Gaining Insights into Key Structural Hotspots within the Allosteric Binding Pockets of Protein Kinases.

Protein kinases are essential regulators of cell function and represent one of the largest and most diverse protein families. They are particularly influential in signal transduction and coordinating complex processes like the cell cycle. Out of the 518 human protein kinases identified, 478 are part of a single superfamily sharing catalytic domains that are related in sequence. The dysregulation of protein kinases due to certain mutations has been associated with various diseases, including cancer. Although most of the protein kinase inhibitors identified as type I or type II primarily target the ATP-binding pockets of kinases, the structural and sequential resemblances among these pockets pose a significant challenge for selective inhibition. Therefore, targeting allosteric pockets that are beside highly conserved ATP pockets has emerged as a promising strategy to prevail current limitations, such as poor selectivity and drug resistance. In this article, we compared the binding pockets of various protein kinases for which allosteric (type III) inhibitors have already been developed. Additionally, understanding the structure and shape of existing ligands could aid in identifying key interaction sites within the allosteric pockets of kinases. This comprehensive review aims to facilitate the design of more effective and selective allosteric inhibitors.

Rational design and synthesis of 2-(1H-indazol-6-yl)-1H-benzo[d]imidazole derivatives as inhibitors targeting FMS-like tyrosine kinase 3 (FLT3) and its mutants.

Fms-like tyrosine kinase 3 (FLT3) has been verified as a therapeutic target for acute myeloid leukaemia (AML). In this study, we report a series of 2-(1H-indazol-6-yl)-1H-benzo[d]imidazol-5-yl benzamide and phenyl urea derivatives as potent FLT3 inhibitors based on the structural optimisation of previous FLT3 inhibitors. Derivatives were synthesised as benzamide 8a-k, 8n-z, and phenyl urea 8l-m, with various substituents. The most potent inhibitor, 8r, demonstrated strong inhibitory activity against FLT3 and FLT3 mutants with a nanomolar IC50 and high selectivity profiles over 42 protein kinases. In addition, these type II FLT3 inhibitors were more potent against FLT3 mutants correlated with drug resistance. Overall, we provide a theoretical basis for the structural optimisation of novel benzimidazole analogues to develop strong inhibitors against FLT3 mutants for AML therapeutics.

Design of Novel IRAK4 Inhibitors Using Molecular Docking, Dynamics Simulation and 3D-QSAR Studies.

Treatment of several autoimmune diseases and types of cancer has been an intense area of research over the past two decades. Many signaling pathways that regulate innate and/or adaptive immunity, as well as those that induce overexpression or mutation of protein kinases, have been targeted for drug discovery. One of the serine/threonine kinases, Interleukin-1 Receptor Associated Kinase 4 (IRAK4) regulates signaling through various Toll-like receptors (TLRs) and interleukin-1 receptor (IL1R). It controls diverse cellular processes including inflammation, apoptosis, and cellular differentiation. MyD88 gain-of-function mutations or overexpression of IRAK4 has been implicated in various types of malignancies such as Waldenström macroglobulinemia, B cell lymphoma, colorectal cancer, pancreatic ductal adenocarcinoma, breast cancer, etc. Moreover, over activation of IRAK4 is also associated with several autoimmune diseases. The significant role of IRAK4 makes it an interesting target for the discovery and development of potent small molecule inhibitors. A few potent IRAK4 inhibitors such as PF-06650833, RA9 and BAY1834845 have recently entered phase I/II clinical trial studies. Nevertheless, there is still a need of selective inhibitors for the treatment of cancer and various autoimmune diseases. A great need for the same intrigued us to perform molecular modeling studies on 4,6-diaminonicotinamide derivatives as IRAK4 inhibitors. We performed molecular docking and dynamics simulation of 50 ns for one of the most active compounds of the dataset. We also carried out MM-PBSA binding free energy calculation to identify the active site residues, interactions of which are contributing to the total binding energy. The final 50 ns conformation of the most active compound was selected to perform dataset alignment in a 3D-QSAR study. Generated RF-CoMFA (q2 = 0.751, ONC = 4, r2 = 0.911) model revealed reasonable statistical results. Overall results of molecular dynamics simulation, MM-PBSA binding free energy calculation and RF-CoMFA model revealed important active site residues of IRAK4 and necessary structural properties of ligand to design more potent IRAK4 inhibitors. We designed few IRAK4 inhibitors based on these results, which possessed higher activity (predicted pIC50) than the most active compounds of the dataset selected for this study. Moreover, ADMET properties of these inhibitors revealed promising results and need to be validated using experimental studies.

Generation of Non-Nucleotide CD73 Inhibitors Using a Molecular Docking and 3D-QSAR Approach.

Radiotherapy and chemotherapy are conventional cancer treatments. Around 60% of all patients who are diagnosed with cancer receive radio- or chemotherapy in combination with surgery during their disease. Only a few patients respond to the blockage of immune checkpoints alone, or in combination therapy, because their tumours might not be immunogenic. Under these circumstances, an increasing level of extracellular adenosine via the activation of ecto-5'-nucleotidase (CD73) and consequent adenosine receptor signalling is a typical mechanism that tumours use to evade immune surveillance. CD73 is responsible for the conversion of adenosine monophosphate to adenosine. CD73 is overexpressed in various tumour types. Hence, targetting CD73's signalling is important for the reversal of adenosine-facilitated immune suppression. In this study, we selected a potent series of the non-nucleotide small molecule inhibitors of CD73. Molecular docking studies were performed in order to examine the binding mode of the inhibitors inside the active site of CD73 and 3D-QSAR was used to study the structure-activity relationship. The obtained CoMFA (q2 = 0.844, ONC = 5, r2 = 0.947) and CoMSIA (q2 = 0.804, ONC = 4, r2 = 0.954) models showed reasonable statistical values. The 3D-QSAR contour map analysis revealed useful structural characteristics that were needed to modify non-nucleotide small molecule inhibitors. We used the structural information from the overall docking and 3D-QSAR results to design new, potent CD73 non-nucleotide inhibitors. The newly designed CD73 inhibitors exhibited higher activity (predicted pIC50) than the most active compound of all of the derivatives that were selected for this study. Further experimental studies are needed in order to validate the new CD73 inhibitors.

Discovery of a Potent and Selective JNK3 Inhibitor with Neuroprotective Effect Against Amyloid ß-Induced Neurotoxicity in Primary Rat Neurons.

As members of the MAPK family, c-Jun-N-terminal kinases (JNKs) regulate the biological processes of apoptosis. In particular, the isoform JNK3 is expressed explicitly in the brain at high levels and is involved in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). In this study, we prepared a series of five 6-dihydroxy-1H-benzo[d]imidazoles as JNK3 inhibitors and found them have potential as neuroprotective agents. Following a previous lead scaffold, benzimidazole moiety was modified with various aryl groups and hydroxylation, and the resulting compounds exhibited JNK3 inhibitory activity with improved potency and selectivity. Out of 37 analogues synthesized, (S)-cyclopropyl(3-((4-(2-(2,3-dihydrobenzo[b][1,4]dioxin -6-yl)-5,6-dihydroxy-1H-benzo[d]imidazol-1-yl)pyrimidin-2-yl)amino) piperidin-1-yl)methanone (35b) demonstrated the highest JNK3 inhibition (IC50 = 9.7 nM), as well as neuroprotective effects against Aß-induced neuronal cell death. As a protein kinase inhibitor, it also showed excellent selectivity over other protein kinases including isoforms JNK1 (>1000 fold) and JNK2 (-10 fold).

Design and Synthesis of a Novel PLK1 Inhibitor Scaffold Using a Hybridized 3D-QSAR Model.

Polo-like kinase 1 (PLK1) plays an important role in cell cycle progression and proliferation in cancer cells. PLK1 also contributes to anticancer drug resistance and is a valuable target in anticancer therapeutics. To identify additional effective PLK1 inhibitors, we performed QSAR studies of two series of known PLK1 inhibitors and proposed a new structure based on a hybridized 3D-QSAR model. Given the hybridized 3D-QSAR models, we designed and synthesized 4-benzyloxy-1-(2-arylaminopyridin-4-yl)-1H-pyrazole-3-carboxamides, and we inspected its inhibitory activities to identify novel PLK1 inhibitors with decent potency and selectivity.

Discovery of 5-methyl-N-(2-arylquinazolin-7-yl)isoxazole-4-carboxamide analogues as highly selective FLT3 inhibitors.

A series of 4-arylamido 5-methylisoxazole derivatives with quinazoline core was designed and synthesised based on conformational rigidification of a previous type II FMS inhibitor. Most of quinazoline analogues displayed activity against FLT3 and FLT3-ITD. Compound 7d, 5-methyl-N-(2-(3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)phenyl)quinazolin-7-yl)isoxazole-4-carboxamide, exhibited the most potent inhibitory activity against FLT3 (IC50= 106 nM) with excellent selectivity profiles over 36 other protein kinases including cKit and FMS kinase. Compound 7d was also active in FLT-ITD, with an IC50 value of 301 nM, and other FLT3 mutants showing potential as an AML therapeutics.

Discovery of 3-alkyl-5-aryl-1-pyrimidyl-1H-pyrazole derivatives as a novel selective inhibitor scaffold of JNK3.

3-alkyl-5-aryl-1-pyrimidyl-1H-pyrazole derivatives were designed and synthesised as selective inhibitors of JNK3, a target for the treatment of neurodegenerative diseases. Following previous studies, we have designed JNK3 inhibitors to reduce the molecular weight and successfully identified a lead compound that exhibits equipotent activity towards JNK3. Kinase profiling results also showed high selectivity for JNK3 among 38 kinases. Among the derivatives, the IC50 value of 8a, (R)-2-(1-(2-((1-(cyclopropanecarbonyl)pyrrolidin-3-yl)amino)pyrimidin-4-yl)-5-(3,4-dichlorophenyl)-1H-pyrazol-3-yl)acetonitrile exhibited 227 nM, showing the highest inhibitory activity against JNK3.

Discovery of 1-Pyrimidinyl-2-Aryl-4,6-Dihydropyrrolo [3,4-d]Imidazole-5(1H)-Carboxamide as a Novel JNK Inhibitor.

We designed and synthesized 1-pyrimidinyl-2-aryl-4, 6-dihydropyrrolo [3,4-d] imidazole-5(1H)-carboxamide derivatives as selective inhibitors of c-Jun-N-terminal Kinase 3 (JNK3), a target for the treatment of neurodegenerative diseases. Based on the compounds found in previous studies, a novel scaffold was designed to improve pharmacokinetic characters and activity, and compound 18a, (R)-1-(2-((1-(cyclopropanecarbonyl)pyrrolidin-3-yl)amino)pyrimidin-4-yl)-2-(3,4-dichlorophenyl)-4,6-dihydro pyrrolo [3,4-d]imidazole-5(1H)-carboxamide, showed the highest IC50 value of 2.69 nM. Kinase profiling results also showed high selectivity for JNK3 among 38 kinases, having mild activity against JNK2, RIPK3, and GSK3ß, which also known to involve in neuronal apoptosis.

Design, synthesis, and in vitro evaluation of N-(3-(3-alkyl-1H-pyrazol-5-yl) phenyl)-aryl amide for selective RAF inhibition.

Notorious oncogenic BRAF V600E plays a significant role in the signal transduction of the MAPK pathway, which is involved in tumor growth, especially in melanoma. Much effort has been made to suppress BRAF V600E through small molecules like vemurafenib and dabrafenib, but the MAPK pathway remains active through paradoxical activation, where CRAF transmits the signal of the MAPK pathway either alone or along with BRAF V600E. Therefore, we designed and synthesized a new series of N-(3-(3-alkyl-1H-pyrazol-5-yl) phenyl)-aryl amide/urea analogues that showed potent inhibitory activities against BRAF V600E and CRAF. Compound 7c exhibited particularly superior selectivity toward BRAF V600E and CRAF over 30 other protein kinases, implying that this chemotype could be investigated as a BRAF paradox breaker. © 2019 Elsevier Ltd. All rights reserved.

Computer-aided design and synthesis of 3-carbonyl-5-phenyl-1H-pyrazole as highly selective and potent BRAFV600E and CRAF inhibitor.

BRAF belongs to the upstream portion of the MAPK pathway, which is involved in cell proliferation and survival. When mutations occur in BRAF, downstream MEK and ERK are phosphorylated irrespective of RAS, resulting in melanoma-like cancer. Over the years, small molecules targeting BRAFV600E have been discovered to be very effective melanoma drugs, but they are known to cause the BRAF paradox. Recently, it was shown that this paradox is caused by the heterodimer phenomenon of BRAF/CRAF. Here, we suggest one method by which paradoxical activation can be avoided by selectively inhibiting BRAFV600E and CRAF but not wild-type BRAF. From previous report of N-(3-(3-alkyl-1H-pyrazol-5-yl) phenyl) aryl amide as a selective inhibitor of BRAFV600E and CRAF, we present compounds that offer enhanced selectivity and efficacy with the aid of molecular modelling.

Conformational restriction of a type II FMS inhibitor leading to discovery of 5-methyl-N-(2-aryl-1H-benzo[d]imidazo-5-yl)isoxazole-4-carboxamide analogues as selective FLT3 inhibitors.

A series of 4-arylamido 5-methylisoxazole derivatives incorporating benzimidazole was designed and synthesised by conformational restriction of an in-house type II FMS inhibitor. Kinase profiling of one compound revealed interesting features, with increased inhibitory potency towards FLT3 and concomitant loss of potency towards FMS. Several benzimidazole derivatives 5a-5g and 6a-6c containing various hydrophobic moieties were synthesised, and their inhibitory activity against FLT3 was evaluated. Specifically, 5a, 5-methyl-N-(2-(3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-5-yl) isoxazole-4-carboxamide, exhibited the most potent inhibitory activity against FLT3 (IC50 = 495 nM), with excellent selectivity profiles.

Discovery of novel 4-aryl-thieno[1,4]diazepin-2-one derivatives targeting multiple protein kinases as anticancer agents.

A series of 4-aryl-thieno[1,4]diazepin-2-one were synthesized and evaluated for their antiproliferative activities against the A375P melanoma and U937 hematopoietic cell lines. Several compounds showed very potent antiproliferative activities toward both cell lines and the activities were better than that of sorafenib, the reference standard. Derivatives were made as amide (8a-8i, 9a-9m) and urea (10a-10d, 11a-11d) with diverse hydrophobic moieties. One of the most potent inhibitor 10d, 1-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-3-(4-(2-oxo-2,3-dihydro-1H-thieno [3,4-b][1,4]diazepin-4-yl)phenyl)urea was found to be very potent inhibitor of multi-protein kinases including FMS kinase (IC50 = 3.73 nM) and is a promising candidate for further development in therapeutics for cancer.

Novel scaffold evolution through combinatorial 3D-QSAR model studies of two types of JNK3 inhibitors.

JNK3 is an emerging target for neurodegenerative diseases including AD and PD, with histological selectivity. Specifically, in AD, JNK3 is the main protein kinase for APP phosphorylation, which is an important mechanism for Aß processing, and a biomarker of Alzheimer's disease. Therefore, targeting JNK3 is a reasonable strategy for drug discovery in neurodegenerative diseases. In order to find a novel scaffold for JNK3 inhibitors, we performed 3D-QSAR modeling studies with two different JNK3 inhibitor series. The CoMFA model was obtained with a q2 value of 0.806 and an r2 value of 0.850. Based on CoMFA and CoMSIA models, rational design was conducted and led to a novel scaffold, N-(thiophen-2-yl)-8H-pyrazolo[1,5-a]pyrido[1,2-c]pyrimidine-10-carboxamide.

Discovery of highly selective CRAF inhibitors, 3-carboxamido-2H-indazole-6-arylamide: In silico FBLD design, synthesis and evaluation.

The recent success of vemurafenib shows the importance of selective BRAF V600E inhibition in melanoma. However, paradoxical activation by structurally diverse ATP-competitive RAF kinase inhibitors strongly suggests that selective CRAF inhibitors, not BRAF inhibitors, would be ideal for some Ras mutation cancer treatment. In this respect, we approached designing selective CRAF inhibitors starting from in silico fragment screening and synthesized a 3-carboxamido-2H-indazole-6-arylamide scaffold. Most of the compounds showed potent antiproliferative activity against the WM3629 melanoma cell line and the most promising compound, compound 10d, was found to be a potent and selective CRAF inhibitor with an IC50 value of 38.6 nM, which shows greater than 270-fold selectivity over BRAF kinase (9.45 µM).

Synthesis of stereochemically diverse cyclic analogs of tubulysins.

The synthesis of tubulysin analogs containing stereochemically diverse cyclic Tuv moieties is described. A tetrahydropyranyl moiety was incorporated into the Tuv unit by enantioselective hetero Diels-Alder reactions of Danishefsky's diene and thiazole aldehyde. Four different stereoisomers of cyclic Tuv units were used as surrogates for the Tuv moiety. The synthesized stereochemically diverse simplified cyclic analogs were evaluated for the inhibition of tubulin polymerization.

Design, synthesis and biological evaluation of benzyl 2-(1H-imidazole-1-yl) pyrimidine analogues as selective and potent Raf inhibitors.

The synthesis of a novel series of (4-aminobenzyl/benzoyl)-1H-imidazol-1-yl pyrimidin-2-yl derivatives 9, 10, 18, 19 and their in vitro antiproliferative activities against the A375P human melanoma cell line and the U937 human leukemic monocyte lymphoma cell line are described. Potent antiproliferative effects were found from 9l, 9s and 10c; 10c was found to be a highly potent and selective BRAF V600E and CRAF inhibitor (IC50=38.3 nM and 8.79 nM).

Click approach to the discovery of 1,2,3-triazolylsalicylamides as potent Aurora kinase inhibitors.

A series of 1,2,3-triazolylsalicylamide derivatives has been developed from the antiproliferative agent 7 and was evaluated for their Aurora kinase inhibitory activity. The novel 1,2,3-triazolylsalicylamide scaffold could be readily assembled by Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition, allowing rapid access to the structurally diverse analogues. The synthesized 1,2,3-triazolylsalicylamide derivatives revealed a significant Aurora kinase inhibitory activity. In particular, 8g inhibited Aurora A with IC50 values of 0.37µM. The critical role of phenolic -OH in the binding was confirmed by a molecular modeling study.

Discovery of N-(5-amido-2-methylphenyl)-5-methylisoxazole-3-carboxamide as dual CSF-1R/c-Kit Inhibitors with improved stability and BBB permeability.

This study explores the potential of CSF-1R inhibitors as therapeutic agents for neurodegenerative diseases. CSF-1R, a receptor tyrosine kinase primarily expressed in macrophage lineages, plays a pivotal role in regulating various cellular processes. Recent research highlights the significance of CSF-1R inhibition in mitigating neuroinflammation, particularly in Alzheimer's disease, where microglial overactivation contributes to neurodegeneration. The research reveals a series of N-(5-amido-2-methylphenyl)-5-methylisoxazole-3-carboxamide CSF-1R inhibitors, where compounds 7d, 7e, and 9a exhibit outstanding inhibitory activities and selectivity, with IC50 values of 33, 31, and 64 nM, respectively. These most promising compounds in this series were profiled for cellular potency and subjected to in vitro pharmacokinetic profiling. These inhibitors exhibit minimal cytotoxicity, even at higher concentrations, and possess promising blood-brain barrier permeability, making them potential candidates for central nervous system diseases. The investigation into the in vitro ADME properties, including plasma and microsomal stability, reveals that these CSF-1R inhibitors maintain their structural integrity and plasma concentration. This resilience positions them for further development as therapeutic agents for neurodegenerative diseases.

Targeting the CSF-1/CSF-1R Axis: Exploring the Potential of CSF1R Inhibitors in Neurodegenerative Diseases.

We report herein the potential of colony-stimulating factor-1 receptor (CSF1R) inhibitors as therapeutic agents in neuroinflammatory diseases, with a focus on Alzheimer's disease (AD). Employing a carefully modified scaffold, N-(4-heterocycloalkyl-2-cycloalkylphenyl)-5-methylisoxazole-3-carboxamide, we identify highly selective and potent CSF1R inhibitors─7dri and 7dsi. Molecular docking studies shed light on the binding modes of these key compounds within the CSF1R binding site. Remarkably, kinome-wide selectivity assessment underscores the impressive specificity of 7dri for CSF-1R. Notably, 7dri emerges as a potent CSF-1R inhibitor with favorable cellular activity and minimal cytotoxicity among the synthesized compounds. Demonstrating efficacy in inhibiting CSF1R phosphorylation in microglial cells and successfully mitigating neuroinflammation in an in vivo LPS-induced model, 7dri establishes itself as a promising antineuroinflammatory agent.