ATP Binding Site Of Kinase Research Articles

Targeted blocking of EGFR and GLUT1 by compound H reveals a new strategy for treatment of triple-negative breast cancer and nasopharyngeal carcinoma

BackgroundCytoplasmic epidermal growth factor receptor (EGFR) is overexpressed in both nasopharyngeal carcinoma (NPC) and triple-negative breast cancer (TNBC), while clinical outcome and prognosis vary greatly among patients treated with gefitinib, and all patients eventually develop resistance to this agent. Therefore, we propose a new concept for synthesizing multitarget compounds and reveal new therapeutic strategies for NPC and TNBC expressing EGFR. MethodsCompound H was synthesized in our previous study. Molecular docking, and cell thermal shift assays (CETSAs) and drug affinity responsive target stability(DARTS) were used to confirm the binding of compound H to EGFR and GLUT1. Methylthiazolyldiphenyl-tetrazolium bromide(MTT), annexin V-PE assays, mitochondrial membrane potential (MMP) assays, and animal models were used to evaluate the inhibitory effect of compound H on TNBC cell lines. Energy metabolism tests, Western blotting, and immunofluorescence staining were performed to evaluate the synergistic effects on EGFR- and glucose transporter type 1(GLUT1)-mediated energy metabolism. ResultsCompound H can simultaneously act on the EGFR tyrosine kinase ATP-binding site and inhibit GLUT1-mediated energy metabolism, resulting in reductions in ATP, MMP, intra-cellular lactic acid, and EGFR nuclear transfer. The anti-tumor activity of compound H is significantly superior to the combination of GLUT1 inhibitor BAY876 and EGFR inhibitor gefitinib. Compound H has remarkable anti-proliferative effects on TNBC MDA-MB231 cells, and importantly, no obvious toxicity effects of compound H were found in vivo. ConclusionsSynergistic effects of inhibition of EGFR- and GLUT1-mediated energy metabolism by compound H may present a new strategy for the treatment of TNBC and NPC.

Catalyst-free late-stage functionalization to assemble α-acyloxyenamide electrophiles for selectively profiling conserved lysine residues

Covalent probes coupled with chemical proteomics represent a powerful method for investigating small molecule and protein interactions. However, the creation of a reactive warhead within various ligands to form covalent probes has been a major obstacle. Herein, we report a convenient and robust process to assemble a unique electrophile, an α-acyloxyenamide, through a one-step late-stage coupling reaction. This procedure demonstrates remarkable tolerance towards other functional groups and facilitates ligand-directed labeling in proteins of interest. The reactive group has been successfully incorporated into a clinical drug targeting the EGFR L858R mutant, erlotinib, and a pan-kinase inhibitor. The resulting probes have been shown to be able to covalently engage a lysine residue proximal to the ATP-binding pocket of the EGFR L858R mutant. A series of active sites, and Mg2+, ATP-binding sites of kinases, such as K33 of CDK1, CDK2, CDK5 were detected. This is the first report of engaging these conserved catalytic lysine residues in kinases with covalent inhibition. Further application of this methodology to natural products has demonstrated its success in profiling ligandable conserved lysine residues in whole proteome. These findings offer insights for the development of new targeted covalent inhibitors (TCIs).

Repurposing of FDA Approved Drugs and Neuropep peptides as Anticancer Agents Against ErbB1 and ErbB2.

ErbB1 and ErbB2 are the most important biological targets in cancer drug discovery and development of dual inhibitors for the cancer therapy. FDA approved drugs and Neuropep peptides were used to fit into the ATP binding site of the tyrosine kinases; ErbB1 and ErbB2 proteins. Cytoscape, iGEMDOCK, HPEPDOCK and DataWarrior softwares were used to study the role of these agents as anticancer drugs. Eleven FDA approved drugs and eleven Neuropep peptides showed the strongest 2D interactions and significant binding energy with the proteins. Invitro MTT anticancer assay revealed that, the test compounds, peptide YSFGL and doxorubicin showed significant IC50 value (µM) of 26.417±0.660 and 7.675±0.278 respectively which are compared with the lapatinib standard IC50 value (µM) of 2.380±0.357 against A549 cells and IC50 value (µM) of 39.047±0.770 and 8.313±0.435 respectively which are compared with the lapatinib standard IC50 value (µM) of 3.026±0.180 against MDA-MB-231 cells.

Utilization of Supervised Machine Learning to Understand Kinase Inhibitor Toxophore Profiles.

There have been more than 70 FDA-approved drugs to target the ATP binding site of kinases, mainly in the field of oncology. These compounds are usually developed to target specific kinases, but in practice, most of these drugs are multi-kinase inhibitors that leverage the conserved nature of the ATP pocket across multiple kinases to increase their clinical efficacy. To utilize kinase inhibitors in targeted therapy and outside of oncology, a narrower kinome profile and an understanding of the toxicity profile is imperative. This is essential when considering treating chronic diseases with kinase targets, including neurodegeneration and inflammation. This will require the exploration of inhibitor chemical space and an in-depth understanding of off-target interactions. We have developed an early pipeline toxicity screening platform that uses supervised machine learning (ML) to classify test compounds' cell stress phenotypes relative to a training set of on-market and withdrawn drugs. Here, we apply it to better understand the toxophores of some literature kinase inhibitor scaffolds, looking specifically at a series of 4-anilinoquinoline and 4-anilinoquinazoline model libraries.

Molecular Recognition of FDA-Approved Small Molecule Protein Kinase Drugs in Protein Kinases.

Protein kinases are key enzymes that catalyze the covalent phosphorylation of substrates via the transfer of the γ-phosphate of ATP, playing a crucial role in cellular proliferation, differentiation, and various cell regulatory processes. Due to their pivotal cellular role, the aberrant function of kinases has been associated with cancers and many other diseases. Consequently, competitive inhibition of the ATP binding site of protein kinases has emerged as an effective means of curing these diseases. Decades of intense development of protein kinase inhibitors (PKIs) resulted in 71 FDA-approved PKI drugs that target dozens of protein kinases for the treatment of various diseases. How do FDA-approved protein kinase inhibitor PKI drugs compete with ATP in their own binding pocket? This is the central question we attempt to address in this work. Based on modes of non-bonded interactions and their calculated interaction strengths by means of the advanced double hybrid DFT method B2PLYP, the molecular recognition of PKI drugs in the ATP-binding pockets was systematically analyzed. It was found that (1) all the FDA-approved PKI drugs studied here form one or more hydrogen bond(s) with the backbone amide N, O atoms in the hinge region of the ATP binding site, mimicking the adenine base; (2) all the FDA-approved PKI drugs feature two or more aromatic rings. The latter reach far and deep into the hydrophobic regions I and II, forming multiple CH-π interactions with aliphatic residues L(3), V(11), A(15), V(36), G(51), L(77) and π-π stacking interactions with aromatic residues F(47) and F(82), but ATP itself does not utilize these regions extensively; (3) all FDA-approved PKI drugs studied here have one thing in common, i.e., they frequently formed non-bonded interactions with a total of 12 residues L(3),V(11), A(15), K(17), E(24),V(36),T(45), F(47), G(51), L(77), D(81) and F(82) in the ATP binding. Many of those 12 commonly involved residues are highly conserved residues with important structural and catalytic functional roles. K(17) and E(24) are the two highly conserved residues crucial for the catalytic function of kinases. D(81) and F(82) belong to the DFG motif; T(45) was dubbed the gate keeper residue. F(47) is located on the hinge region and G(51) sits on the linker that connects the hinge to the αD-helix. It is this targeting of highly conserved residues in protein kinases that led to promiscuous PKI drugs that lack selectivity. Although the formation of hydrogen bond(s) with the backbone of the hinge gives PKI drugs the added binding affinity and the much-needed directionality, selectivity is sacrificed. That is why so many FDA-approved PKI drugs are known to have multiple targets. Moreover, off-target-mediated toxicity caused by a lack of selectivity was one of the major challenges facing the PKI drug discovery community. This work suggests a road map for future PKI drug design, i.e., targeting non-conserved residues in the ATP binding pocket to gain better selectivity so as to avoid off-target-mediated toxicity.

Deciphering the kinome of Theileria annulata for identification of drug targets and anti-theilerial drug

Tropical theileriosis is one of the major parasitic diseases of ruminants. It is a tick-borne disease caused by an apicomplexan parasite, Theileria annulata. In the infected cells, these parasites induce phenotypes similar to cancerous cells. Among the most critical changes induced by the parasite are immortalization, hyperproliferation, and dissemination. The proliferative signal in the T. annulata transformed cells are provided by different kinases such as mitogen-activated protein kinases, SRC family kinases, casein kinase-2, and phosphatidylinositide 3-kinase. Deregulation of protein kinases in cancer is also well known. Targeting protein kinases in a cancerous cell is one of the most common methods in cancer therapy. Here, we revisited the kinome of T. annulata and studied its evolutionary relationship with other piroplasms. This analysis revealed that T. annulata kinome encodes 54 protein kinases. Based on our analysis, 12 of these 54 kinases were identified for the first time in the T. annulata proteome. Three protein kinases, TA16570, TA09820, and TA07000, had <40% identity with Bos taurus and >40% identity with the previously identified potential drug targets present in the Therapeutic Target Database (TTD). These 3 proteins were predicted to be essential for the survival of T. annulata and were selected as drug targets. Screening these drug targets in the Protein Kinase Inhibitor Database (PKID) led to shortlisting of 5 drugs. Only Dabrafenib, out of these 5 drugs, could bind to the ATP binding site (in silico) of the Calcium Dependent Protein Kinase 3 of both T. annulata and Theileria parva. Further, dabrafenib could inhibit the proliferation of T. annulata infected bovine leucocytes in 6 days proliferation assay with the IC50 value of 0.66 µM. Also, this drug did not have a cytotoxic effect on bovine peripheral blood mononuclear cells. In summary, the analysis of T. annulata kinome led to the identification of dabrafenib as a potential drug for treating theileriosis.

One of the active ingredients in Paeoniae Radix Alba functions as JAK1 inhibitor in rheumatoid arthritis.

Objective: We aimed to explore and verify the mechanism underlying the action of the active ingredients of Paeoniae Radix Alba (PRA) in the treatment of rheumatoid arthritis (RA). Methods: The protein targets of PRA’s six active ingredients and RA were identified. Then, the intersection of the two groups was studied. The drug–target network was constructed, visualized, and analyzed by Cytoscape software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed to analyze these genes. Furthermore, we validated our predictions of the potential targets through a docking study. Finally, the anti-inflammatory effect of Palbinone (PB), one of the active ingredients of PRA, was tested by conducting in vitro and in vivo studies. Results: Six active ingredients of PRA were identified, and 103 overlapping genes were discovered. Functional enrichment analysis indicated that the genes are mostly enriched in IL-17 signaling pathway, Th17 cell differentiation, and the FoxO, ErbB, and TNF signaling pathways. 10 hub genes and two gene cluster modules were identified by Cytoscape. Molecular docking analysis proved that PB was able to bind to the ATP binding site of Janus kinase (JAK)1, thereby acting as a potential inhibitor of JAK1. In vitro and in vivo studies demonstrated that PB exerts its anti-inflammatory role via the inhibition of JAK1. Conclusion: We constructed a multitarget pharmacological network of PRA in RA treatment. PB, one of the active compounds of PRA, was demonstrated to be a promising inhibitor of JAK1.

Identification of Novel GSK-3β Hits Using Competitive Biophysical Assays

Glycogen synthase kinase 3 beta (GSK-3β) is an evolutionarily conserved serine-threonine kinase dysregulated in numerous pathologies, such as Alzheimer’s disease and cancer. Even though GSK-3β is a validated pharmacological target most of its inhibitors have two main limitations: the lack of selectivity due to the high homology that characterizes the ATP binding site of most kinases, and the toxicity that emerges from GSK-3β complete inhibition which translates into the impairment of the plethora of pathways GSK-3β is involved in. Starting from a 1D 19F NMR fragment screening, we set up several biophysical assays for the identification of GSK-3β inhibitors capable of binding protein hotspots other than the ATP binding pocket or to the ATP binding pocket, but with an affinity able of competing with a reference binder. A phosphorylation activity assay on a panel of several kinases provided selectivity data that were further rationalized and corroborated by structural information on GSK-3β in complex with the hit compounds. In this study, we identified promising fragments, inhibitors of GSK-3β, while proposing an alternative screening workflow that allows facing the flaws that characterize the most common GSK-3β inhibitors through the identification of selective inhibitors and/or inhibitors able to modulate GSK-3β activity without leading to its complete inhibition.

The Druggability of the ATP Binding Site of Glycogen Phosphorylase Kinase Probed by Coumarin Analogues

The Druggability of the ATP Binding Site of Glycogen Phosphorylase Kinase Probed by Coumarin Analogues

The druggability of the ATP binding site of glycogen phosphorylase kinase probed by coumarin analogues

Glycogen phosphorylase kinase (PhK) converts by phosphorylation, the inactive glycogen phosphorylase (GPb) into active GPa in the glycogenolytic pathway. It is a complex enzyme comprising of the catalytic (γ) and three regulatory subunits (α, β, δ) forming a hexadecamer with stoichiometry (αβγδ)4. Several studies have indicated PhK as a promising target for the development of antihyperglycemics as its inhibition blocks glycogenolysis in liver and a potential therapeutic target for cancer against pathological angiogenesis and tumor progression. The identification of compounds that inhibit the kinase through their direct binding to its catalytic site is an effective approach to identify bioactive molecules of therapeutic significance. Towards this, the structure of the N-terminal kinase domain (residues 1–298) of the catalytic γ subunit of PhK (PhKγtrnc) has been determined by X-ray crystallography while staurosporine and indirubin analogues have been characterized as potent inhibitors targeting the ATP binding site. In this study, a series of 38 synthetic analogues of naturally occurring coumarins were screened for inhibition of PhKγtrnc, in vitro, using a photometric assay. The IC50 values of the two most potent compounds were determined for PhKγtrnc and the pharmacologically relevant target, human liver isoform (PHKG2A). Their cellular efficacy and toxicity in HepG2 cells were further assessed ex vivo. Docking experiments and the structural comparison with previously described inhibitors reveal the binding mode of the coumarin scaffold at a no hinge region of the ATP site of PhK and the role of a conserved β3-Lys in binding. The experimental findings provide structural insights with implications to the kinase targeting and drug design.

Chemical Modification of Phage-Displayed Helix-Loop-Helix Peptides to Construct Kinase-Focused Libraries.

Conformationally constrained peptides hold promise as molecular tools in chemical biology and as a new modality in drug discovery. The construction and screening of a target‐focused library could be a promising approach for the generation of de novo ligands or inhibitors against target proteins. Here, we have prepared a protein kinase‐focused library by chemically modifying helix‐loop‐helix (HLH) peptides displayed on phage and subsequently tethered to adenosine. The library was screened against aurora kinase A (AurA). The selected HLH peptide Bip‐3 retained the α‐helical structure and bound to AurA with a K D value of 13.7 μM. Bip‐3 and the adenosine‐tethered peptide Bip‐3‐Adc provided IC50 values of 103 μM and 7.7 μM, respectively, suggesting that Bip‐3‐Adc bivalently inhibited AurA. In addition, the selectivity of Bip‐3‐Adc to several protein kinases was tested, and was highest against AurA. These results demonstrate that chemical modification can enable the construction of a kinase‐focused library of phage‐displayed HLH peptides.

Fragment-based lead discovery to identify novel inhibitors that target the ATP binding site of pyruvate dehydrogenase kinases

A fragment-based lead discovery approach was applied to Pyruvate Dehydrogenase Kinases (PDHKs) to discover inhibitors against the ATP binding site with novel chemotypes. X-ray fragment screening toward PDHK4 provided a fragment hit 1 with a characteristic interaction in a deep pocket of the ATP binding site. While known inhibitors utilize several water molecules in a deep pocket to form water-mediated hydrogen bond interactions, the fragment hit binds deeper in the pocket with a hydrophobic group. Displacement of a remaining water molecule in the pocket led to the identification of lead compound 7 with a notable improvement in inhibition potency. This lead compound possessed high ligand efficiency (LE) and showed decent selectivity profile. Two additional lead compounds 10 and 13 with new scaffolds with tricyclic and bicyclic cores were generated by merging structural information of another fragment hit 2. The characteristic interaction of these novel inhibitors in a deep pocket provides new structural insights about PDHKs ATP binding site and opens a novel direction for the development of PDHKs inhibitors.

Aromatic Rings as Molecular Determinants for the Molecular Recognition of Protein Kinase Inhibitors.

Protein kinases are key enzymes in many signal transduction pathways, and play a crucial role in cellular proliferation, differentiation, and various cell regulatory processes. However, aberrant function of kinases has been associated with cancers and many other diseases. Consequently, competitive inhibition of the ATP binding site of protein kinases has emerged as an effective means of curing these diseases. Over the past three decades, thousands of protein kinase inhibitors (PKIs) with varying molecular frames have been developed. Large-scale data mining of the Protein Data Bank resulted in a database of 2139 non-redundant high-resolution X-ray crystal structures of PKIs bound to protein kinases. This provided us with a unique opportunity to study molecular determinants for the molecular recognition of PKIs. A chemoinformatic analysis of 2139 PKIs resulted in findings that PKIs are “flat” molecules with high aromatic ring counts and low fractions of sp3 carbon. All but one PKI possessed one or more aromatic rings. More importantly, it was found that the average weighted hydrogen bond count is inversely proportional to the number of aromatic rings. Based on this linear relationship, we put forward the exchange rule of hydrogen bonding interactions and non-bonded π-interactions. Specifically, a loss of binding affinity caused by a decrease in hydrogen bonding interactions is compensated by a gain in binding affinity acquired by an increase in aromatic ring-originated non-bonded interactions (i.e., π–π stacking interactions, CH–π interactions, cation–π interactions, etc.), and vice versa. The very existence of this inverse relationship strongly suggests that both hydrogen bonding and aromatic ring-originated non-bonded interactions are responsible for the molecular recognition of PKIs. As an illustration, two representative PKI–kinase complexes were employed to examine the relative importance of different modes of non-bonded interactions for the molecular recognition of PKIs. For this purpose, two FDA-approved PKI drugs, ibrutinib and lenvatinib, were chosen. The binding pockets of both PKIs were thoroughly examined to identify all non-bonded intermolecular interactions. Subsequently, the strengths of interaction energies between ibrutinib and its interacting residues in tyrosine kinase BTK were quantified by means of the double hybrid DFT method B2PLYP. The resulting energetics for the binding of ibrutinib in tyrosine kinase BTK showed that CH–π interactions and π–π stacking interactions between aromatic rings of the drug and hydrophobic residues in its binding pocket dominate the binding interactions. Thus, this work establishes that, in addition to hydrogen bonding, aromatic rings function as important molecular determinants for the molecular recognition of PKIs. In conclusion, our findings support the following pharmacophore model for ATP-competitive kinase inhibitors: a small molecule features a scaffold of one or more aromatic rings which is linked with one or more hydrophilic functional groups. The former has the structural role of acting as a scaffold and the functional role of participating in aromatic ring-originated non-bonded interactions with multiple hydrophobic regions in the ATP binding pocket of kinases. The latter ensure water solubility and form hydrogen bonds with the hinge region and other hydrophilic residues of the ATP binding pocket.

A New Series of Antileukemic Agents: Design, Synthesis, In Vitro and In Silico Evaluation of Thiazole-Based ABL1 Kinase Inhibitors.

After the approval of imatinib, more than 25 antitumor agents targeting kinases have been approved, and several promising candidates are at various stages of clinical evaluation. Due to the importance of the thiazole scaffold in targeted anticancer drug discovery, the goal of this work is to identify new thiazolyl hydrazones as potent ABL1 kinase inhibitors for the management of Chronic Myeloid Leukemia (CML). New thiazolyl hydrazones (2a-p) were synthesized and investigated for their cytotoxic effects on the K562 CML cell line. Compounds 2h, 2j and 2l showed potent anticancer activity against K562 cell line. The cytotoxic effects of these compounds on other leukemia (HL-60, MT-2 and Jurkat) and HeLa human cervical carcinoma cell lines were also investigated. Furthermore, their cytotoxic effects on Mitogen-Activated Peripheral Blood Mononuclear Cells (MA-PBMCs) were evaluated to determine their selectivity. Due to its selective and potent anticancer activity, compound 2j was benchmarked for its apoptosis-inducing potential on K562 cell line and inhibitory effects on eight different Tyrosine Kinases (TKs), including ABL1 kinase. In order to investigate the binding mode of compound 2j into the ATP binding site of ABL1 kinase (PDB: 1IEP), a molecular docking study was conducted using MOE 2018.01 program. The QikProp module of Schrödinger's Molecular modelling package was used to predict the pharmacokinetic properties of compounds 2a-p. 4-(4-(Methylsulfonyl)phenyl)-2-[2-((1,3-benzodioxol-4-yl)methylene)hydrazinyl]thiazole (2j) showed antiproliferative activity against K562 cell line with an IC50 value of 8.87±1.93 μM similar to imatinib (IC50= 6.84±1.11μM). Compound 2j was found to be more effective than imatinib on HL-60, Jurkat and MT-2 cells. Compound 2j also showed cytotoxic activity against HeLa cell line similar to imatinib. The higher selectivity index value of compound 2j than imatinib indicated that its antiproliferative activity was selective. Compound 2j also induced apoptosis in K562 cell line more than imatinib. Among eight TKs, compound 2j showed the strongest inhibitory activity against ABL1 kinase enzyme (IC50= 5.37±1.17μM). According to molecular docking studies, compound 2j exhibited high affinity to the ATP binding site of ABL1 kinase, forming significant intermolecular interactions. On the basis of in silico studies, this compound did not violate Lipinski's rule of five and Jorgensen's rule of three. Compound 2j stands out as a potential orally bioavailable ABL1 kinase inhibitor for the treatment of CML.

Abstract 4040: A cell-based screening assay to identify novel kinase inhibitors

Abstract The majority of the FDA-approved kinase inhibitors target ATP binding sites of kinases. Despite their activity against tumorigenesis, these therapies are often non-specific and are susceptible to resistance mechanisms. One method of enhancing the selectivity of these compounds and overcoming potential chemoresistance is to better identify novel compounds that are highly specific. In this study, we introduce one such strategy by identifying kinase inhibitors that target kinase-substrate interactions. c-MYC overexpression and deregulation has been implicated in the development and progression of a number of malignancies. We have previously identified that OCT4 binds to the MYC promoter/enhancer region to transcriptionally activate c-MYC. Novel OCT4 binding sites in the c-MYC promoter region were identified, and kinases (DNA-PKcs for the current study) that mediate the phosphorylation of specific amino acid residues of OCT4 were determined. We then identified the domains of DNA-PKcs that are critical to bind and phosphorylate OCT4. The goal of the current project is to develop an assay to identify the compounds that interferes the interaction between OCT4 and DNA-PKcs and to validate the identified "hits." In order to selectively inhibit the interaction of protein-kinase related to c-MYC transcriptional activation, we co-expressed the fragments of OCT4 required for c-MYC expression and the fragments DNA-PKcs required to activate OCT4 to 1) confirm the interaction between DNA-PKcs and OCT4, and 2) develop a cell-based assay that can screen novel compounds that prevent this interaction. After confirming the kinase-substrate interaction, we co-transduced the crucial fragments of DNA-PKcs and OCT4 tagged with luminescent probes in HEK 293FT cells. We then screened a chemical library of compounds to identify hits that inhibited luminescence, and thus our kinase-substrate protein interaction. In our screening assay, 67 compounds were found to inhibit the luminescence interaction between the tagged OCT4 and DNA-PKcs protein fragments. After the initial screening, we conducted a two-step hit validation for the 67 hits identified that consisted of: 1) the decrease in pOCT4S93 expression along with the reduction in c-MYC expression: 2) and the inhibition of binding between DNA-PKcs and OCT4 by co-immunoprecipitation. Additional experiments to validate the hits include kinase activity assays and immunoblotting to determine if the screened hits inhibited DNA-PKcs kinase activity. In conclusion, our screening assay and validation experiments identified 7 novel compounds that demonstrated: 1) impairment of DNA-PKcs-mediated phosphorylation of OCT4; and 2) inhibition of the DNA-PKcs-OCT4 kinase-substrate interaction. These newly identified compounds have the potential to oppose aberrant c-MYC expression and warrant further investigation in order to determine their activity in high c-MYC-expressing malignancies. Citation Format: Ismail S. Mohiuddin, Sung J. Wei, Inhyoung Yang, Gloria Martinez, Hwangeui Cho, Min H. Kang. A cell-based screening assay to identify novel kinase inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4040.

Design, Synthesis, Molecular Docking, and Anticancer Evaluation of Pyrazole Linked Pyrazoline Derivatives with Carbothioamide Tail as EGFR Kinase Inhibitors.

The Epidermal Growth Factor Receptor (known as EGFR) induces cell differentiation and proliferation upon activation through the binding of its ligands. Since EGFR is thought to be involved in the development of cancer, the identification of new target inhibitors is the most viable approach, which recently gained momentum as a potential anticancer therapy. To assess various pyrazole linked pyrazoline derivatives with carbothioamide for EGFR kinase inhibitory as well as anti-proliferative activity against human cancer cell lines viz. A549 (non-small cell lung tumor), MCF-7 (breast cancer cell line), SiHa (cancerous tissues of the cervix uteri), and HCT-116 (colon cancer cell line). In vitro EGFR kinase assay, in vitro MTT assay, Lactate dehydrogenase release, nuclear staining (DAPI), and flow cytometry cell analysis. Compounds 6h and 6j inhibited EGFR kinase at concentrations of 1.66μM and 1.9μM, respectively. Furthermore, compounds 6h and 6j showed the most potent anti-proliferative results against the A549 KRAS mutation cell line (IC50 = 9.3 & 10.2μM). Through DAPI staining and phase contrast microscopy, it was established that compounds 6h and 6j also induced apoptotic activity in A549 cells. This activity was further confirmed by FACS using Annexin-V-FITC and Propidium Iodide (PI) labeling. Molecular docking studies performed on 6h and 6j suggested that the compounds can bind to the hinge region of ATP binding site of EGFR tyrosine kinase in a similar pose as that of the standard drug gefitinib. The potential anticancer activity of compounds 6h and 6j was confirmed and need further exploration in cancer cell lines of different tissue origin and signaling pathways, as well as in animal models of cancer development.

Investigating Protein-Protein Allosteric Network using Current-Flow Scheme.

Protein dynamic network analysis provides a powerful tool for investigating protein allosteric regulation. We recently developed a current-flow betweenness scheme for protein network analysis and demonstrated that this method, that is, using current-flow betweenness as edge weights, is more appropriate and more robust for investigating the signal transmission between two predefined protein residues or domains as compared with direct usage of correlation scores as edge weights. Here we seek to expand the current-flow scheme to study allosteric regulations involving protein-protein binding. Specifically, we investigated three gain-of-function mutations located at the binding interface of ALK2 (also known as ACVR1) kinase and its inhibitory protein FKBP12. We first searched for the optimal smoothing function for contact network construction and then calculated the subnetwork between FKBP12 protein and the kinase ATP binding site using current-flow betweenness. By comparing the networks between the wild-type and three mutants, we have identified statistically significant changes in the protein-protein networks that are common among all three mutants that allosterically shift the kinase toward a catalytically competent configuration. © 2019 Wiley Periodicals, Inc.

Identification of a New Inhibitor That Stabilizes Interleukin-2-Inducible T-Cell Kinase in Its Inactive Conformation

Interleukin-2-inducible T-cell kinase (ITK) plays an important role in T-cell signaling and is considered a promising drug target. As the ATP binding sites of protein kinases are highly conserved, the design of selective kinase inhibitors remains a challenge. Targeting inactive kinase conformations can address the issue of kinase inhibitor selectivity. It is important for selectivity considerations to identify compounds that stabilize inactive conformations from the primary screen hits. Here we screened a library of 390,000 compounds with an ADP-Glo assay using dephosphorylated ITK. After a surface plasmon resonance (SPR) assay was used to filter out promiscuous inhibitors, 105 hits were confirmed. Next, we used a fluorescent biosensor to enable the detection of conformational changes to identify inactive conformation inhibitors. A single-cysteine-substituted ITK mutant was labeled with acrylodan, and fluorescence emission was monitored. Using a fluorescent biosensor assay, we identified 34 inactive conformation inhibitors from SPR hits. Among them, one compound was bound to a site other than the ATP pocket and exhibited excellent selectivity against a kinase panel. Overall, (1) biochemical screening using dephosphorylated kinase, (2) hit confirmation by SPR assay, and (3) fluorescent biosensor assay that can distinguish inactive compounds provide a useful platform and offer opportunities to identify selective kinase inhibitors.

Targeting Dynamic ATP-Binding Site Features Allows Discrimination between Highly Homologous Protein Kinases.

ATP-competitive inhibitors that demonstrate exquisite selectivity for specific members of the human kinome have been developed. Despite this success, the identification of highly selective inhibitors is still very challenging, and it is often not possible to rationally engineer selectivity between the ATP-binding sites of kinases, especially among closely related family members. Src-family kinases (SFKs) are a highly homologous family of eight multidomain, nonreceptor tyrosine kinases that play general and specialized roles in numerous cellular processes. The high sequence and functional similarities between SFK members make it hard to rationalize how selectivity can be gained with inhibitors that target the ATP-binding site. Here, we describe the development of a series of inhibitors that are highly selective for the ATP-binding sites of the SFKs Lyn and Hck over other SFKs. By biochemically characterizing how these selective ATP-competitive inhibitors allosterically influence the global conformation of SFKs, we demonstrate that they most likely interact with a binding pocket created by the movement of the conformationally flexible helix αC in the ATP-binding site. With a series of sequence swap experiments, we show that sensitivity to this class of selective inhibitors is due to the identity of residues that control the conformational flexibility of helix αC rather than any specific ATP-binding site interactions. Thus, the ATP-binding sites of highly homologous kinases can be discriminated by targeting heterogeneity within conformationally flexible regions.

Rethinking to riluzole mechanism of action: the molecular link among protein kinase CK1δ activity, TDP-43 phosphorylation, and amyotrophic lateral sclerosis pharmacological treatment.

Rethinking to riluzole mechanism of action: the molecular link among protein kinase CK1δ activity, TDP-43 phosphorylation, and amyotrophic lateral sclerosis pharmacological treatment.