Kinase Crystal Structure Research Articles

Synthesis, anticancer evaluation, molecular docking and ADME study of novel pyrido[4ʹ,3ʹ:3,4]pyrazolo[1,5-a]pyrimidines as potential tropomyosin receptor kinase A (TrKA) inhibitors

The starting compound 3-amino-1,7-dihydro-4H-pyrazolo[4,3-c]pyridine-4,6(5H)-dione (1) is reacted with each of diketone and β-ketoester, forming pyridopyrazolo[1,5-a]pyrimidines 4a,b and 14a,b, respectively. The compounds 4 and 14 reacted with each of aromatic aldehyde and arenediazonium salt to give the respective arylidenes and arylhydrazo derivatives, respectively. The structure of the new products was established using spectroscopic techniques. The cytotoxic activity of selected targets was tested in vitro against three cancer cell lines MCF7, HepG2 and HCT116. The data obtained from enzymatic assays of TrKA indicated that compounds 7b and 16c have the strongest inhibitory effects on TrKA with IC50 = 0.064 ± 0.0037 μg/ml and IC50 = 0.047 ± 0.0027 μg/ml, respectively, compared to the standard drug Larotrectinib with IC50 = 0.034 ± 0.0021 μg/ml for the HepG2 cancer cell line. In cell cycle analysis, compounds 7b, 15b, 16a and 16c caused the greatest arrest in cell cycle at the G2/M phase. In addition, compound 15b has a higher apoptosis-inducing effect (36.72%) than compounds 7b (34.70%), 16a (21.14) and 16c (26.54%). Compounds 7b, 16a and 16c were shown fit tightly into the active site of the TrKA kinase crystal structure (PDB: 5H3Q). Also, ADME study was performed on some selected potent anticancer compounds described in this study.

Drugging the Undruggable: How Isoquinolines and PKA Initiated the Era of Designed Protein Kinase Inhibitor Therapeutics.

In 1984, Japanese researchers led by the biochemist Hiroyoshi Hidaka described the first synthetic protein kinase inhibitors based on an isoquinoline sulfonamide structure (Hidaka et al. Biochemistry, 1984 Oct 9; 23(21): 5036-41. doi: 10.1021/bi00316a032). These led to the first protein kinase inhibitor approved for medical use (fasudil), an inhibitor of the AGC subfamily Rho kinase. With potencies strong enough to compete against endogenous ATP, the isoquinoline compounds established the druggability of the ATP binding site. Crystal structures of their protein kinase complexes, including with cAMP-dependent protein kinase (PKA), showed interactions that, on the one hand, could mimic ATP but, on the other hand, could be optimized for high potency binding, kinase selectivity, and diversification away from adenosine. They also showed the flexibility of the glycine-rich loop, and PKA became a major prototype for crystallographic and nuclear magnetic resonance (NMR) studies of protein kinase mechanism and dynamic activity control. Since fasudil, more than 70 kinase inhibitors have been approved for clinical use, involving efforts that progressively have introduced new paradigms of data-driven drug discovery. Publicly available data alone comprise over 5000 protein kinase crystal structures and hundreds of thousands of binding data. Now, new methods, including artificial intelligence techniques and expansion of protein kinase targeting approaches, together with the expiration of patent protection for optimized inhibitor scaffolds, promise even greater advances in drug discovery. Looking back to the time of the first isoquinoline hinge binders brings the current state-of-the-art into stark contrast. Appropriately for this Perspective article, many of the milestone papers during this time were published in Biochemistry (now ACS Biochemistry).

Kinases and pseudokinases online symposium

The Biochemical Society’s Kinases and pseudokinases online symposium took place on 26 May, with 110 participants joining from around the globe. The event was convened to celebrate the 30th anniversary of the publication of the first ever protein kinase crystal structure by Professor Susan Taylor. Professor Taylor joined us as a keynote speaker, along with Professor Stefan Knapp from Frankfurt. Professor Taylor presented a tour de force covering her career, explaining how structural and functional analyses of kinases, including neglected PKA isoforms within the ‘dark kinome’, are advancing knowledge of the molecular basis of signal transduction. Professor Knapp discussed new insights into technologies, tools and challenges in studying pseudokinase structure and function.The remainder of the program focused on 25 emerging researchers, who presented short and flash talks, with contributions from the USA, Australia, Europe, India and Japan. PhD students, post-docs and early career faculty gave engaging presentations spanning microbial, plant, yeast and mammalian kinases and pseudokinases, impinging on host defence, cell death, oncogenesis, chemical biology and beyond. These ‘taster’ presentations now set the scene for what promises to be an outstanding 88th Harden Conference on kinases and pseudokinases, scheduled for May 2022 in Warwickshire, UK. Our thanks go to our sponsors, the Biochemical Journal and SVI Phosphocellulose, for supporting our speaker prizes, which were awarded to Dr Florentine Rutaganira (UC Berkeley, USA) and Dr Chris Horne (WEHI, Australia).

Reduction of Feedback Inhibition in Homoserine Kinase (ThrB) of Corynebacterium glutamicum Enhances l-Threonine Biosynthesis.

l-Threonine is an important supplement in the food industry. It is currently produced through fermentation of Escherichia coli but requires additional purification steps to remove E. coli endotoxin. To avoid these steps, it is desirable to use Corynebacterium glutamicum, a microorganism generally regarded as safe. Engineering of C. glutamicum to increase production of l-threonine has mainly focused on gene regulation as well as l-threonine export or carbon flux depletion. In this study, we focus on the negative feedback inhibition produced by l-threonine on the enzyme homoserine kinase (ThrB). Although l-threonine binds to allosteric sites of aspartate kinase (LysC) and homoserine dehydrogenase (Hom), serving as a noncompetitive inhibitor, it acts as a competitive inhibitor on ThrB. This is problematic when attempting to engineer enzymes that are nonresponsive to increasing cellular concentrations of l-threonine. Using primary structure alignment as well as analysis of the Methanocaldococcus jannaschii ThrB (MjaThrB) active site in complex with l-threonine (inhibitor of ThrB) and l-homoserine (substrate of ThrB), a conserved active-site alanine residue (A20) in C. glutamicum ThrB (CglThrB) was predicted to be important for differential interactions with l-threonine and l-homoserine. Through site-directed mutagenesis, we show that one variant of C. glutamicum ThrB, CglThrB-A20G, retains wild-type enzymatic activity, with dramatically decreased feedback inhibition by l-threonine. Additionally, by solving the first Corynebacterium X-ray crystal structure of homoserine kinase, we can confirm that the changes in l-threonine affinity to the CglThrB-A20G active site derive from loss of van der Waals interactions.

Structural and developmental expression of Ss-riok-2, an RIO protein kinase encoding gene of Strongyloides stercoralis

RIO kinases are essential atypical protein kinases in diverse prokaryotic and eukaryotic organisms, playing significant roles in yeast and humans. However, little is known about their functions in parasitic nematodes. In the present study, we have isolated and characterized the full-length cDNA, gDNA and a putative promoter of a RIOK-2 protein kinase (Ss-RIOK-2) encoding gene (Ss-riok-2) from Strongyloides stercoralis, a medically important parasitic nematode (Order Rhabditida). A three-dimensional structure (3D) model of Ss-RIOK-2 was generated using the Chaetomium thermophilum RIOK-2 protein kinase (Ct-RIOK-2) crystal structure 4GYG as a template. A docking study revealed some critical sites for ATP binding and metal binding. The putative promoter of Ss-riok-2 contains a number of conserved elements. RNAseq analysis revealed the highest levels of the Ss-riok-2 transcript in free-living females and parasitic females. To identify anatomical patterns of Ss-riok-2 expression in S. stercoralis, we observed expression patterns of a transgene construct encoding green fluorescent protein under the Ss-riok-2 promoter in post free-living S. stercoralis. Expression driven by this promoter predominated in intestinal cells. This study demonstrates significant advancement in molecular and cellular biological study of S. stercoralis and of parasitic nematodes generally, and provides a foundation for further functional genomic studies.

Classifying kinase conformations using a machine learning approach

BackgroundSignaling proteins such as protein kinases adopt a diverse array of conformations to respond to regulatory signals in signaling pathways. Perhaps the most fundamental conformational change of a kinase is the transition between active and inactive states, and defining the conformational features associated with kinase activation is critical for selectively targeting abnormally regulated kinases in diseases. While manual examination of crystal structures have led to the identification of key structural features associated with kinase activation, the large number of kinase crystal structures (~3,500) and extensive conformational diversity displayed by the protein kinase superfamily poses unique challenges in fully defining the conformational features associated with kinase activation. Although some computational approaches have been proposed, they are typically based on a small subset of crystal structures using measurements biased towards the active site geometry.ResultsWe utilize an unbiased informatics based machine learning approach to classify all eukaryotic protein kinase conformations deposited in the PDB. We show that the orientation of the activation segment, measured by φ, ψ, χ1, and pseudo-dihedral angles more accurately classify kinase crystal conformations than existing methods. We show that the formation of the K-E salt bridge is statistically dependent upon the activation segment orientation and identify evolutionary differences between the activation segment conformation of tyrosine and serine/threonine kinases. We provide evidence that our method can identify conformational changes associated with the binding of allosteric regulatory proteins, and show that the greatest variation in inactive structures comes from kinase group and family specific side chain orientations.ConclusionWe have provided the first comprehensive machine learning based classification of protein kinase active/inactive conformations, taking into account more structures and measurements than any previous classification effort. Further, our unbiased classification of inactive structures reveals residues associated with kinase functional specificity. To enable classification of new crystal structures, we have made our classifier publicly accessible through a stand-alone program housed at https://github.com/esbg/kinconform [DOI:10.5281/zenodo.249090].

The juxtamembrane region of TrkA kinase is critical for inhibitor selectivity

Although numerous crystal structures for protein kinases have been reported, many include only the kinase domain but not the juxtamembrane (JM) region, a critical activity-controlling segment of receptor tyrosine kinases (RTKs). In this study, we determined the X-ray crystal structure of the tropomyosin receptor kinase (Trk) A selective inhibitor A1 complexed with the TrkA kinase domain and the JM region. This structure revealed that the unique inhibitor-binding pocket created by a novel JM configuration yields significant potency and high selectivity against TrkB and TrkC. Moreover, we validated the importance of the JM region for the potency of A1 using in vitro assays. The introduction of moieties that interact with the JM region will be one of the most effective strategies for producing highly selective RTK inhibitors.

NMR Experiments on Wild-Type and Mutant Fibroblast Growth Factor Receptor Kinases Reveal Conformational Dynamics Associated with Enzyme Activation

The kinase domain of receptor tyrosine kinases (RTKs) is responsible for phosphorylating intracellular substrates upon hormone binding to the extracellular ectodomain. The resulting signal cascade is essential for several biological processes in human biology, including development and metabolism. Mutations in the kinase domain of RTKs can enhance the intrinsic activity of the enzyme and over-stimulate the resulting signal cascade leading to a wide variety of cancers and growth defects. Although a plethora of kinase crystal structures have been resolved, the dynamics associated with pathogenic mutations remain poorly understood. Here we analyzed the dynamics of pathogenic mutations within the fibroblast growth factor receptor kinase domain (FGFRK) as probed using Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments. Previous work showed that mutations at a conserved lysine residue in the activation loop bypassed the need for activation loop phosphorylation by shifting the population of the kinase from an autoinhibited state toward an active conformation. Interestingly, the degree of active state directly correlated with the kinase activity both in vitro and in human disease. The most prominent gain-of-function mutant was a lysine to glutamic acid substitution, which was found to stabilize the active conformation of the activation loop without the need for tyrosine phosphorylation (i.e., normal activation). The observed dispersions acquired on these pathogenic kinases using the CPMG experiments reveal conformational dynamics of the kinase in switching between active and inactive conformations and thus enabled us along with kinase crystal structures to identify an allosteric pathway spanning from the activation loop to the kinase hinge. These data agree well with the structural differences observed between kinases harboring different activating mutations, thus explaining how intrinsically activating mutations propagate their effects through the kinase.

Discovery of Akt kinase inhibitors through structure-based virtual screening and their evaluation as potential anticancer agents.

Akt acts as a pivotal regulator in the PI3K/Akt signaling pathway and represents a potential drug target for cancer therapy. To search for new inhibitors of Akt kinase, we performed a structure-based virtual screening using the DOCK 4.0 program and the X-ray crystal structure of human Akt kinase. From the virtual screening, 48 compounds were selected and subjected to the Akt kinase inhibition assay. Twenty-six of the test compounds showed more potent inhibitory effects on Akt kinase than the reference compound, H-89. These 26 compounds were further evaluated for their cytotoxicity against HCT-116 human colon cancer cells and HEK-293 normal human embryonic kidney cells. Twelve compounds were found to display more potent or comparable cytotoxic activity compared to compound H-89 against HCT-116 colon cancer cells. The best results were obtained with Compounds a46 and a48 having IC50 values (for HCT-116) of 11.1 and 9.5 µM, respectively, and selectivity indices (IC50 for HEK-293/IC50 for HCT-116) of 12.5 and 16.1, respectively. Through structure-based virtual screening and biological evaluations, we have successfully identified several new Akt inhibitors that displayed cytotoxic activity against HCT-116 human colon cancer cells. Especially, Compounds a46 and a48 may serve as useful lead compounds for further development of new anticancer agents.

Large-scale docking approaches to the kinome

Docking nowadays is a widely used tool and has successfully and repeatedly been applied to identify hits with new chemical scaffolds [1]. Despite its successful applications, still suffers mainly form its scoring functions, which tend to be optimized for speed to the disadvantage of accuracy. Because of that, successfully enriches active molecules compared to inactive ones, however the false positive rate in runs is still very high. Several attempts have been made to improve by applying consensus scoring approaches using multiple structures or softwares or by rescoring compounds using normalization procedures [2-4]. At the same time, the number of large datasets of activity data derived from high-throughput screening methods has strongly increased during the last years, especially for kinases, allowing re-evaluation of these techniques [5-7]. We herein carried out a large-scale approach using 650 different kinase crystal structures. To the best of our knowledge, this is the largest number of targets docked to at the same time. Subsequently, different normalization techniques were re-evaluated using the experimental activity data as target function. Furthermore, we investigated the similarity of the kinase structures within the docking universe and correlated these similarities with other similarity metrices.

Conformational Stability of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Dictates Its Substrate Selectivity

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) converts inositol 1,3,4,5,6-pentakisphosphate(IP5) to inositol hexakisphosphate (IP6). IPK1 shares structural similarity with protein kinases and is suspected to employ a similar mechanism of activation. Previous studies revealed roles for the 1- and 3-phosphates of IP5 in IPK1 activation and revealed that the N-lobe of IPK1 is unstable in the absence of inositol phosphate (IP). Here, we demonstrate the link between IPK1 substrate specificity and the stability of its N-lobe. Limited proteolysis of IPK1 revealed that N-lobe stability is dependent on the presence of the 1-phosphate of the substrate, whereas overall stability of IPK1 was increased in ternary complexes with nucleotide and IPs possessing 1- and 3-phosphates that engage the N-lobe of IPK1. Thus, the 1- and 3-phosphates possess dual roles in both IPK1 activation and IPK1 stability. To test whether kinase stability directly contributed to substrate selectivity of the kinase, we engineered IPK1 mutants with disulfide bonds that artificially stabilized the N-lobe in an IP-independent manner thereby mimicking its substrate-bound state in the absence of IP. IPK1 E82C/S142C exhibited a DTT-sensitive 5-fold increase in kcat for 3,4,5,6-inositol tetrakisphosphate (3,4,5,6-IP4) as compared with wild-type IPK1. The crystal structure of the IPK1 E82C/S142C mutant confirmed the presence of the disulfide bond and revealed a small shift in the N-lobe. Finally, we determined that IPK1 E82C/S142C is substantially more stable than wild-type IPK1 under nonreducing conditions, revealing that increased stability of IPK1 E82C/S142C correlates with changes in substrate specificity by allowing IPs lacking the stabilizing 1-phosphate to be used. Taken together, our results show that IPK1 substrate selection is linked to the ability of each potential substrate to stabilize IPK1.

Roles of Phosphate Recognition in Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Substrate Binding and Activation

Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) to yield a group of small signaling molecules involved in diverse cellular processes. IPK1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) phosphorylates inositol 1,3,4,5,6-pentakisphosphate to inositol 1,2,3,4,5,6-hexakisphosphate; however, the mechanism of IP recognition employed by IPK1 is currently unresolved. We demonstrated previously that IPK1 possesses an unstable N-terminal lobe in the absence of IP, which led us to propose that the phosphate profile of the IP was linked to stabilization of IPK1. Here, we describe a systematic study to determine the roles of the 1-, 3-, 5-, and 6-phosphate groups of inositol 1,3,4,5,6-pentakisphosphate in IP binding and IPK1 activation. The 5- and 6-phosphate groups were the most important for IP binding to IPK1, and the 1- and 3-phosphate groups were more important for IPK1 activation than the others. Moreover, we demonstrate that there are three critical residues (Arg-130, Lys-170, and Lys-411) necessary for IPK1 activity. Arg-130 is the only substrate-binding N-terminal lobe residue that can render IPK1 inactive; its 1-phosphate is critical for full IPK1 activity and for stabilization of the active conformation of IPK1. Taken together, our results support the model for recognition of the IP substrate by IPK1 in which (i) the 4-, 5-, and 6-phosphates are initially recognized by the C-terminal lobe, and subsequently, (ii) the interaction between the 1-phosphate and Arg-130 stabilizes the N-terminal lobe and activates IPK1. This model of IP recognition, believed to be unique among IPKs, could be exploited for selective inhibition of IPK1 in future studies that investigate the role of higher IPs.

In silico design: Extended molecular dynamic simulations of a new series of dually acting inhibitors against EGFR and HER2

Based on the hit structures that have been identified in our previous studies against EGFR and HER2, new potential inhibitors that share the same scaffold of the hit structures are designed and screened in silico. Insights into understanding the potential inhibitory effect of the new inhibitors against both EGFR and HER2 receptors is obtained using extended molecular dynamics (MD) simulations and different scoring techniques. The binding mechanisms and dynamics are detailed with respect to two approved inhibitors against EGFR (lapatinib) and HER2 (SYR127063). The best scoring inhibitor (T9) is chosen for additional in silico investigation against both the wild-type and T790M mutant strain of EGFR and the wild-type HER2. The results reveal that certain substitution patterns increase the stability and assure stronger binding and higher H-bond occupancy of the conserved water molecule that is commonly observed with kinase crystal structures. Furthermore, the new inhibitor (T9) forms stable interactions with the mutant strain as a direct consequence of the enhanced ability to form additional hydrogen bonding interactions with binding site residues.

Abstract SY20-02: Discovering tool compounds and chemistry leads by highly accurate kinase virtual screening.

Abstract SY20-02: Discovering tool compounds and chemistry leads by highly accurate kinase virtual screening.

Transitions to catalytically inactive conformations in EGFR kinase

The epidermal growth factor receptor (EGFR) is a key protein in cellular signaling, and its kinase domain (EGFR kinase) is an intensely pursued target of small-molecule drugs. Although both catalytically active and inactive conformations of EGFR kinase have been resolved crystallographically, experimental characterization of the transitions between these conformations remains difficult. Using unbiased, all-atom molecular dynamics simulations, we observed EGFR kinase spontaneously transition from the active to the so-called "Src-like inactive" conformation by way of two sets of intermediate conformations: One corresponds to a previously identified locally disordered state and the other to previously undescribed "extended" conformations, marked by the opening of the ATP-binding site between the two lobes of the kinase domain. We also simulated the protonation-dependent transition of EGFR kinase to another ["Asp-Phe-Gly-out" ("DFG-out")] inactive conformation and observed similar intermediate conformations. A key element observed in the simulated transitions is local unfolding, or "cracking," which supports a prediction of energy landscape theory. We used hydrogen-deuterium (H/D) exchange measurements to corroborate our simulations and found that the simulated intermediate conformations correlate better with the H/D exchange data than existing active or inactive EGFR kinase crystal structures. The intermediate conformations revealed by our simulations of the transition process differ significantly from the existing crystal structures and may provide unique possibilities for structure-based drug discovery.

Mechanistic Insights Revealed by the Crystal Structure of a Histidine Kinase with Signal Transducer and Sensor Domains

Author SummaryTwo-component signal transduction systems (TCSs) are promising targets for new antimicrobial research because they help bacteria and fungi adapt and survive. One of the main components of TCSs is a sensor histidine kinase (SK), which relays extracellular signals to intracellular pathways. Despite intensive research, a full-length structure of an SK has yet to be solved. In this study, we report the first crystal structure of the complete cytoplasmic region of VicK, an important SK in the tooth decay pathogen S. mutans. VicK is composed of several domains (HAMP, PAS, DHp, and catalytic and ATP binding domain [CA]) in addition to a short transmembrane domain. We find that the dimeric VicK protein has an elegant rod-shaped structure with the domains linearly connected like beads on a string. The structure suggests that VicK kinase activates itself by helical bending of the DHp domain and coordinated swinging around of the catalytic CA domain to engage with the target histidine. Structure-based mutagenesis experiments also helped us to identify key residues that are required for VicK's opposing phosphatase activity. Our studies of the multi-modular VicK protein suggest a sequential kinase activation model that may involve helical bending of the DHp domain and repositioning of the CA domains.

Identification of a hidden strain switch provides clues to an ancient structural mechanism in protein kinases

The protein kinase catalytic domain contains several conserved residues of unknown functions. Here, using a combination of computational and experimental approaches, we show that the function of some of these residues is to maintain the backbone geometry of the active site in a strained conformation. Specifically, we find that the backbone geometry of the catalytically important HRD motif deviates from ideality in high-resolution structures and the strained geometry results in favorable hydrogen bonds with conserved noncatalytic residues in the active site. In particular, a conserved aspartate in the F-helix hydrogen bonds to the strained HRD backbone in diverse eukaryotic and eukaryotic-like protein kinase crystal structures. Mutations that alter this hydrogen-bonding interaction impair catalytic activity in Aurora kinase. Although the backbone strain is present in most active conformations, several inactive conformations lack the strain because of a peptide flip in the HRD backbone. The peptide flip is correlated with loss of hydrogen bonds with the F-helix aspartate as well as with other interactions associated with kinase regulation. Within protein kinases that are regulated by activation loop phosphorylation, the strained residue is an arginine, which coordinates with the activation loop phosphate. Based on analysis of strain across the protein kinase superfamily, we propose a model in which backbone strain co-evolved with conserved residues for allosteric control of catalytic activity. Our studies provide new clues for the design of allosteric protein kinase inhibitors.

X‐Ray Crystal Structure of Bone Marrow Kinase in the X Chromosome: A Tec Family Kinase

Bone marrow kinase in the X chromosome, a member of the Tec family of tyrosine kinases, plays a role in both monocyte/macrophage trafficking as well as cytokine secretion. Although the structures of Tec family kinases Bruton's tyrosine kinase and IL-2-inducible T-cell kinase are known, the crystal structures of other Tec family kinases have remained elusive. We report the X-ray crystal structures of bone marrow kinase in the X chromosome in complex with dasatinib at 2.4 Å resolution and PP2 at 1.9 Å resolution. The bone marrow kinase in the X chromosome structures reveal a typical kinase protein fold; with well-ordered protein conformation that includes an open/extended activation loop and a stabilized DFG-motif rendering the kinase in an inactive conformation. Dasatinib and PP2 bind to bone marrow kinase in the X chromosome in the ATP binding pocket and display similar binding modes to that observed in other Tec and Src protein kinases. The bone marrow kinase in the X chromosome structures identify conformational elements of the DFG-motif that could potentially be utilized to design potent and/or selective bone marrow kinase in the X chromosome inhibitors.

Comparison of ATP binding sites using structure-based similarity methods and molecular interaction fields

Protein kinases represent one of the largest group of drug targets in humans. All kinase enzymes share a catalytic domain that binds ATP. In recent years, many small molecule ATP-competitive kinase inhibitors have been developed. However, the evolutionary relatedness and structural conservation of these targets often lead to unforeseen cross reactivity. The goal of this work was to develop structure based ATP binding site descriptors that reflect the pharmacological profile of a predefined set of inhibitor molecules. For this purpose, a 3-step procedure was applied. First, a multiple sequence alignment generated by Buzko and Shokat [1] was adopted and binding site residues were identified. Second, kinase crystal structures were collected from SwissProt searches with EC numbers and superimposed based on the aligned binding site C-alpha atoms. Finally, molecular interaction field (MIF) descriptors were calculated and compared using a fuzzy Dice coefficient modified for 3D Euclidean space. For reasons of comparability with MIF descriptors, sequence descriptors based on the alignment were calculated. Moreover, kinase-ligand interaction fingerprints based on the superimposition of co-crystallized X‑ray structures were computed and mapped on the alignment. A dataset of 38 kinase inhibitors with experimental data against 90 human kinases was used to assess the performance of the developed descriptors [2]. Performance was measured by ROC enrichments for sampled kinase structures and Neighborhood Behavior criteria [3] for the complete kinase panel. Here, the performance is based on a comparison of the pairwise distances of the binding site descriptors to the pairwise distances of the inhibitor’s pharmacological profiles. Overall, classification based on the sequence of the binding pocket performed best. However, sequence-based methods cannot detect unrelated kinases with similar pharmacological profile. This in turn is the strength of the MIF-based descriptors since these descriptors characterize the geometry of hot spots on the protein and are independent of sequence information. Visualization of binding site hot spots makes this method suitable to rationally optimize the selectivity profile of a compound.

Optimisation of ITK inhibitors through successive iterative design cycles

Based on a hit cluster of compounds inhibiting interleukin-2 inducible T-cell kinase (ITK) in the submicromolar range a series of ITK inhibitor libraries were synthesized. Through iterative design cycles including kinase crystal structure information, indolylindazole libraries were identified which showed low nanomolar activity in enzymatic and cellular assays. The potential of these novel lead series was confirmed through in vivo tests in an anti-CD3-IL2 mouse model. The intravenous administration of highly potent ITK inhibitor 11o resulted in dose-dependent, efficient suppression of IL-2.