Modulation of ERK5 Activity as a Therapeutic Anti-Cancer Strategy.

The extracellular signal-regulated kinase 5 (ERK5) signaling pathway is one of four conventional mitogen-activated protein (MAP) kinase pathways. Genetic perturbation of ERK5 has suggested that modulation of ERK5 activity may have therapeutic potential in cancer chemotherapy. This Miniperspective examines the evidence for ERK5 as a drug target in cancer, the structure of ERK5, and the evolution of structurally distinct chemotypes of ERK5 kinase domain inhibitors. The emerging complexities of ERK5 pharmacology are discussed, including the confounding phenomenon of paradoxical ERK5 activation by small-molecule ERK5 inhibitors. The impact of the recent development and biological evaluation of potent and selective bifunctional degraders of ERK5 and future opportunities in ERK modulation are also explored.

Advanced approaches of developing targeted covalent drugs.

In recent years, the development of targeted covalent inhibitors has gained popularity around the world. Specific groups (electrophilic warheads) form irreversible bonds with the side chain of nucleophilic amino acid residues, thus changing the function of biological targets such as proteins. Since the first targeted covalent inhibitor was disclosed in the 1990s, great efforts have been made to develop covalent ligands from known reversible leads or drugs by addition of tolerated electrophilic warheads. However, high reactivity and "off-target" toxicity remain challenging issues. This review covers the concept of targeted covalent inhibition to diseases, discusses traditional and interdisciplinary strategies of cysteine-focused covalent drug discovery, and exhibits newly disclosed electrophilic warheads majorly targeting the cysteine residue. Successful applications to address the challenges of designing effective covalent drugs are also introduced.

Mapping Ligand Interactions of Bromodomains BRD4 and ATAD2 with FragLites and PepLites─Halogenated Probes of Druglike and Peptide-like Molecular Interactions.

The development of ligands for biological targets is critically dependent on the identification of sites on proteins that bind molecules with high affinity. A set of compounds, called FragLites, can identify such sites, along with the interactions required to gain affinity, by X-ray crystallography. We demonstrate the utility of FragLites in mapping the binding sites of bromodomain proteins BRD4 and ATAD2 and demonstrate that FragLite mapping is comparable to a full fragment screen in identifying ligand binding sites and key interactions. We extend the FragLite set with analogous compounds derived from amino acids (termed PepLites) that mimic the interactions of peptides. The output of the FragLite maps is shown to enable the development of ligands with leadlike potency. This work establishes the use of FragLite and PepLite screening at an early stage in ligand discovery allowing the rapid assessment of tractability of protein targets and informing downstream hit-finding.

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.

An Alkynylpyrimidine-Based Covalent Inhibitor That Targets a Unique Cysteine in NF-κB-Inducing Kinase.

NF-κB-inducing kinase (NIK) is a key enzyme in the noncanonical NF-κB pathway, of interest in the treatment of a variety of diseases including cancer. Validation of NIK as a drug target requires potent and selective inhibitors. The protein contains a cysteine residue at position 444 in the back pocket of the active site, unique within the kinome. Analysis of existing inhibitor scaffolds and early structure-activity relationships (SARs) led to the design of C444-targeting covalent inhibitors based on alkynyl heterocycle warheads. Mass spectrometry provided proof of the covalent mechanism, and the SAR was rationalized by computational modeling. Profiling of more potent analogues in tumor cell lines with constitutively activated NIK signaling induced a weak antiproliferative effect, suggesting that kinase inhibition may have limited impact on cancer cell growth. This study shows that alkynyl heterocycles are potential cysteine traps, which may be employed where common Michael acceptors, such as acrylamides, are not tolerated.

Structure-Based Design of Potent and Orally Active Isoindolinone Inhibitors of MDM2-p53 Protein-Protein Interaction.

Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.

Chronic glucokinase activator treatment activates liver Carbohydrate response element binding protein and improves hepatocyte ATP homeostasis during substrate challenge.

AIM: To test the hypothesis that glucokinase activators (GKAs) induce hepatic adaptations that alter intra-hepatocyte metabolite homeostasis. METHODS: C57BL/6 mice on a standard rodent diet were treated with a GKA (AZD1656) acutely or chronically. Hepatocytes were isolated from the mice after 4 or 8 weeks of treatment for analysis of cellular metabolites and gene expression in response to substrate challenge. RESULTS: Acute exposure of mice to AZD1656 or a liver-selective GKA (PF-04991532), before a glucose tolerance test, or challenge of mouse hepatocytes with GKAs ex vivo induced various Carbohydrate response element binding protein (ChREBP) target genes, including Carbohydrate response element binding protein beta isoform (ChREBP-ß), Gckr and G6pc. Both glucokinase activation and ChREBP target gene induction by PF-04991532 were dependent on the chirality of the molecule, confirming a mechanism linked to glucokinase activation. Hepatocytes from mice treated with AZD1656 for 4 or 8 weeks had lower basal glucose 6-phosphate levels and improved ATP homeostasis during high substrate challenge. They also had raised basal ChREBP-ß mRNA and AMPK-α mRNA (Prkaa1, Prkaa2) and progressively attenuated substrate induction of some ChREBP target genes and Prkaa1 and Prkaa2. CONCLUSIONS: Chronic GKA treatment of C57BL/6 mice for 8 weeks activates liver ChREBP and improves the resilience of hepatocytes to compromised ATP homeostasis during high-substrate challenge. These changes are associated with raised mRNA levels of ChREBP-ß and both catalytic subunits of AMP-activated protein kinase.

Nek7 conformational flexibility and inhibitor binding probed through protein engineering of the R-spine.

Nek7 is a serine/threonine-protein kinase required for proper spindle formation and cytokinesis. Elevated Nek7 levels have been observed in several cancers, and inhibition of Nek7 might provide a route to the development of cancer therapeutics. To date, no selective and potent Nek7 inhibitors have been identified. Nek7 crystal structures exhibit an improperly formed regulatory-spine (R-spine), characteristic of an inactive kinase. We reasoned that the preference of Nek7 to crystallise in this inactive conformation might hinder attempts to capture Nek7 in complex with Type I inhibitors. Here, we have introduced aromatic residues into the R-spine of Nek7 with the aim to stabilise the active conformation of the kinase through R-spine stacking. The strong R-spine mutant Nek7SRS retained catalytic activity and was crystallised in complex with compound 51, an ATP-competitive inhibitor of Nek2 and Nek7. Subsequently, we obtained the same crystal form for wild-type Nek7WT in apo form and bound to compound 51. The R-spines of the three well-ordered Nek7WT molecules exhibit variable conformations while the R-spines of the Nek7SRS molecules all have the same, partially stacked configuration. Compound 51 bound to Nek2 and Nek7 in similar modes, but differences in the precise orientation of a substituent highlights features that could be exploited in designing inhibitors that are selective for particular Nek family members. Although the SRS mutations are not required to obtain a Nek7-inhibitor structure, we conclude that it is a useful strategy for restraining the conformation of a kinase in order to promote crystallogenesis.

2-Arylamino-6-ethynylpurines are cysteine-targeting irreversible inhibitors of Nek2 kinase.

Renewed interest in covalent inhibitors of enzymes implicated in disease states has afforded several agents targeted at protein kinases of relevance to cancers. We now report the design, synthesis and biological evaluation of 6-ethynylpurines that act as covalent inhibitors of Nek2 by capturing a cysteine residue (Cys22) close to the catalytic domain of this protein kinase. Examination of the crystal structure of the non-covalent inhibitor 3-((6-cyclohexylmethoxy-7H-purin-2-yl)amino)benzamide in complex with Nek2 indicated that replacing the alkoxy with an ethynyl group places the terminus of the alkyne close to Cys22 and in a position compatible with the stereoelectronic requirements of a Michael addition. A series of 6-ethynylpurines was prepared and a structure activity relationship (SAR) established for inhibition of Nek2. 6-Ethynyl-N-phenyl-7H-purin-2-amine [IC50 0.15 µM (Nek2)] and 4-((6-ethynyl-7H-purin-2-yl)amino)benzenesulfonamide (IC50 0.14 µM) were selected for determination of the mode of inhibition of Nek2, which was shown to be time-dependent, not reversed by addition of ATP and negated by site directed mutagenesis of Cys22 to alanine. Replacement of the ethynyl group by ethyl or cyano abrogated activity. Variation of substituents on the N-phenyl moiety for 6-ethynylpurines gave further SAR data for Nek2 inhibition. The data showed little correlation of activity with the nature of the substituent, indicating that after sufficient initial competitive binding to Nek2 subsequent covalent modification of Cys22 occurs in all cases. A typical activity profile was that for 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide [IC50 0.06 µM (Nek2); GI50 (SKBR3) 2.2 µM] which exhibited >5-10-fold selectivity for Nek2 over other kinases; it also showed > 50% growth inhibition at 10 µM concentration against selected breast and leukaemia cell lines. X-ray crystallographic analysis confirmed that binding of the compound to the Nek2 ATP-binding site resulted in covalent modification of Cys22. Further studies confirmed that 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide has the attributes of a drug-like compound with good aqueous solubility, no inhibition of hERG at 25 µM and a good stability profile in human liver microsomes. It is concluded that 6-ethynylpurines are promising agents for cancer treatment by virtue of their selective inhibition of Nek2.

Selective DNA-PKcs inhibition extends the therapeutic index of localized radiotherapy and chemotherapy.

Potentiating radiotherapy and chemotherapy by inhibiting DNA damage repair is proposed as a therapeutic strategy to improve outcomes for patients with solid tumors. However, this approach risks enhancing normal tissue toxicity as much as tumor toxicity, thereby limiting its translational impact. Using NU5455, a newly identified highly selective oral inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity, we found that it was indeed possible to preferentially augment the effect of targeted radiotherapy on human orthotopic lung tumors without influencing acute DNA damage or a late radiation-induced toxicity (fibrosis) to normal mouse lung. Furthermore, while NU5455 administration increased both the efficacy and the toxicity of a parenterally administered topoisomerase inhibitor, it enhanced the activity of doxorubicin released locally in liver tumor xenografts without inducing any adverse effect. This strategy is particularly relevant to hepatocellular cancer, which is treated clinically with localized drug-eluting beads and for which DNA-PKcs activity is reported to confer resistance to treatment. We conclude that transient pharmacological inhibition of DNA-PKcs activity is effective and tolerable when combined with localized DNA-damaging therapies and thus has promising clinical potential.

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.

Recent advances in CDK inhibitors for cancer therapy.

Inhibition of CDKs is an attractive approach to cancer therapy due to their vital role in cell growth and transcription. Pan-CDK inhibitors have shown some clinical benefit, and trials are ongoing. Selective CDK4 and CDK6 inhibitors have been licensed for the treatment of hormone responsive, RB-positive breast cancer in combination with antihormonal agents. Selective inhibitors of CDKs 5, 7, 8, 9 and 12 have been identified across a range of chemotypes.

Validating and enabling phosphoglycerate dehydrogenase (PHGDH) as a target for fragment-based drug discovery in PHGDH-amplified breast cancer.

3-Phosphoglycerate dehydrogenase (PHGDH) has recently been identified as an attractive target in cancer therapy as it links upregulated glycolytic flux to increased biomass production in cancer cells. PHGDH catalyses the first step in the serine synthesis pathway and thus diverts glycolytic flux into serine synthesis. We have used siRNA-mediated suppression of PHGDH expression to show that PHGDH is a potential therapeutic target in PHGDH-amplified breast cancer. Knockdown caused reduced proliferation in the PHGDH-amplified cell line MDA-MB-468, whereas breast cancer cells with low PHGDH expression or with elevated PHGDH expression in the absence of genomic amplification were not affected. As a first step towards design of a chemical probe for PHGDH, we report a fragment-based drug discovery approach for the identification of PHGDH inhibitors. We designed a truncated PHGDH construct that gave crystals which diffracted to high resolution, and could be used for fragment soaking. 15 fragments stabilising PHGDH were identified using a thermal shift assay and validated by X-ray crystallography and ITC competition experiments to exhibit 1.5-26.2 mM affinity for PHGDH. A structure-guided fragment growing approach was applied to the PHGDH binders from the initial screen, yielding greater understanding of the binding site and suggesting routes to achieve higher affinity NAD-competitive inhibitors.

Identification of a novel ligand for the ATAD2 bromodomain with selectivity over BRD4 through a fragment growing approach.

ATAD2 is an ATPase that is overexpressed in a variety of cancers and associated with a poor patient prognosis. This protein has been suggested to function as a cofactor for a range of transcription factors, including the proto-oncogene MYC and the androgen receptor. ATAD2 comprises an ATPase domain, implicated in chromatin remodelling, and a bromodomain which allows it to interact with acetylated histone tails. Dissection of the functional roles of these two domains would benefit from the availability of selective, cell-permeable pharmacological probes. An in silico evaluation of the 3D structures of various bromodomains suggested that developing small molecule ligands for the bromodomain of ATAD2 is likely to be challenging, although recent reports have shown that ATAD2 bromodomain ligands can be identified. We report a structure-guided fragment-based approach to identify lead compounds for ATAD2 bromodomain inhibitor development. Our findings indicate that the ATAD2 bromodomain can accommodate fragment hits (Mr < 200) that yield productive structure-activity relationships, and structure-guided design enabled the introduction of selectivity over BRD4.

Structural insights into the enzymatic activity and potential substrate promiscuity of human 3-phosphoglycerate dehydrogenase (PHGDH).

Cancer cells reprogram their metabolism and energy production to sustain increased growth, enable metastasis and overcome resistance to cancer treatments. Although primary roles for many metabolic proteins have been identified, some are promiscuous in regards to the reaction they catalyze. To efficiently target these enzymes, a good understanding of their enzymatic function and structure, as well as knowledge regarding any substrate or catalytic promiscuity is required. Here we focus on the characterization of human 3-phosphoglycerate dehydrogenase (PHGDH). PHGDH catalyzes the NAD+-dependent conversion of 3-phosphoglycerate to phosphohydroxypyruvate, which is the first step in the de novo synthesis pathway of serine, a critical amino acid for protein and nucleic acid biosynthesis. We have investigated substrate analogues to assess whether PHGDH might possess other enzymatic roles that could explain its occasional over-expression in cancer, as well as to help with the design of specific inhibitors. We also report the crystal structure of the catalytic subunit of human PHGDH, a dimer, solved with bound cofactor in one monomer and both cofactor and L-tartrate in the second monomer. In vitro enzyme activity measurements show that the catalytic subunit of PHGDH is still active and that PHGDH activity could be significantly inhibited with adenosine 5'-diphosphoribose.

Targeting DNA-Dependent Protein Kinase for Cancer Therapy.

The catalytic activity of DNA-dependent protein kinase (DNA-PK) is critical to its ability to repair lethal DNA double-strand breaks (DSBs). This includes repair of DSB lesions resulting from oxidative stress, oncogene-induced transcription, or following therapeutic treatment of cancer cells. Armed with this knowledge, many attempts have been made to identify small-molecule inhibitors of DNA-PK activity as an approach to induce tumour chemo- and radiosensitisation. This review examines the structures of known reversible and irreversible inhibitors, including those based on chromen-4-one, arylmorpholine, and benzaldehyde scaffolds. DNA-PK catalytic inhibitors, such as VX-984 (8-[(1S)-2-[[6-(4,6-dideuterio-2-methylpyrimidin-5-yl)pyrimidin-4-yl]amino]-1-methylethyl]quinoline-4-carboxamide) and M3814 ((S)-[2-chloro-4-fluoro-5-(7-morpholinoquinazolin-4-yl)phenyl]-(6-methoxypyridazin-3-yl)methanol), have now progressed into clinical development which should help to further advance our understanding of whether this approach is a promising therapeutic strategy for the treatment of cancer.

Structure-guided design of purine-based probes for selective Nek2 inhibition.

Nek2 (NIMA-related kinase 2) is a cell cycle-dependent serine/threonine protein kinase that regulates centrosome separation at the onset of mitosis. Overexpression of Nek2 is common in human cancers and suppression can restrict tumor cell growth and promote apoptosis. Nek2 inhibition with small molecules, therefore, offers the prospect of a new therapy for cancer. To achieve this goal, a better understanding of the requirements for selective-inhibition of Nek2 is required. 6-Alkoxypurines were identified as ATP-competitive inhibitors of Nek2 and CDK2. Comparison with CDK2-inhibitor structures indicated that judicious modification of the 6-alkoxy and 2-arylamino substituents could achieve discrimination between Nek2 and CDK2. In this study, a library of 6-cyclohexylmethoxy-2-arylaminopurines bearing carboxamide, sulfonamide and urea substituents on the 2-arylamino ring was synthesized. Few of these compounds were selective for Nek2 over CDK2, with the best result being obtained for 3-((6-(cyclohexylmethoxy)-9H-purin-2-yl)amino)-N,N-dimethylbenzamide (CDK2 IC50 = 7.0 µM; Nek2 IC50 = 0.62 µM) with >10-fold selectivity. Deletion of the 6-substituent abrogated activity against both Nek2 and CDK2. Nine compounds containing an (E)-dialkylaminovinyl substituent at C-6, all showed selectivity for Nek2, e.g. (E)-6-(2-(azepan-1-yl)vinyl)-N-phenyl-9H-purin-2-amine (CDK2 IC50 = 2.70 µM; Nek2 IC50 = 0.27 µM). Structural biology of selected compounds enabled a partial rationalization of the observed structure activity relationships and mechanism of Nek2 activation. This showed that carboxamide 11 is the first reported inhibitor of Nek2 in the DFG-in conformation.

A new tool for the chemical genetic investigation of the Plasmodium falciparum Pfnek-2 NIMA-related kinase.

BACKGROUND: Examining essential biochemical pathways in Plasmodium falciparum presents serious challenges, as standard molecular techniques such as siRNA cannot be employed in this organism, and generating gene knock-outs of essential proteins requires specialized conditional approaches. In the study of protein kinases, pharmacological inhibition presents a feasible alternative option. However, as in mammalian systems, inhibitors often lack the desired selectivity. Described here is a chemical genetic approach to selectively inhibit Pfnek-2 in P. falciparum, a member of the NIMA-related kinase family that is essential for completion of the sexual development of the parasite. RESULTS: Introduction of a valine to cysteine mutation at position 24 in the glycine rich loop of Pfnek-2 does not affect kinase activity but confers sensitivity to the protein kinase inhibitor 4-(6-ethynyl-9H-purin-2-ylamino) benzene sulfonamide (NCL-00016066). Using a combination of in vitro kinase assays and mass spectrometry, (including phosphoproteomics) the study shows that this compound acts as an irreversible inhibitor to the mutant Pfnek2 likely through a covalent link with the introduced cysteine residue. In particular, this was shown by analysis of total protein mass using mass spectrometry which showed a shift in molecular weight of the mutant kinase in the presence of the inhibitor to be precisely equivalent to the molecular weight of NCL-00016066. A similar molecular weight shift was not observed in the wild type kinase. Importantly, this inhibitor has little activity towards the wild type Pfnek-2 and, therefore, has all the properties of an effective chemical genetic tool that could be employed to determine the cellular targets for Pfnek-2. CONCLUSIONS: Allelic replacement of wild-type Pfnek-2 with the mutated kinase will allow for targeted inhibition of Pfnek-2 with NCL-00016066 and hence pave the way for comparative studies aimed at understanding the biological role and transmission-blocking potential of Pfnek-2.

Combined PI3K and CDK2 inhibition induces cell death and enhances in vivo antitumour activity in colorectal cancer.

BACKGROUND: The phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway is commonly deregulated in human cancer, hence many PI3K and mTOR inhibitors have been developed and have now reached clinical trials. Similarly, CDKs have been investigated as cancer drug targets. METHODS: We have synthesised and characterised a series of 6-aminopyrimidines identified from a kinase screen that inhibit PI3K and/or mTOR and/or CDK2. Kinase inhibition, tumour cell growth, cell cycle distribution, cytotoxicity and signalling experiments were undertaken in HCT116 and HT29 colorectal cancer cell lines, and in vivo HT29 efficacy studies. RESULTS: 2,6-Diaminopyrimidines with an O(4)-cyclohexylmethyl substituent and a C-5-nitroso or cyano group (1,2,5) induced cell cycle phase alterations and were growth inhibitory (GI50<20 µM). Compound 1, but not 2 or 5, potently inhibits CDK2 (IC50=0.1 nM) as well as PI3K, and was cytotoxic at growth inhibitory concentrations. Consistent with kinase inhibition data, compound 1 reduced phospho-Rb and phospho-rS6 at GI50 concentrations. Combination of NU6102 (CDK2 inhibitor) and pictilisib (GDC-0941; pan-PI3K inhibitor) resulted in synergistic growth inhibition, and enhanced cytotoxicity in HT29 cells in vitro and HT29 tumour growth inhibition in vivo. CONCLUSIONS: These studies identified a novel series of mixed CDK2/PI3K inhibitors and demonstrate that dual targeting of CDK2 and PI3K can result in enhanced antitumour activity.

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.