A beginner's guide to current synthetic linker strategies towards VHL-recruiting PROTACs.

Over the last two decades, proteolysis targeting chimeras (PROTACs) have been revolutionary in drug development rendering targeted protein degradation (TPD) as an emerging therapeutic modality. These heterobifunctional molecules are comprised of three units: a ligand for the protein of interest (POI), a ligand for an E3 ubiquitin ligase, and a linker that tethers the two motifs together. Von Hippel-Lindau (VHL) is one of the most widely employed E3 ligases in PROTACs development due to its prevalent expression across tissue types and well-characterised ligands. Linker composition and length has proven to play an important role in determining the physicochemical properties and spatial orientation of the POI-PROTAC-E3 ternary complex, thus influencing the bioactivity of degraders. Numerous articles and reports have been published showcasing the medicinal chemistry aspects of the linker design, but few have focused on the chemistry around tethering linkers to E3 ligase ligands. In this review, we focus on the current synthetic linker strategies employed in the assembly of VHL-recruiting PROTACs. We aim to cover a range of fundamental chemistries used to incorporate linkers of varying length, composition and functionality.

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.

Cyclizations and fragmentations in the alkylation of 6-chloro-5-hydroxy-4-aminopyrimidines with aminoalkyl chlorides.

Substituted aminopyrimidines are an important class of compounds, in part because they frequently show biological activity. Facile synthesis of polysubstituted aminopyrimidines is highly desirable for the synthesis of screening libraries. We describe a route to 4,6-diamino-5-alkoxypyrimidines via a SNAr-alkylation-SNAr sequence from readily available 4,6-dichloro-5-methoxypyrimidine, which allows the synthesis of such compounds with regiochemical control. The extension of this approach to alkylating agents bearing amino substituents led to unexpected and, in some cases, unprecedented products resulting from intramolecular SNAr cyclization and subsequent fragmentation.

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.

Human Toxicity Caused by Indole and Indazole Carboxylate Synthetic Cannabinoid Receptor Agonists: From Horizon Scanning to Notification.

BACKGROUND: The emergence of novel psychoactive substances (NPS), particularly synthetic cannabinoid receptor agonists (SCRA), has involved hundreds of potentially harmful chemicals in a highly dynamic international market challenging users', clinicians', and regulators' understanding of what circulating substances are causing harm. We describe a toxicovigilance system for NPS that predicted the UK emergence and identified the clinical toxicity caused by novel indole and indazole carboxylate SCRA. METHODS: To assist early accurate identification, we synthesized 5 examples of commercially unavailable indole and indazole carboxylate SCRA (FUB-NPB-22, 5F-NPB-22, 5F-SDB-005, FUB-PB-22, NM-2201). We analyzed plasma and urine samples from 160 patients presenting to emergency departments with severe toxicity after suspected NPS use during 2015 to 2016 for these and other NPS using data-independent LC-MS/MS. RESULTS: We successfully synthesized 5 carboxylate SCRAs using established synthetic and analytical chemistry methodologies. We identified at least 1 SCRA in samples from 49 patients, including an indole or indazole carboxylate SCRA in 17 (35%), specifically 5F-PB-22 (14%), FUB PB-22 (6%), BB-22 (2%), 5F NPB-22 (20%), FUB NPB-22 (2%), and 5F-SDB-005 (4%). In these 17 patients, there was analytical evidence of other substances in 16. Clinical features included agitation and aggression (82%), reduced consciousness (76%), acidosis (47%), hallucinations and paranoid features (41%), tachycardia (35%), hypertension (29%), raised creatine kinase (24%), and seizures (12%). CONCLUSIONS: This toxicovigilance system predicted the emergence of misuse of indole and indazole carboxylate SCRA, documented associated clinical harms, and notified relevant agencies. Toxicity appears consistent with other SCRA, including mental state disturbances and reduced consciousness.

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.

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.

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.

Trifluoroacetic acid in 2,2,2-trifluoroethanol facilitates S(N)Ar reactions of heterocycles with arylamines.

Small-molecule drug discovery requires reliable synthetic methods for attaching amino compounds to heterocyclic scaffolds. Trifluoroacetic acid-2,2,2-trifluoroethanol (TFA-TFE) is as an effective combination for achieving SN Ar reactions between anilines and heterocycles (e.g., purines and pyrimidines) substituted with a leaving group (fluoro-, chloro-, bromo- or alkylsulfonyl). This method provides a variety of compounds containing a "kinase-privileged fragment" associated with potent inhibition of kinases. TFE is an advantageous solvent because of its low nucleophilicity, ease of removal and ability to solubilise polar substrates. Furthermore, TFE may assist the breakdown of the Meisenheimer-Jackson intermediate by solvating the leaving group. TFA is a necessary and effective acidic catalyst, which activates the heterocycle by N-protonation without deactivating the aniline by conversion into an anilinium species. The TFA-TFE methodology is compatible with a variety of functional groups and complements organometallic alternatives, which are often disadvantageous because of the expense of reagents, the frequent need to explore diverse sets of reaction conditions, and problems with product purification. In contrast, product isolation from TFA-TFE reactions is straightforward: evaporation of the reaction mixture, basification and chromatography affords analytically pure material. A total of 45 examples are described with seven discrete heterocyclic scaffolds and 2-, 3- and 4-substituted anilines giving product yields that are normally in the range 50-90 %. Reactions can be performed with either conventional heating or microwave irradiation, with the latter often giving improved yields.

Model system for irreversible inhibition of Nek2: thiol addition to ethynylpurines and related substituted heterocycles.

Recent studies have shown that irreversible inhibition of Nek2 kinase [(Never in mitosis gene a)-related kinase 2], overexpression of which is observed in several cancers, can be achieved using Michael acceptors containing an ethynyl group, which target the enzyme's cysteine 22 residue lying near the catalytic site. The model studies described herein demonstrate an analogous capture of the ethynyl moiety in a series of ethynyl-heterocycles (e.g. 6-ethynyl-N-phenyl-9H-purin-2-amine) by N-acetylcysteine methyl ester in the presence of 1,4-diazabicyclo[2.2.2]octane in either dimethyl sulfoxide or N,N-dimethylformamide. Kinetic studies showed a 50-fold range in reactivity with 7-ethynyl-N-phenyl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine being the most reactive compound, whereas 4-ethynyl-N-phenyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine was the least reactive. Studies of the isomeric compounds, 2-(3-((6-ethynyl-7-methyl-7H-purin-2-yl)amino)phenyl)acetamide and 2-(3-((6-ethynyl-9-methyl-9H-purin-2-yl)amino)phenyl)acetamide, revealed the N(7)-methyl isomer to be 5-fold more reactive than the 9-methyl isomer, which is ascribed to a buttressing effect in the N(7)-methyl compound. Comparison of the crystal structures of these isomers showed that the ethynyl group is significantly displaced away from the methyl group exclusively in the N(7)-methyl isomer with an sp(2) bond angle of 124°, whereas the corresponding angle in the N(9)-methyl isomer was the expected 120°. The results of this study indicate heterocyclic scaffolds that are likely to be more promising for inhibition of Nek2 and other kinases containing a reactive cysteine.

Trifluoroethanol solvent facilitates selective N-7 methylation of purines.

Purines protected at N-9 by p-methoxybenzyl are methylated or ethylated in 2,2,2-trifluoroethanol at N-7 by trimethyl- or triethyl-oxonium borofluorate, respectively. Subjecting the resulting cationic species to microwave irradiation releases an N(7)-methyl- or ethyl-purine. This one-pot procedure is an efficient regiospecific method applicable to diverse substrates.

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.

Potent enantioselective inhibition of DNA-dependent protein kinase (DNA-PK) by atropisomeric chromenone derivatives.

Substitution at the 7-position of the chromen-4-one pharmacophore of 8-(dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one NU7441, a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor, with allyl, n-propyl or methyl enabled the resolution by chiral HPLC of atropisomers. Biological evaluation against DNA-PK of each pair of atropisomers showed a marked difference in potency, with biological activity residing exclusively in the laevorotatory enantiomer.

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.

DNA-dependent protein kinase (DNA-PK) inhibitors: structure-activity relationships for O-alkoxyphenylchromen-4-one probes of the ATP-binding domain.

Introduction of an O-alkoxyphenyl substituent at the 8-position of the 2-morpholino-4H-chromen-4-one pharmacophore enabled regions of the ATP-binding site of DNA-dependent protein kinase (DNA-PK) to be probed further. Structure-activity relationships have been elucidated for inhibition of DNA-PK and PI3K (p110α), with N-(2-(cyclopropylmethoxy)-4-(2-morpholino-4-oxo-4H-chromen-8-yl)phenyl)-2-morpholinoacetamide 11a being identified as a potent and selective DNA-PK inhibitor (IC(50)=8 nM).

Versatile synthesis of functionalised dibenzothiophenes via Suzuki coupling and microwave-assisted ring closure.

Amino-substituted biphenyls were obtained by Suzuki cross-coupling of 2,6-dibromoaniline with a phenylboronic acid (substituted with Me, NO(2), OH, OMe or Cl) preferably assisted by microwave irradiation. Conversion of the amino group into a thiol preceded a base-induced intramolecular substitution, also facilitated by microwave heating, to generate the second C-S bond of the target dibenzothiophene. The 1-, 2-, 3- or 4-substituted 6-halodibenzothiophenes obtained were subjected to a palladium-mediated coupling with 2-morpholin-4-yl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4H-chromen-4-one to give the respective 6-, 7-, 8- or 9-substituted dibenzothiophen-4-ylchromenones. These compounds were evaluated as inhibitors of DNA-dependent protein kinase (DNA-PK) and compared to the parent 8-(dibenzo[b,d]thiophen-4-yl)-2-morpholin-4-yl-4H-chromen-4-one. Notably, derivatives bearing hydroxy or methoxy substituents at C-8 or C-9 retained activity, whereas substitution at C-7 lowered activity. Substitution with chloro at C-6 was not detrimental to activity, but a chloro group at C-7 or C-8 reduced potency. The data indicate permissive elaboration of hydroxyl at C-8 or C-9, enabling the possibility of improved pharmaceutical properties, whilst retaining potency against DNA-PK.

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.