Good Selectivity Profile Research Articles

Systematic diversification of benzylidene heterocycles yields novel inhibitor scaffolds selective for Dyrk1A, Clk1 and CK2

The dual-specificity tyrosine-regulated kinase 1A (Dyrk1A) has gathered much interest as a pharmacological target in Alzheimer's disease (AD), but it plays a role in malignant brain tumors as well. As both diseases are multi-factorial, further protein kinases, such as Clk1 and CK2, were proposed to contribute to the pathogenesis. We designed a new class of α-benzylidene–γ-butyrolactone inhibitors that showed low micromolar potencies against Dyrk1A and/or Clk1 and a good selectivity profile among the most frequently reported off-target kinases. A systematic replacement of the heterocyclic moiety gave access to further inhibitor classes with interesting selectivity profiles, demonstrating that the benzylidene heterocycles provide a versatile tool box for developing inhibitors of the CMGC kinase family members Dyr1A/1B, Clk1/4 and CK2. Efficacy for the inhibition of Dyrk1A–mediated tau phosphorylation was demonstrated in a cell-based assay. Multi-targeted but not non-specific kinase inhibitors were also obtained, that co-inhibited the lipid kinases PI3Kα/γ. These compounds were shown to inhibit the proliferation of U87MG cells in the low micromolar range. Based on the molecular properties, the inhibitors described here hold promise for CNS activity.

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.

Abstract C190: Potent and selective inhibition of CDK7 by novel covalent inhibitors

Abstract Cyclin-dependent kinase 7 (CDK7) is an important constituent of the cellular transcriptional machinery, where it phosphorylates the C-terminal domain (CTD) of RNAP polymerase II (RNAPII). Because many tumor types are critically dependent on transcription for maintenance of their oncogenic state, pharmacological modulation of CDK7 kinase activity is considered as an approach to treat cancer. Multiple series of CDK7 inhibitors were identified by iterative medicinal chemistry efforts and SAR based approach. Early compounds were optimized towards attaining good physicochemical properties, high potency, good selectivity and desirable pharmacokinetic profile to achieve anti-tumor activity. We have identified compounds from two distinct chemical series that are highly potent in inhibiting CDK7 in biochemical assays. These inhibitors demonstrate time-dependent inhibition of CDK7 indicating covalent nature of binding. The compounds showed potent anti-proliferative activity in cell lines derived from various tumor types and this was accompanied by CDK7 modulation in cells as monitored by pS5RNAPII levels. They have excellent drug-like characteristics including solubility, permeability, metabolic stability and good oral bioavailability. In a broad panel of kinases (332 kinase), selected compounds from both series showed good selectivity profile. Tolerability and efficacy studies are ongoing with selected early leads to test their impact on tumor growth inhibition in xenograft models. We have identified novel and selective CDK7 covalent inhibitors from two series with desirable drug-like properties, which are being evauated for anti-tumor activity in xenograft models. Citation Format: Ramulu Poddutoori, Leena K. Satyam, Girish Daginakatte, Subhendu Mukherjee, Sivapriya Marappan, Sreevalsam Gopinath, Raghuveer Ramachandra, Anirudha Lakshminarasimhan, Manoj Pothuganti, Shilpa Nayak, Nandish C, Chandranath Naik, Ravindra MV, Madhu Dabbeeru, Thomas Antony, Chetan Pandit, Murali Ramachandra, Shekar Chelur, Susanta Samajdar. Potent and selective inhibition of CDK7 by novel covalent inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C190.

Development of new highly potent imidazo[1,2-b]pyridazines targeting Toxoplasma gondii calcium-dependent protein kinase 1

Using a structure-based design approach, we have developed a new series of imidazo[1,2-b]pyridazines, targeting the calcium-dependent protein kinase-1 (CDPK1) from Toxoplasma gondii. Twenty derivatives were thus synthesized. Structure-activity relationships and docking studies confirmed the binding mode of these inhibitors within the ATP binding pocket of TgCDPK1. Two lead compounds (16a and 16f) were then identified, which were able to block TgCDPK1 enzymatic activity at low nanomolar concentrations, with a good selectivity profile against a panel of mammalian kinases. The potential of these inhibitors was confirmed in vitro on T. gondii growth, with EC50 values of 100 nM and 70 nM, respectively. These best candidates also displayed low toxicity to mammalian cells and were selected for further in vivo investigations on murine model of acute toxoplasmosis.

2,4-Diarylamino-pyrimidines as kinase inhibitors co-targeting IGF1R and EGFR(L⁸⁵⁸R/T⁷⁹⁰M).

IGF1R amplification was recently implied to be related to the secondary acquired resistance against the 2nd or 3rd generation EGFR inhibitor therapies. We have successfully identified a series of 2,4-diarylamino-pyrimidines as new IGF1R/EGFR(L858R/T790M) co-targeting agents. One of the most promising compounds 8g potently inhibits both kinases with low nanomolar IC50 values, but is significantly less potent in inhibiting the wild type EGFR. The compound also displays a good kinase selectivity profile against a panel of 468 kinases. Moreover, 8g strongly suppresses the proliferation of CO-1686-resistant H1975-IGF1R cancer cells, suggesting its promising potential as a new lead compound for future anticancer drug discovery.

Optimization and Evaluation of 5-Styryl-Oxathiazol-2-one Mycobacterium tuberculosis Proteasome Inhibitors as Potential Antitubercular Agents.

This is the first report of 5-styryl-oxathiazol-2-ones as inhibitors of the Mycobacterium tuberculosis (Mtb) proteasome. As part of the study, the structure–activity relationship of oxathiazolones as Mtb proteasome inhibitors has been investigated. Furthermore, the prepared compounds displayed a good selectivity profile for Mtb compared to the human proteasome. The 5-styryl-oxathiazol-2-one inhibitors identified showed little activity against replicating Mtb, but were rapidly bactericidal against nonreplicating bacteria. (E)-5-(4-Chlorostyryl)-1,3,4-oxathiazol-2-one) was most effective, reducing the colony-forming units (CFU)/mL below the detection limit in only seven days at all concentrations tested. The results suggest that this new class of Mtb proteasome inhibitors has the potential to be further developed into novel antitubercular agents for synergistic combination therapies with existing drugs.

Disarming Pseudomonas aeruginosa Virulence Factor LasB by Leveraging a Caenorhabditis elegans Infection Model

The emergence of antibiotic resistance places a sense of urgency on the development of alternative antibacterial strategies, of which targeting virulence factors has been regarded as a "second generation" antibiotic approach. In the case of Pseudomonas aeruginosa infections, a proteolytic virulence factor, LasB, is one such target. Unfortunately, we and others have not been successful in translating invitro potency of LasB inhibitors to invivo efficacy in an animal model. To overcome this obstacle, we now integrate insilico and invitro identification of the mercaptoacetamide motif as an effective class of LasB inhibitors with full invivo characterization of mercaptoacetamide prodrugs using Caenorhabditis elegans. We show that one of our mercaptoacetamide prodrugs has a good selectivity profile and high invivo efficacy, and confirm that LasB is a promising target for the treatment of bacterial infections. In addition, our work highlights that the C.elegans infection model is a user-friendly and cost-effective translational tool for the development of anti-virulence compounds.

Structure-based design of inhibitors of coagulation factor XIa with novel P1 moieties.

Compound 2 was previously identified as a potent inhibitor of factor XIa lacking oral bioavailability. A structure-based approach was used to design analogs of 2 with novel P1 moieties with good selectivity profiles and oral bioavailability. Further optimization of the P1 group led to the identification of a 4-chlorophenyltetrazole P1 analog, which when combined with further modifications to the linker and P2' group provided compound 32 with FXIa Ki=6.7 nM and modest oral exposure in dogs.

Pyrrolo[3,2-b]quinoxaline derivatives as types I1/2 and II Eph tyrosine kinase inhibitors: structure-based design, synthesis, and in vivo validation.

The X-ray crystal structures of the catalytic domain of the EphA3 tyrosine kinase in complex with two type I inhibitors previously discovered in silico (compounds A and B) were used to design type I1/2 and II inhibitors. Chemical synthesis of about 25 derivatives culminated in the discovery of compounds 11d (type I1/2), 7b, and 7g (both of type II), which have low-nanomolar affinity for Eph kinases in vitro and a good selectivity profile on a panel of 453 human kinases (395 nonmutant). Surface plasmon resonance measurements show a very slow unbinding rate (1/115 min) for inhibitor 7m. Slow dissociation is consistent with a type II binding mode in which the hydrophobic moiety (trifluoromethyl-benzene) of the inhibitor is deeply buried in a cavity originating from the displacement of the Phe side chain of the so-called DFG motif as observed in the crystal structure of compound 7m. The inhibitor 11d displayed good in vivo efficacy in a human breast cancer xenograft.

Fragment-Based Discovery of Type I Inhibitors of Maternal Embryonic Leucine Zipper Kinase

Fragment-based drug design was successfully applied to maternal embryonic leucine zipper kinase (MELK). A low affinity (160 μM) fragment hit was identified, which bound to the hinge region with an atypical binding mode, and this was optimized using structure-based design into a low-nanomolar and cell-penetrant inhibitor, with a good selectivity profile, suitable for use as a chemical probe for elucidation of MELK biology.

Lead identification of novel and selective TYK2 inhibitors

A therapeutic rationale is proposed for the treatment of inflammatory diseases, such as psoriasis and inflammatory bowel diseases (IBD), by selective targeting of TYK2. Hit triage, following a high-throughput screen for TYK2 inhibitors, revealed pyridine 1 as a promising starting point for lead identification. Initial expansion of 3 separate regions of the molecule led to eventual identification of cyclopropyl amide 46, a potent lead analog with good kinase selectivity, physicochemical properties, and pharmacokinetic profile. Analysis of the binding modes of the series in TYK2 and JAK2 crystal structures revealed key interactions leading to good TYK2 potency and design options for future optimization of selectivity.

Discovery of Tyrosine Kinase Inhibitors by Docking into an Inactive Kinase Conformation Generated by Molecular Dynamics

Several small molecules that bind to the inactive DFG-out conformation of tyrosine kinases (called type II inhibitors) have shown a good selectivity profile over other kinase targets. To obtain a set of DFG-out structures, we performed an explicit solvent molecular dynamics (MD) simulation of the complex of the catalytic domain of a tyrosine kinase receptor, ephrin type-A receptor 3 (EphA3), and a manually docked type II inhibitor. Automatic docking of four previously reported type II inhibitors was used to select a single snapshot from the MD trajectory for virtual screening. High-throughput docking of a pharmacophore-tailored library of 175,000 molecules resulted in about 4 million poses, which were further filtered by van der Waals efficiency and ranked according to a force-field-based energy function. Notably, around 20 % of the compounds with predicted binding energy smaller than -10 kcal mol(-1) are known type II inhibitors. Moreover, a series of 5-(piperazine-1-yl)isoquinoline derivatives was identified as a novel class of low-micromolar inhibitors of EphA3 and unphosphorylated Abelson tyrosine kinase (Abl1). The in silico predicted binding mode of the new inhibitors suggested a similar affinity to the gatekeeper mutant T315I of Abl1, which was verified in vitro by using a competition binding assay. Additional evidence for the type II binding mode was obtained by two 300 ns MD simulations of the complex between N-(3-chloro-4-(difluoromethoxy)phenyl)-2-(4-(8-nitroisoquinolin-5-yl)piperazin-1-yl)acetamide and EphA3.

Lead optimisation of selective non-zinc binding inhibitors of MMP13. Part 2

Directed screening has identified a novel series of non-zinc binding MMP13 inhibitors that possess good levels of activity whilst demonstrating excellent selectivity over related MMPs. A lead optimisation campaign has delivered compounds with enhanced MMP13 potency, good selectivity and acceptable bioavailability profiles leading to a predicted twice-a-day dosing regimen in man.

Triazole ring-opening leads to the discovery of potent nonsteroidal 17β-hydroxysteroid dehydrogenase type 2 inhibitors

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) catalyzes the oxidation of the highly potent steroids: the estrogen estradiol (E2) and the androgen testosterone (T) to the less active estrone and androstenedione, respectively. Inhibition of this enzyme may help maintain the local E2 level in bone tissue when the circulating E2 level drops and is therefore a novel and promising approach for the treatment of osteoporosis. In this work, a series of new nonsteroidal and achiral 17β-HSD2 inhibitors, namely N-benzyl-diphenyl-3(or 4)-carboxamide and N-benzyl-5-phenyl-thiophene-2-carboxamide was designed and the compounds were synthesized in a two to three steps reaction. A small library was built applying parallel synthesis. Highly potent 17β-HSD2 inhibitors could be identified in the thiophene-2-carboxamide class with IC 50 in the low nanomolar range. These compounds also showed a good selectivity profile toward 17β-HSD1 and toward the estrogen receptors α and β. The most interesting 17β-HSD2 inhibitor identified in this study is the 5-(2-fluoro-3-methoxyphenyl)- N-(3-hydroxybenzyl)- N-methylthiophene-2-carboxamide 6w displaying an IC 50 of 61 nM and a selectivity factor of 73 toward 17β-HSD1.

Discovery and Structure−Activity Relationship of 3-Aminopyrid-2-ones as Potent and Selective Interleukin-2 Inducible T-Cell Kinase (Itk) Inhibitors

Interleukin-2 inducible T-cell kinase (Itk) plays a role in T-cell functions, and its inhibition potentially represents an attractive intervention point to treat autoimmune and allergic diseases. Herein we describe the discovery of a series of potent and selective novel inhibitors of Itk. These inhibitors were identified by structure-based design, starting from a fragment generated de novo, the 3-aminopyrid-2-one motif. Functionalization of the 3-amino group enabled rapid enhancement of the inhibitory activity against Itk, while introduction of a substituted heteroaromatic ring in position 5 of the pyridone fragment was key to achieving optimal selectivity over related kinases. A careful analysis of the hydration patterns in the kinase active site was necessary to fully explain the observed selectivity profile. The best molecule prepared in this optimization campaign, 7v, inhibits Itk with a K(i) of 7 nM and has a good selectivity profile across kinases.

Inhibitors of the tyrosine kinase EphB4. Part 4: Discovery and optimization of a benzylic alcohol series

Optimization of our bis-anilino-pyrimidine series of EphB4 kinase inhibitors led to the discovery of compound 12 which incorporates a key m-hydroxymethylene group on the C4 aniline. 12 displays a good kinase selectivity profile, good physical properties and pharmacokinetic parameters, suggesting it is a suitable candidate to investigate the therapeutic potential of EphB4 kinase inhibitors.

High‐Throughput Virtual Screening Using Quantum Mechanical Probes: Discovery of Selective Kinase Inhibitors

A procedure based on semi-empirical quantum mechanical (QM) calculations of interaction energy is proposed for the rapid screening of compound poses generated by high-throughput docking. Small molecules (consisting of 2-10 atoms and termed "probes") are overlapped with polar groups in the binding site of the protein target. The interaction energy values between each compound pose and the probes, calculated by a semi-empirical Hamiltonian, are used as filters. The QM probe method does not require fixed partial charges and takes into account polarization and charge-transfer effects which are not captured by conventional force fields. The procedure is applied to screen approximately 100 million poses (of 2.7 million commercially available compounds) obtained by high-throughput docking in the ATP binding site of the tyrosine kinase erythropoietin-producing human hepatocellular carcinoma receptor B4 (EphB4). Three QM probes on the hinge region and one at the entrance pocket are employed to select for binding affinity, while a QM probe on the side chain of the so-called gatekeeper residue (a hypervariable residue in the kinome) is used to enforce selectivity. The poses with favorable interactions with the five QM probes are filtered further for hydrophobic matching and low ligand strain. In this way, a single-digit micromolar inhibitor of EphB4 with a relatively good selectivity profile is identified in a multimillion-compound library upon experimental tests of only 23 molecules.

In vivo and in vitro SAR of tetracyclic MAPKAP-K2 (MK2) inhibitors. Part I

Pyrrolo[2,3-f]isoquinoline based amino acids, tetracyclic lactams and cyclic ketone analogues are described as novel MK2 inhibitors with IC50 as low as 5nM and good selectivity profiles against a number of related kinases including ERK, p38α and JNKs. TNFα release was suppressed from human peripheral blood mononuclear cells (hPBMCs), and a representative compound inhibited LPS induced TNFα release in mice illustrating the potential of this series to provide orally active MK2 inhibitors.

Amicarbazone, a New Photosystem II Inhibitor

Amicarbazone is a new triazolinone herbicide with a broad spectrum of weed control. The phenotypic responses of sensitive plants exposed to amicarbazone include chlorosis, stunted growth, tissue necrosis, and death. Its efficacy as both a foliar- and root-applied herbicide suggests that absorption and translocation of this compound is very rapid. This new herbicide is a potent inhibitor of photosynthetic electron transport, inducing chlorophyll fluorescence and interrupting oxygen evolution ostensibly via binding to the QB domain of photosystem II (PSII) in a manner similar to the triazines and the triazinones classes of herbicides. As a result, its efficacy is susceptible to the most common form of resistance to PSII inhibitors. Nonetheless, amicarbazone has a good selectivity profile and is a more potent herbicide than atrazine, which enables its use at lower rates than those of traditional photosynthetic inhibitors.

The discovery of thienopyridine analogues as potent IκB kinase β inhibitors. Part II

An SAR study that identified a series of thienopyridine-based potent IκB Kinase β (IKKβ) inhibitors is described. With focuses on the structural optimization at C 4 and C 6 of structure 1 (Fig. 1), the study reveals that small alkyl and certain aromatic groups are preferred at C 4, whereas polar groups with proper orientation at C 6 efficiently enhance compound potency. The most potent analogues inhibit IKKβ with IC 50s as low as 40 nM, suppress LPS-induced TNF-α production in vitro and in vivo, display good kinase selectivity profiles, and are active in a HeLa cell NF-κB reporter gene assay, demonstrating that they directly interfere with the NF-κB signaling pathway.