-a]isoquinolin-4-one pharmacophore, with only a 2-morpholino or 2-(2'-methylmorpholino) group being tolerated at this position. More detailed biological studies conducted with the most potent inhibitor NU7163 (48; IC(50) = 0.19 microM) demonstrated ATP-competitive DNA-PK inhibition, with a K(i) value of 24 nM, and 48 exhibited selectivity for DNA-PK compared with the related enzymes ATM, ATR, mTOR, and PI 3-K (p110alpha). Compound 48 sensitized the HeLa human tumor cell line to the cytotoxic effects of ionizing radiation in vitro, a dose modification factor of 2.3 at 10% survival being observed with an inhibitor concentration of 5 microM. This study identified these structural classes as novel DNA-PK inhibitors and delineated initial structure-activity relationships against DNA-PK.

Identification of a highly potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) by screening of chromenone libraries.

A solution-phase multiple-parallel synthesis approach was employed for the preparation of 6-, 7- and 8-aryl-substituted chromenone libraries, which were screened as inhibitors of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). These studies resulted in the identification of 8-dibenzothiophen-4-yl-2-morpholin-4-yl-chromen-4-one (NU7441) as a highly potent and selective DNA-PK inhibitor (IC50=14 nM), exhibiting ATP-competitive inhibition kinetics.

2,6-disubstituted pyran-4-one and thiopyran-4-one inhibitors of DNA-Dependent protein kinase (DNA-PK).

6-aryl-2-morpholin-4-yl-4H-pyran-4-ones and 6-aryl-2-morpholin-4-yl-4H-thiopyran-4-ones were synthesised and evaluated as potential inhibitors of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). Several compounds in each series exhibited superior activity to the chromenone LY294002, and were of comparable potency to the benzochromenone NU7026 (IC(50)=0.23 microM). Importantly, members of both structural classes were found to be selective inhibitors of DNA-PK over related phosphatidylinositol 3-kinase-related kinase (PIKK) family members. A multiple-parallel synthesis approach, employing Suzuki cross-coupling methodology, was utilised to prepare libraries of thiopyran-4-ones with a range of aromatic groups at the 3'- and 4'-positions on the thiopyran-4-one 6-aryl ring. Screening of the libraries resulted in the identification of 6-aryl-2-morpholin-4-yl-4H-thiopyran-4-ones bearing naphthyl or benzo[b]thienyl substituents at the 4'-position, as potent DNA-PK inhibitors with IC(50) values in the 0.2-0.4 microM range.