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.

Quantitation of ERK1/2 inhibitor cellular target occupancies with a reversible slow off-rate probe.

Target engagement is a key concept in drug discovery and its direct measurement can provide a quantitative understanding of drug efficacy and/or toxicity. Failure to demonstrate target occupancy in relevant cells and tissues has been recognised as a contributing factor to the low success rate of clinical drug development. Several techniques are emerging to quantify target engagement in cells; however, in situ measurements remain challenging, mainly due to technical limitations. Here, we report the development of a non-covalent clickable probe, based on SCH772984, a slow off-rate ERK1/2 inhibitor, which enabled efficient pull down of ERK1/2 protein via click reaction with tetrazine tagged agarose beads. This was used in a competition setting to measure relative target occupancy by selected ERK1/2 inhibitors. As a reference we used the cellular thermal shift assay, a label-free biophysical assay relying solely on ligand-induced thermodynamic stabilization of proteins. To validate the EC50 values measured by both methods, the results were compared with IC50 data for the phosphorylation of RSK, a downstream substrate of ERK1/2 used as a functional biomarker of ERK1/2 inhibition. We showed that a slow off-rate reversible probe can be used to efficiently pull down cellular proteins, significantly extending the potential of the approach beyond the need for covalent or photoaffinity warheads.

Highly Potent Clickable Probe for Cellular Imaging of MDM2 and Assessing Dynamic Responses to MDM2-p53 Inhibition.

MDM2 is a key negative regulator of the p53 tumor suppressor. Direct binding of MDM2 to p53 represses the protein's transcriptional activity and induces its polyubiquitination, targeting it for degradation by the proteasome. Consequently, small molecule inhibitors that antagonize MDM2-p53 binding, such as RG7388, have progressed into clinical development aiming to reactivate p53 function in TP53 wild-type tumors. Here, we describe the design, synthesis, and biological evaluation of a trans-cyclooctene tagged derivative of RG7388, RG7388-TCO, which showed high cellular potency and specificity for MDM2. The in-cell reaction of RG7388-TCO with a tetrazine-tagged BODIPY dye enabled fluorescence imaging of endogenous MDM2 in SJSA-1 and T778 tumor cells. RG7388-TCO was also used to pull down MDM2 by reaction with tetrazine-tagged agarose beads in SJSA-1 lysates. The data presented show that RG733-TCO enables precise imaging of MDM2 in cells and can permit a relative assessment of target engagement and MDM2-p53 antagonism in vitro.

Protein degradation: a validated therapeutic strategy with exciting prospects.

In a time of unprecedented challenges in developing potent, selective and well-tolerated protein inhibitors as therapeutics, drug hunters are increasingly seeking alternative modalities to modulate pharmacological targets. Selective inhibitors are achievable for only a fraction of the proteome, and are not guaranteed to elicit the desired response in patients, especially when pursuing targets identified through genetic knockdown. Targeted protein degradation holds the potential to expand the range of proteins that can be effectively modulated. Drugs inducing protein degradation through misfolding or by modulating cereblon (CRBN) substrate recognition are already approved for treatment of cancer patients. The last decade has seen the development of proteolysis targeting chimeras (PROTACs), small molecules that elicit proteasomal degradation by causing protein polyubiquitination. These have been used to degrade a range of disease-relevant proteins in cells, and some show promising efficacy in preclinical animal models, although their clinical efficacy and tolerability is yet to be proven. This review introduces current strategies for protein degradation with an emphasis on PROTACs and the role of click chemistry in PROTAC research through the formation of libraries of preclicked PROTACs or in-cell click-formed PROTACs (CLIPTACs).

Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe.

The RAS-RAF-MEK-ERK pathway has been intensively studied in oncology, with RAS known to be mutated in ∼30% of all human cancers. The recent emergence of ERK1/2 inhibitors and their ongoing clinical investigation demands a better understanding of ERK1/2 behavior following small-molecule inhibition. Although fluorescent fusion proteins and fluorescent antibodies are well-established methods of visualizing proteins, we show that ERK1/2 can be visualized via a less-invasive approach based on a two-step process using inverse electron demand Diels-Alder cycloaddition. Our previously reported trans-cyclooctene-tagged covalent ERK1/2 inhibitor was used in a series of imaging experiments following a click reaction with a tetrazine-tagged fluorescent dye. Although limitations were encountered with this approach, endogenous ERK1/2 was successfully imaged in cells, and "on-target" staining was confirmed by over-expressing DUSP5, a nuclear ERK1/2 phosphatase that anchors ERK1/2 in the nucleus.

In-gel activity-based protein profiling of a clickable covalent ERK1/2 inhibitor.

In-gel activity-based protein profiling (ABPP) offers rapid assessment of the proteome-wide selectivity and target engagement of a chemical tool. Here we demonstrate the use of the inverse electron demand Diels Alder (IEDDA) click reaction for in-gel ABPP by evaluating the selectivity profile and target engagement of a covalent ERK1/2 probe tagged with a trans-cyclooctene group. The chemical probe was shown to bind covalently to Cys166 of ERK2 using protein MS and X-ray crystallography, and displayed submicromolar GI50s in A375 and HCT116 cells. In both cell lines, the probe demonstrated target engagement and a good selectivity profile at low concentrations, which was lost at higher concentrations. The IEDDA cycloaddition enabled fast and quantitative fluorescent tagging for readout with a high background-to-noise ratio and thereby provides a promising alternative to the commonly used copper catalysed alkyne-azide cycloaddition.

Protein Degradation by In-Cell Self-Assembly of Proteolysis Targeting Chimeras.

Selective degradation of proteins by proteolysis targeting chimeras (PROTACs) offers a promising potential alternative to protein inhibition for therapeutic intervention. Current PROTAC molecules incorporate a ligand for the target protein, a linker, and an E3 ubiquitin ligase recruiting group, which bring together target protein and ubiquitinating machinery. Such hetero-bifunctional molecules require significant linker optimization and possess high molecular weight, which can limit cellular permeation, solubility, and other drug-like properties. We show here that the hetero-bifunctional molecule can be formed intracellularly by bio-orthogonal click combination of two smaller precursors. We designed a tetrazine tagged thalidomide derivative which reacts rapidly with a trans-cyclo-octene tagged ligand of the target protein in cells to form a cereblon E3 ligase recruiting PROTAC molecule. The in-cell click-formed proteolysis targeting chimeras (CLIPTACs) were successfully used to degrade two key oncology targets, BRD4 and ERK1/2. ERK1/2 degradation was achieved using a CLIPTAC based on a covalent inhibitor. We expect this approach to be readily extendable to other inhibitor-protein systems because the tagged E3 ligase recruiter is capable of undergoing the click reaction with a suitably tagged ligand of any protein of interest to elicit its degradation.

Discovery of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one (AZD8835): A potent and selective inhibitor of PI3Kα and PI3Kδ for the treatment of cancers.

Starting from potent inhibitors of PI3Kα having poor general kinase selectivity (e.g., 1 and 2), optimisation of this series led to the identification of 25, a potent inhibitor of PI3Kα (wild type, E545K and H1047R mutations) and PI3Kδ, selective versus PI3Kß and PI3Kγ, with excellent general kinase selectivity. Compound 25 displayed low metabolic turnover and suitable physical properties for oral administration. In vivo, compound 25 showed pharmacodynamic modulation of AKT phosphorylation and near complete inhibition of tumour growth (93% tumour growth inhibition) in a murine H1047R PI3Kα mutated SKOV-3 xenograft tumour model after chronic oral administration at 25mg/kg b.i.d. Compound 25, also known as AZD8835, is currently in phase I clinical trials.

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.