ATP Site Research Articles

Nifuroxazide as JAK2 inhibitor: A binding mode proposal and Hel cell proliferation assay

Nifuroxazide has been employed as an anti-diarrheic agent since 1966, but in the last decade has brought to the research spotlight again due to its recently described antitumoral activity through the JAK2 inhibitory potential. Since 2008, more than 70 papers have been published about the issue and more are expected to the following years. Herein we discuss the findings of molecular modelling studies which were performed to elucidate the potential binding mode of this drug into the JAK2 ATP recognition site and also into the allosteric region near the catalytic site. Molecular modelling followed by dynamics simulations indicated the NFZ could bind at both sites, such as a Type II kinase inhibitor since residues from both ATP and modulatory site would exhibit contacts with the drug when in a stable complex. Synthesis of NFZ and its sulfur bioisosteric analogue GPQF-63 were performed and experimental assays against HEL cells indicate the potential of NFZ and, mainly of its analogue GPQF-63 in acting as inhibitors of cell growth. HEL-cells present the JAK2 V617F mutation which leads to an enhanced JAK/STAT pathway and they have never been tested by the NFZ activity before. A mechanistic approach was also performed and revealed that both compounds induce cell apoptosis.Taken together, both the theoretical and experimental approaches point out the N-acylhydrazones as good starting points in the search for JAK2 modulatory small molecules which could then, be studied as promising leads toward new alternatives to control the JAK-STAT pathway related pathologies. This is the first study, as far as we have known, to propose a potential binding mode for NFZ as well as reporting the activity of this drug against HEL cells, which are a usual cellular model to human erythroleukemia and other myeloproliferative diseases.

Biochemical Characterization of Recombinant UDPG-Dependent IAA Glucosyltransferase from Maize (Zea mays).

Here, we report a biochemical characterization of recombinant maize indole-3-acetyl-β-d-glucose (IAGlc) synthase which glucosylates indole-3-acetic acid (IAA) and thus abolishes its auxinic activity affecting plant hormonal homeostasis. Substrate specificity analysis revealed that IAA is a preferred substrate of IAGlc synthase; however, the enzyme can also glucosylate indole-3-butyric acid and indole-3-propionic acid with the relative activity of 66% and 49.7%, respectively. KM values determined for IAA and UDP glucose are 0.8 and 0.7 mM, respectively. 2,4-Dichlorophenoxyacetic acid is a competitive inhibitor of the synthase and causes a 1.5-fold decrease in the enzyme affinity towards IAA, with the Ki value determined as 117 μM, while IAA–Asp acts as an activator of the synthase. Two sugar-phosphate compounds, ATP and glucose-1-phosphate, have a unique effect on the enzyme by acting as activators at low concentrations and showing inhibitory effect at higher concentrations (above 0.6 and 4 mM for ATP and glucose-1-phosphate, respectively). Results of molecular docking revealed that both compounds can bind to the PSPG (plant secondary product glycosyltransferase) motif of IAGlc synthase; however, there are also different potential binding sites present in the enzyme. We postulate that IAGlc synthase may contain more than one binding site for ATP and glucose-1-phosphate as reflected in its activity modulation.

Selectivity and potency of natural product PIM kinase inhibitors identified by in silico docking

PIM3 (Proviral Integration site for Maloney murine leukemia virus kinase 3) is a proto-oncogene with serine/threonine kinase activity that prevents apoptosis, promotes cell survival, and stimulates protein translation. In addition, PIM3 functions in inflammation and immunity pathways. PIM3 inhibitors are being developed to treat cancer and inflammation-related disorders. Here we screen a 98,000 compound virtual library of natural products to identify those that are predicted to fit in the ATP site of PIM3. Since the structure of PIM3 has not been determined experimentally, we performed molecular structure prediction using the SWISS-MODEL tool to generate a PIM3 model structure for in silico screening. Compounds predicted to fit the ATP binding site of PIM3 were validated using biochemical assays, revealing activity against PIM3 for all eight candidates, with potencies mostly in the micromolar range. We tested several analogs of two validated candidates, the diosgenin glycoside dioscin and the biflavonoid hinokiflavone. Among five dioscin analogs, three exhibit similar potency against PIM3, and with some selectivity for PIM3 versus PIM1 and 2. Meanwhile, three of seven biflavonoid analogs exhibit sub-micromolar IC50 potency against PIM3, but with less selectivity for PIM3 versus PIM1 and 2.

Abstract PS17-59: Comparative study of breast tumors with differential expression of intracellular energy sensor adenosine monophosphate kinase

Abstract Adenosine monophosphate kinase (AMPK), encoded by mammalian gene PRKAA1 is a central energy sensor which acts as a metabolic switch and directs cellular response based on AMP/ATP ratio in cells. AMPK has been shown to be involved in tumorigenesis as part of metabolic reprogramming. Further, chronic metabolic disorders like obesity and insulin dependent diabetes are linked to increased cancer recurrence and poor prognosis. Interestingly, AMPK expression and activity are affected in both obesity and insulin dependent diabetes. Structurally, AMPK is a heterotrimeric protein with alpha (α), beta (β) and gamma (γ) subunits. AMPKα subunit is the catalytic subunit, AMPKβ is the scaffold subunit and AMPKγ subunit provides the competitive binding site for AMP or ATP. In case of nutrient/ energy deprivation, AMP/ATP cellular ratio increases, and AMPK is activated to suppress anabolic pathways and promote catabolic pathways. On the other hand, under excess nutrient, AMP/ATP cellular ratio is decreased, and AMPK function is impeded leading to promotion of anabolic pathways over catabolic pathways. In metabolic disorders like obesity and insulin dependent diabetes, AMPK expression and function are suppressed. Current literature and our research indicate that AMPK can be the bridge between understanding the links between metabolic disorders and cancer. In the current study, we explored the clinical and molecular differences between breast tumors expressing relatively low and high AMPK, indicated as AMPKlow and AMPKhi breast tumors. The cBioPortal (http://cbioportal.org) for Cancer Genomics platform was used for this study. The data mining was focused on METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) Breast cancer study, which reports clinical and molecular data for over 2500 breast cancer patients. Among the 2500 breast cancer tumors, AMPKhi and AMPKlow expressing breast tumors were 310 and 311 in numbers, respectively. The AMPKhi and AMPKlow expressing breast tumors in RNA-seq dataset were queried using cBioPortal embedded SQL feature as PRKAA1: EXP>1 and PRKAA1: EXP<-1, respectively. Comparative analysis of AMPKlow and AMPKhi expressing breast tumors was performed for clinical impact and mRNA expression profiles. The mRNA data was further analyzed for integrated networks using Reactome. The query for AMPKhi and AMPKlow resulted in non-overlapping cohorts of breast tumors. Analysis revealed that significantly higher percentage (65.45%) of AMPKlow breast tumors were positive for Estrogen receptor with high proliferative index. Overall patient survival (months) is significantly more in patients with AMPKhi breast tumors compared to patients with AMPKlow breast tumors. Microarray mRNA expression data revealed unique profile of mRNA expression in both the cohorts. The top 10 most significant differentially expressed genes are KLHL2, COX6C, SLC29A3, SPRY2, RAMP1, ANXA3, HAGH, TLCD3B, KLHL24 and ACVR2A. Pathway enrichment analysis with the top 100 differentially expressed mRNA using Reactome suggested significant enrichment of Wnt, Notch, MAPK and ESR signaling pathways. The molecular profile of AMPKhi and AMPKlow expressing breast tumors are unique and have different mRNA expression signatures. In addition, cancer stem cell homeostasis signaling pathways seem to be critical in the molecular functioning of the two groups. Clinical and molecular data suggests that further investigations into the two molecular subtypes of breast cancer can provide useful insight towards the links between metabolic disorders and cancer. Citation Format: Roxanne L. Lopez, Dorothy Dean, Sushmita Nandy. Comparative study of breast tumors with differential expression of intracellular energy sensor adenosine monophosphate kinase [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-59.

Amentoflavone Ameliorates Memory Deficits and Abnormal Autophagy in Aβ25-35-Induced Mice by mTOR Signaling.

Alzheimer's disease (AD) is a neurodegenerative disease in which autophagy plays a crucial role. Amentoflavone is a flavonoid obtained from various plants and has been shown to have AD-resistant neuroprotective effects. This study investigated the role of amentoflavone on memory impairment and abnormal autophagy in amyloid-β25-35 (Aβ25-35)-induced mice to elucidate the mechanisms by which it exerts neuroprotective effects. In this experiment, the AD mouse model was established by intracerebroventricular (ICV) injection of Aβ25-35 peptides, and amentoflavone was administered orally for 4 weeks. Behavioral changes in mice and pathological changes in the hippocampus were observed, and levels of inflammation, oxidative stress, and autophagy in the brain were detected and analyzed. PC-12 and APPswe-N2a cells were used in vitro to further investigate the effect of amentoflavone on the level of intracellular autophagy. Molecular docking was used to determine the action sites of amentoflavone. The results showed that amentoflavone improved memory function, eased anxiety symptoms in Aβ25-35-induced mice, and reduced atrophic degeneration of neurons in the hippocampus. Moreover, amentoflavone lessened the oxidative stress and inflammation in the brains of mice. Through in vivo and in vitro experiments, we found that amentoflavone may enhance autophagy, by way of binding to the ATP site of the mTOR protein kinase domain. Amentoflavone not only interacted with mTOR, but also improved Aβ25-35-induced cognitive dysfunction in mice by enhancing autophagy, attenuating levels of inflammation and oxidative stress, and reducing apoptosis in brain cells.

Structural insights into the inhibition of bacterial RecA by naphthalene polysulfonated compounds.

SummaryAs a promising target for alternative antimicrobials, bacterial recombinase A (RecA) protein has attracted much attention for its roles in antibiotic-driven SOS response and mutagenesis. Naphthalene polysulfonated compounds (NPS) such as suramin have previously been explored as antibiotic adjuvants targeting RecA, although the underlying structural bases for RecA-ligand interactions remain obscure. Based on our in silico predictions and documented activity of NPS in vitro, we conclude that the analyzed NPS likely interact with Tyr103 (Y103) and other key residues in the ATPase activity center (pocket A). For validation, we generated recombinant RecA proteins (wild-type versus Y103 mutant) to determine the binding affinities for RecA protein interactions with suramin and underexamined NPS in isothermal titration calorimetry. The corresponding dissociation constants (Kd) ranged from 11.5 to 18.8 μM, and Y103 was experimentally shown to be critical to RecA-NPS interactions.

Positioning of an unprecedented spiro[5.5]undeca ring system into kinase inhibitor space

In-house 1,5-oxaza spiroquinone 1, with spiro[5.5]undeca ring system, was announced as an unprecedented anti-inflammatory scaffold through chemistry-oriented synthesis (ChOS), a chemocentric approach. Herein, we studied how to best position the spiro[5.5]undeca ring system in kinase inhibitor space. Notably, late-stage modification of the scaffold 1 into compounds 2a-r enhanced kinase-likeness of the scaffold 1. The improvement could be depicted with (1) selectivity with target shift (from JNK-1 into GSK-3) and (2) potency (> 20-fold). In addition, ATP independent IC50 of compound 2j suggested a unique binding mode of this scaffold between ATP site and substrate site, which was explained by docking based optimal site selection and molecular dynamic simulations of the optimal binding site. Despite the shift of kinase profiling, the anti-inflammatory activity of compounds 2a-r could be retained in hyperactivated microglial cells.

Efficacy and Safety Results from ASCEMBL, a Multicenter, Open-Label, Phase 3 Study of Asciminib, a First-in-Class STAMP Inhibitor, vs Bosutinib (BOS) in Patients (Pts) with Chronic Myeloid Leukemia in Chronic Phase (CML-CP) Previously Treated with ≥2 Tyrosine Kinase Inhibitors (TKIs

Efficacy and Safety Results from ASCEMBL, a Multicenter, Open-Label, Phase 3 Study of Asciminib, a First-in-Class STAMP Inhibitor, vs Bosutinib (BOS) in Patients (Pts) with Chronic Myeloid Leukemia in Chronic Phase (CML-CP) Previously Treated with ≥2 Tyrosine Kinase Inhibitors (TKIs

Solution structure of the Hop TPR2A domain and investigation of target druggability by NMR, biochemical and in silico approaches

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an important role in tumour biology by promoting the stabilisation and activity of oncogenic ‘client’ proteins. Inhibition of Hsp90 by small-molecule drugs, acting via its ATP hydrolysis site, has shown promise as a molecularly targeted cancer therapy. Owing to the importance of Hop and other tetratricopeptide repeat (TPR)-containing cochaperones in regulating Hsp90 activity, the Hsp90-TPR domain interface is an alternative site for inhibitors, which could result in effects distinct from ATP site binders. The TPR binding site of Hsp90 cochaperones includes a shallow, positively charged groove that poses a significant challenge for druggability. Herein, we report the apo, solution-state structure of Hop TPR2A which enables this target for NMR-based screening approaches. We have designed prototype TPR ligands that mimic key native ‘carboxylate clamp’ interactions between Hsp90 and its TPR cochaperones and show that they block binding between Hop TPR2A and the Hsp90 C-terminal MEEVD peptide. We confirm direct TPR-binding of these ligands by mapping 1H–15N HSQC chemical shift perturbations to our new NMR structure. Our work provides a novel structure, a thorough assessment of druggability and robust screening approaches that may offer a potential route, albeit difficult, to address the chemically challenging nature of the Hop TPR2A target, with relevance to other TPR domain interactors.

Dimerization Induced by C-Terminal 14-3-3 Binding Is Sufficient for BRAF Kinase Activation.

The Ras-RAF-MEK-ERK signaling axis, commonly mutated in human cancers, is highly regulated to prevent aberrant signaling in healthy cells. One of the pathway modulators, 14-3-3, a constitutive dimer, induces RAF dimerization and activation by binding to a phosphorylated motif C-terminal to the RAF kinase domain. Recent work has suggested that a C-terminal "DTS" region in BRAF is necessary for this 14-3-3-mediated activation. We show that the catalytic activity and ATP binding affinity of the BRAF:14-3-3 complex is insensitive to the presence or absence of the DTS, while the ATP sites of both BRAF molecules are identical and available for binding. We also present a crystal structure of the apo BRAF:14-3-3 complex showing that the DTS is not required to attain the catalytically active conformation of BRAF. Rather, BRAF dimerization induced by 14-3-3 is the key step in activation, allowing the active BRAF:14-3-3 tetramer to achieve catalytic activity comparable to the constitutively active oncogenic BRAF V600E mutant.

Structure-guided optimization and mechanistic study of a class of quinazolinone-threonine hybrids as antibacterial ThrRS inhibitors.

Aminoacyl-tRNA synthetases (aaRSs) are an attractive class of antibacterial drug targets due to their essential roles in protein translation. While most traditional aaRS inhibitors target the binding pockets of substrate amino acids and/or ATP, we recently developed a class of novel tRNA-amino acid dual-site inhibitors including inhibitor 3 ((2S,3R)-2-amino-N-((E)-4-(6,7-dichloro-4-oxoquinazolin-3(4H)-yl)but-2-en-1-yl)-3-hydroxybutanamide) against threonyl-tRNA synthetase (ThrRS). Here, the binding modes and structure-activity relationships (SARs) of these inhibitors were analyzed by the crystal structures of Salmonella enterica ThrRS (SeThrRS) in complex with three of them. Based on the cocrystal structures, twelve quinazolinone-threonine hybrids were designed and synthesized, and their affinities, enzymatic inhibitory activities, and cellular potencies were evaluated. The best derivative 8g achieved a Kd value of 0.40μM, an IC50 value of 0.50μM against SeThrRS and MIC values of 16-32μg/mL against the tested bacterial strains. The cocrystal structure of the SeThrRS-8g complex revealed that 8g induced a bended conformation for Met332 by forming hydrophobic interactions, which better mimicked the binding of tRNAThr to ThrRS. Moreover, the inhibitory potency of 8g was less impaired than a reported ATP competitive inhibitor at high concentrations of ATP, supporting our hypothesis that tRNA site inhibitors are likely superior to ATP site inhibitors invivo, where ATP typically reaches millimolar concentrations.

Identification of benzimidazole containing 4H–chromen–4–one derivative as potential MAP kinase inhibitors by in-silico approaches

MAP kinase is one of the important targets in the treatment of osteoarthritis, inflammation and cancer. Many p38 inhibitors with diverse chemical structures and modes of protein interaction have been designed on the basis of their ability to compete with ATP site or allosteric site for binding to MAP Kinase. This study involves the molecular docking of benzimidazoles containing 4H–chrome–4–one derivatives as potent inhibitors of the MAP kinase enzyme. The compounds were computationally designed and optimized with the molecular docking to investigate the interactions between the target compounds and the amino acid residues of the MAP Kinase. The inhibitory activities against human MAP kinase enzyme were investigated by molecular docking using the Autodock and discovery studio software. All the designed compounds were shown good binding energy when compared with the binging energies of standard drug Imatinib (anti-cancer). Among all the designed compounds, compound D1 and D6 have higher binding energy values when compared to standard drug. Here we also studied the molecular properties of designed compound using Molinspiration software. Further, we planned to synthesis these benzimidazole derivatives and screen for in-vitro and in-vivo of anti-cancer activity.

Corrigendum: Addressing the Glycine-Rich Loop of Protein Kinases by a Multi-Facetted Interaction Network: Inhibition of PKA and a PKB Mimic.

Protein kinases continue to be hot targets in drug discovery research, as they are involved in many essential cellular processes and their deregulation can lead to a variety of diseases. A series of 32 enantiomerically pure inhibitors was synthesized and tested towards protein kinase A (PKA) and protein kinase B mimic PKAB3 (PKA triple mutant). The ligands bind to the hinge region, ribose pocket, and glycine-rich loop at the ATP site. Biological assays showed high potency against PKA, with Ki values in the low nanomolar range. The investigation demonstrates the significance of targeting the often neglected glycine-rich loop for gaining high binding potency. X-ray co-crystal structures revealed a multi-facetted network of ligand-loop interactions for the tightest binders, involving orthogonal dipolar contacts, sulfur and other dispersive contacts, amide-π stacking, and H-bonding to organofluorine, besides efficient water replacement. The network was analyzed in a computational approach.

Structural Insights into Pseudokinase Domains of Receptor Tyrosine Kinases

Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.

A Photo-clickable ATP-Mimetic Reveals Nucleotide Interactors in the Membrane Proteome

ATP is an important energy metabolite and allosteric signal in health and disease. ATP-interacting proteins, such as P2 receptors, control inflammation, cell death, migration, and wound healing. However, identification of allosteric ATP sites remains challenging, and our current inventory of ATP-controlled pathways is likely incomplete. Here, we develop and verify mipATP as a minimally invasive photoaffinity probe for ATP-interacting proteins. Its N6 functionalization allows target enrichment by UV crosslinking and conjugation to reporter tags by "click" chemistry. The additions are compact, allowing mipATP to completely retain the calcium signaling responses of native ATP invitro and invivo. mipATP specifically enriched for known nucleotide binders in A549 cell lysates and membrane fractions. In addition, it retrieved unannotated ATP interactors, such as the FAS receptor, CD44, and various SLC transporters. Thus, mipATP is a promising tool to identify allosteric ATP sites in the proteome.

Metadynamics as a Postprocessing Method for Virtual Screening with Application to the Pseudokinase Domain of JAK2.

With standard scoring methods, top-ranked compounds from virtual screening by docking often turn out to be inactive. For this reason, metadynamics, a method used to sample rare events, was studied to further evaluate docking poses with the aim of reducing false positives. Specifically, virtual screening was performed with Glide SP to seek potential molecules to bind to the ATP site in the pseudokinase domain of JAK2 kinase, and promising compounds were selected from the top-ranked 1000 based on visualization. Rescoring with Glide XP, GOLD, and MM/GBSA was unable to differentiate well between active and inactive compounds. Metadynamics was then used to gauge the relative binding affinity from the required time or the potential of mean force needed to dissociate the ligand from the bound complex. With consideration of previously known binders of varying affinities, metadynamics was able to differentiate between the most active compounds and inactive or weakly active ones, and it could identify correctly most of the selected virtual screening compounds as false positives. Thus, metadynamics has the potential to be a viable postprocessing method for virtual screening, minimizing the expense of buying or synthesizing inactive compounds.

Treating Cancer by Spindle Assembly Checkpoint Abrogation: Discovery of Two Clinical Candidates, BAY 1161909 and BAY 1217389, Targeting MPS1 Kinase.

Inhibition of monopolar spindle 1 (MPS1) kinase represents a novel approach to cancer treatment: instead of arresting the cell cycle in tumor cells, cells are driven into mitosis irrespective of DNA damage and unattached/misattached chromosomes, resulting in aneuploidy and cell death. Starting points for our optimization efforts with the goal to identify MPS1 inhibitors were two HTS hits from the distinct chemical series "triazolopyridines" and "imidazopyrazines". The major initial issue of the triazolopyridine series was the moderate potency of the HTS hits. The imidazopyrazine series displayed more than 10-fold higher potencies; however, in the early project phase, this series suffered from poor metabolic stability. Here, we outline the evolution of the two hit series to clinical candidates BAY 1161909 and BAY 1217389 and reveal how both clinical candidates bind to the ATP site of MPS1 kinase, while addressing different pockets utilizing different binding interactions, along with their synthesis and preclinical characterization in selected in vivo efficacy models.

Comparative analysis of the dual EGFR-DNA targeting and growth inhibitory properties of 6-mono-alkylamino- and 6,6-dialkylaminoquinazoline-based type II combi-molecules

Over the past decade, we described a novel tumour targeted approach that sought to design “combi-molecules” to hit two distinct targets in tumour cells. Here, to generate small combi-molecules with strong DNA damaging potential while retaining EGFR inhibitory potency, we developed the first synthetic strategy to access the 6-N, N-disubstituted quinazoline scaffold and designed JS61 to possess a nitrogen mustard function directly attached to the 6-position of the quinazoline ring. We compared its biological activity with that of structures containing either a hemi mustard or a non-alkylating substituent. Surprisingly, the results showed that JS61, while capable of inducing strong DNA damage, exhibited moderate EGFR inhibitory potency. In contrast, “combi-molecules” with no bulky substituent at the N-6 position (e.g. ZR2002 and JS84) showed stronger EGFR and growth inhibitory potency than JS61 in a panel of lung cancer cells. To rationalize these results, X-ray crystallography and molecular modeling studies were undertaken, and the data obtained indicated that bulkiness of the 6-N,N-disubstituted moieties hinder its binding to the ATP site and affects binding reversibility.

Unexpected CK2β-antagonistic functionality of bisubstrate inhibitors targeting protein kinase CK2.

Protein kinase CK2, a heterotetrameric holoenzyme composed of two catalytic chains (CK2α) attached to a homodimer of regulatory subunits (CK2β), is a target for drug development for cancer therapy. Here, we describe the tetraiodobenzimidazole derivative ARC-3140, a bisubstrate inhibitor addressing the ATP site and the substrate-binding site of CK2 with extraordinary affinity (Ki=84pM). In a crystal structure of ARC-3140 in complex with CK2α, three copies of the inhibitor are visible, one of them at the CK2β interface of CK2α. Subsequent interaction studies based on microscale thermophoresis and fluorescence anisotropy changes revealed a significant impact of ARC-3140 and of its tetrabromo equivalent ARC-1502 on the CK2α/CK2β interaction. A structural inspection revealed that ARC-3140, unlike CK2β antagonists described so far, interferes with both sub-interfaces of the bipartite CK2α/CK2β interaction. Thus, ARC-3140 is a lead for the further development of highly effective compounds perturbating the quaternary structure of the CK2α2β2 holoenzyme.

Druggable exosites of the human kino-pocketome

Small molecules binding at any of the multiple regulatory sites on the molecular surface of a protein kinase may stabilize or disrupt the corresponding interaction, leading to consequent modulation of the kinase cellular activity. As such, each of these sites represents a potential drug target. Even targeting sites outside the immediate ATP site, the so-called exosites, may cause desirable biological effects through an allosteric mechanism. Targeting exosites can alleviate adverse effects and toxicity that is common when ATP-site compounds bind promiscuously to many other types of kinases. In this study we have identified, catalogued, and annotated all potentially druggable exosites on the protein kinase domains within the existing structural human kinome. We then priority-ranked these exosites by those most amenable to drug design. In order to identify pockets that are either consistent across the kinome, or unique and specific to a particular structure, we have also implemented a normalized representation of all pockets, and displayed these graphically. Finally, we have built a database and designed a web-based interface for users interested in accessing the 3-dimensional representations of these pockets. We envision this information will assist drug discovery efforts searching for untargeted binding pockets in the human kinome.