Design Of Kinase Inhibitors Research Articles

Design and synthesis of novel selective anaplastic lymphoma kinase inhibitors.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase belonging to the insulin receptor superfamily. Expression of ALK in normal human tissues is only found in a subset of neural cells, however it is involved in the genesis of several cancers through genetic aberrations involving translocation of the kinase domain with multiple fusion partners (e.g., NPM-ALK in anaplastic large cell lymphoma ALCL or EML4-ALK in non-small cell lung cancer) or activating mutations in the full-length receptor resulting in ligand-independent constitutive activation (e.g., neuroblastoma). Here we are reporting the discovery of novel and selective anaplastic lymphoma kinase inhibitors from specific modifications of the 2,4-diaminopyridine core present in TAE684 and LDK378. Synthesis, structure activity relationships (SAR), absorption, distribution, metabolism, and excretion (ADME) profile, and in vivo efficacy in a mouse xenograft model of anaplastic large cell lymphoma are described.

The Structural Basis for Activation and Inhibition of ZAP-70 Kinase Domain.

ZAP–70 (Zeta-chain-associated protein kinase 70) is a tyrosine kinase that interacts directly with the activated T-cell receptor to transduce downstream signals, and is hence a major player in the regulation of the adaptive immune response. Dysfunction of ZAP–70 causes selective T cell deficiency that in turn results in persistent infections. ZAP–70 is activated by a variety of signals including phosphorylation of the kinase domain (KD), and binding of its regulatory tandem Src homology 2 (SH2) domains to the T cell receptor. The present study investigates molecular mechanisms of activation and inhibition of ZAP–70 via atomically detailed molecular dynamics simulation approaches. We report microsecond timescale simulations of five distinct states of the ZAP–70 KD, comprising apo, inhibited and three phosphorylated variants. Extensive analysis of local flexibility and correlated motions reveal crucial transitions between the states, thus elucidating crucial steps in the activation mechanism of the ZAP–70 KD. Furthermore, we rationalize previously observed staurosporine-bound crystal structures, suggesting that whilst the KD superficially resembles an “active-like” conformation, the inhibitor modulates the underlying protein dynamics and restricts it in a compact, rigid state inaccessible to ligands or cofactors. Finally, our analysis reveals a novel, potentially druggable pocket in close proximity to the activation loop of the kinase, and we subsequently use its structure in fragment-based virtual screening to develop a pharmacophore model. The pocket is distinct from classical type I or type II kinase pockets, and its discovery offers promise in future design of specific kinase inhibitors, whilst mutations in residues associated with this pocket are implicated in immunodeficiency in humans.

Design of substrate-based BCR-ABL kinase inhibitors using the cyclotide scaffold.

The constitutively active tyrosine kinase BCR-ABL is the underlying cause of chronic myeloid leukemia (CML). Current CML treatments rely on the long-term use of tyrosine kinase inhibitors (TKIs), which target the ATP binding site of BCR-ABL. Over the course of treatment, 20–30% of CML patients develop TKI resistance, which is commonly attributed to point mutations in the drug-binding region. We design a new class of peptide inhibitors that target the substrate-binding site of BCR-ABL by grafting sequences derived from abltide, the optimal substrate of Abl kinase, onto a cell-penetrating cyclotide MCoTI-II. Three grafted cyclotides show significant Abl kinase inhibition in vitro in the low micromolar range using a novel kinase inhibition assay. Our work also demonstrates that a reengineered MCoTI-II with abltide sequences grafted in both loop 1 and 6 inhibits the activity of [T315I]Abl in vitro, a mutant Abl kinase harboring the “gatekeeper” mutation which is notorious for being multidrug resistant. Results from serum stability and cell internalization studies confirm that the MCoTI-II scaffold provides enzymatic stability and cell-penetrating properties to the lead molecules. Taken together, our study highlights that reengineered cyclotides incorporating abltide-derived sequences are promising substrate-competitive inhibitors for Abl kinase and the T315I mutant.

Structure-based de novo design and synthesis of aminothiazole-based p38 MAP kinase inhibitors.

p38 mitogen-activated protein kinase (MAPK) is a promising target for the development of therapeutics for various immunological diseases. We designed and synthesized aminothiazole-based p38 MAPK inhibitors of with IC50 values ranging from 0.1 to 2 μM by means of the structure-based de novo design of phenyl-(2-phenylamino-thiazol-5-yl)-methanone scaffold. Because these newly identified inhibitors were also screened for having desirable physicochemical properties as a drug candidate, they deserve consideration for further investigation as anti-inflammatory drugs. Structural features relevant to the stabilization of the newly identified inhibitors in the ATP-binding site of p38 MAPK are discussed in detail.

Abstract 2827: Discovery of AP26113, a potent, orally active inhibitor of anaplastic lymphoma kinase and clinically relevant mutants

Abstract Phosphorus, despite its abundance in the human body, is rarely found in drug molecules, with clinical utility limited to a few phosphonic or bisphosponic acid-based medicines and several phosphonate or phosphate-containing prodrugs. Concerns about poor cell penetration, low oral bioavailability, or biological instability have limited application of these functional classes in drug design. In our efforts to discover pharmaceuticals with novel functionality, we introduced a neutral, stable phosphine oxide moiety as a unique hydrogen-bond acceptor in the design of anaplastic lymphoma kinase (ALK) inhibitors. ALK is a receptor tyrosine kinase first identified as a chromosomal rearrangement (NPM-ALK fusion gene) in anaplastic large cell lymphoma (ALCL) and subsequently detected as an alternate fusion oncogene (EML4-ALK) in a subset of non-small cell lung cancers (NSCLC). Crizotinib, the first approved ALK inhibitor, has demonstrated impressive clinical benefit in EML4-ALK (ALK+) NSCLC patients. Drug resistance, however, emerges rapidly and point mutations within the kinase domain have been identified as a major resistance mechanism. With the specific objective to identify more potent ALK inhibitors with pan-inhibitory activity against crizotinib-resistant ALK mutants, we designed and tested a series of phosphine oxide-based compounds culminating in the identification of the clinical candidate AP26113. AP26113 exhibited 10-fold greater potency than crizotinib against ALK-positive ALCL and NSCLC cell lines, and effectively inhibited clinically relevant crizotinib-resistant mutants. AP26113 displayed ∼100-fold increased selectivity for ALK-positive cells over ALK-negative cell lines while maintaining selectivity over insulin receptor tyrosine kinase receptors. Consistent with the in vitro profile, AP26113 demonstrated oral efficacy in multiple ALK+ mouse models including Karpas-299 (ALCL), H3122 (NSCLC), and Ba/F3 cells expressing crizotinib resistant mutants including G1269S and L1196M. Finally, AP26113 exhibited excellent drug-like properties including high cell permeability and solubility, moderate cross-species protein binding, and inactivity toward major cytochrome P450 iso-enzymes and hERG ion channels. Medicinal chemistry efforts leading to the discovery of this potent pan-ALK inhibitor, including design strategy, chemical series evolution, and DMPK optimization will be presented. AP26113 is currently in a global phase 2 registration trial (NCT02094573) in patients with locally advanced or metastatic NSCLC who test positive for the ALK oncogene and were previously treated with crizotinib. Citation Format: Wei-Sheng Huang, Feng Li, Lisi Cai, Yongjin Xu, Sen Zhang, Scott D. Wardwell, Yaoyu Ning, Anna Kohlmann, Tianjun Zhou, Emily Y. Ye, Xiaotian Zhu, Narayana I. Narasimhan, Tim Clackson, Victor M. Rivera, David Dalgarno, William C. Shakespeare. Discovery of AP26113, a potent, orally active inhibitor of anaplastic lymphoma kinase and clinically relevant mutants. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2827. doi:10.1158/1538-7445.AM2015-2827

Structural Biology Insight for the Design of Sub-type Selective Aurora Kinase Inhibitors.

Aurora kinase A, B and C, are key regulators of mitosis and are over expressed in many of the human cancers, making them an ideal drug target for cancer chemotherapy. Currently, over a dozen of Aurora kinase inhibitors are in various phases of clinical development. The majority of the inhibitors (VX-680/MK-0457, PHA-739358, CYC116, SNS-314, AMG 900, AT-9283, SCH- 1473759, ABT-348, PF-03814735, R-763/AS-703569, KW-2449 and TAK-901) are pan-selective (isoform non-selective) and few are Aurora A (MLN8054, MLN8237, VX-689/MK5108 and ENMD 2076) and Aurora B (AZD1152 and GSK1070916) sub-type selective. Despite the intensive research efforts in the past decade, no Aurora kinase inhibitor has reached the market. Recent evidence suggests that the sub-type selective Aurora kinase A inhibitor could possess advantages over pan-selective Aurora inhibitors, by avoiding Aurora B mediated neutropenia. However, sub-type selective Aurora kinase A inhibitor design is very challenging due to the similarity in the active site among the isoforms. Structural biology and computational aspects pertaining to the design of Aurora kinase inhibitors were analyzed and found that a possible means to develop sub-type selective inhibitor is by targeting Aurora A specific residues (Leu215, Thr217 and Arg220) or Aurora B specific residues (Arg159, Glu161 and Lys164), near the solvent exposed region of the protein. Particularly, a useful strategy for the design of sub-type selective Aurora A inhibitor could be by targeting Thr217 residue as in the case of MLN8054. Further preclinical and clinical studies with the sub-type selective Aurora inhibitors could help bring them to the market for the treatment of cancer.

3D-QSAR and virtual screening studies in identification of new Rho kinase inhibitors with different scaffolds

Among the most well-known members of the serine/threonine (Ser/Thr) protein kinase family are Rho kinases. They are potential targets to treat some diseases, such as cancer, cardiovascular diseases and multiple sclerosis, because of their key roles in the organization of the actin cytoskeleton. In this study, 3D-QSAR (quantitative structure–activity relationship) techniques using popular CoMFA and CoMSIA tools as well as recently developed automated grid potential analysis (AutoGPA) based on pharmacophore alignment were applied on 41 urea-based derivatives as Rho kinase inhibitors, which were split into 32 and 9 compounds as train and test sets using Kennard and Stone algorithm. The statistical parameters of AutoGPA-based 3D-QSAR model were q 2 = 0.506, $$R_{\text{pred}}^{ 2}$$ = 0.844, $$R_{\text{m}}^{2}$$ = 0.806 which show its acceptable prediction reliability. The verified model was further utilized to search novel hits from ZINC database in virtual screening task. The obtained compounds were subjected to Lipinski filter, then their activities were predicted by AutoGPA model and docked with CDOCKER algorithm to discover potent hits. In silico ADME and toxicity risk assessment analyses were carried out on the seven hits with highest CDOCKER scores. Six out of the seven hits have diverse structures and are reported as new scaffold candidates for design of new Rho kinase inhibitors.

Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic.

Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors.

Design, Synthesis and Inhibitory Activity of Photoswitchable RET Kinase Inhibitors.

REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase required for normal development and maintenance of neurons of the central and peripheral nervous systems. Deregulation of RET and hyperactivity of the RET kinase is intimately connected to several types of human cancers, most notably thyroid cancers, making it an attractive therapeutic target for small-molecule kinase inhibitors. Novel approaches, allowing external control of the activity of RET, would be key additions to the signal transduction toolbox. In this work, photoswitchable RET kinase inhibitors based on azo-functionalized pyrazolopyrimidines were developed, enabling photonic control of RET activity. The most promising compound displays excellent switching properties and stability with good inhibitory effect towards RET in cell-free as well as live-cell assays and a significant difference in inhibitory activity between its two photoisomeric forms. As the first reported photoswitchable small-molecule kinase inhibitor, we consider the herein presented effector to be a significant step forward in the development of tools for kinase signal transduction studies with spatiotemporal control over inhibitor concentration in situ.

Luciferin and derivatives as a DYRK selective scaffold for the design of protein kinase inhibitors

D-Luciferin is widely used as a substrate in luciferase catalysed bioluminescence assays for in vitro studies. However, little is known about cross reactivity and potential interference of D-luciferin with other enzymes. We serendipitously found that firefly luciferin inhibited the CDK2/Cyclin A protein kinase. Inhibition profiling of D-luciferin over a 103-protein kinase panel showed significant inhibition of a small set of protein kinases, in particular the DYRK-family, but also other members of the CMGC-group, including ERK8 and CK2. Inhibition profiling on a 16-member focused library derived from D-luciferin confirms that D-luciferin represents a DYRK-selective chemotype of fragment-like molecular weight. Thus, observation of its inhibitory activity and the initial SAR information reported here promise to be useful for further design of protein kinase inhibitors with related scaffolds.

A new “angle” on kinase inhibitor design: Prioritizing amphosteric activity above kinase inhibition

The MYCN oncoprotein has remained an elusive target for decades. We recently reported a new class of kinase inhibitors designed to disrupt the conformation of Aurora kinase A enough to block its kinase-independent interaction with MYCN, resulting in potent degradation of MYCN. These studies provide proof-of-principle for a new method of targeting enzyme activity-independent functions of kinases and other enzymes.

Abstract B49: Investigating kinase inhibitor resistance mechanisms through quantitative network mapping of the human kinase interactome

Abstract The human kinases have become one of the most important classes of drug targets in the pharmaceutical industry. More than 20 drugs targeting one or more kinases have been approved for clinical use in a variety of cancers, including lung, breast, melanoma, colorectal, pancreatic, and prostate cancers. However, patients treated with those kinase inhibitors eventually develop resistance, and their prolong survivals are typically only a few months. So far, no general model has emerged to address the kinase inhibitor resistance. In this study, we constructed a systems biology-based framework to catalogue the human kinome (kinases and their interactions), including 538 kinase genes, in the broader context of the human interactome. Specifically, we constructed three networks: a kinase-substrate interaction network containing 7,346 pairs connecting 379 kinases to 36,576 phosphorylation sites in 1,961 substrates, a protein-protein interaction network (PPIN) containing 92,699 pairs, and an atomic resolution PPIN containing 4,278 pairs. We identified the conserved regulatory phosphorylation motifs (e.g., Ser/Thr-Pro) using a sequence logo analysis. We found the typical anticancer target selection strategy that uses network hubs as drug targets might lead to a high adverse drug reaction risk. Furthermore, we found the distinct network centrality of kinases creates a high anticancer drug resistance risk by feedback or crosstalk mechanisms within cellular networks. This notion is supported by the systematic network and pathway analyses that anticancer drug resistance genes are significantly enriched as hubs and heavily participate in multiple signaling pathways. Collectively, this comprehensive human kinome map sheds light on anticancer drug resistance mechanisms and provides an innovative resource for rational kinase inhibitor design. Note: This abstract was not presented at the conference. Citation Format: Feixiong Cheng, Zhongming Zhao. Investigating kinase inhibitor resistance mechanisms through quantitative network mapping of the human kinase interactome. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B49.

Design and synthesis of VEGFR-2 tyrosine kinase inhibitors as potential anticancer agents by virtual based screening

Design and synthesis of VEGFR-2 tyrosine kinase inhibitors as potential anticancer agents has been done by a virtual based screening approach.

Fragment-based design of kinase inhibitors: a practical guide.

Fragment-based drug design has become an important strategy for drug design and development over the last decade. It has been used with particular success in the development of kinase inhibitors, which are one of the most widely explored classes of drug targets today. The application of fragment-based methods to discovering and optimizing kinase inhibitors can be a complicated and daunting task; however, a general process has emerged that has been highly fruitful. Here a practical outline of the fragment process used in kinase inhibitor design and development is laid out with specific examples. A guide to the overall process from initial discovery through fragment screening, including the difficulties in detection, to the computational methods available for use in optimization of the discovered fragments is reported.

Protein Kinase Inhibitor Design and Screening - A GTCBIO conference. Philadelphia, Pennsylvania, USA - September 10-11, 2015

Protein Kinase Inhibitor Design and Screening - A GTCBIO conference. Philadelphia, Pennsylvania, USA - September 10-11, 2015

Protein kinase inhibitor design and screening - GTCBio a GTCbio conference, - Philadelphia, PA, USA, September 10-11, 2015

Protein kinase inhibitor design and screening - GTCBio a GTCbio conference, - Philadelphia, PA, USA, September 10-11, 2015

Overcoming myelosuppression due to synthetic lethal toxicity for FLT3-targeted acute myeloid leukemia therapy.

Activating mutations in FLT3 confer poor prognosis for individuals with acute myeloid leukemia (AML). Clinically active investigational FLT3 inhibitors can achieve complete remissions but their utility has been hampered by acquired resistance and myelosuppression attributed to a 'synthetic lethal toxicity' arising from simultaneous inhibition of FLT3 and KIT. We report a novel chemical strategy for selective FLT3 inhibition while avoiding KIT inhibition with the staurosporine analog, Star 27. Star 27 maintains potency against FLT3 in proliferation assays of FLT3-transformed cells compared with KIT-transformed cells, shows no toxicity towards normal human hematopoiesis at concentrations that inhibit primary FLT3-mutant AML blast growth, and is active against mutations that confer resistance to clinical inhibitors. As a more complete understanding of kinase networks emerges, it may be possible to define anti-targets such as KIT in the case of AML to allow improved kinase inhibitor design of clinical agents with enhanced efficacy and reduced toxicity.

Structure-based design of low-nanomolar PIM kinase inhibitors

PIM kinases are implicated in variety of cancers by promoting cell survival and proliferation and are targets of interest for therapeutic intervention. We have identified a low-nanomolar pan-PIM inhibitor (PIM1/2/3 potency 5:14:2nM) using structure based modeling. The crystal structure of this compound with PIM1 confirmed the predicted binding mode and protein–ligand interactions except those in the acidic ribose pocket. We show the SAR suggesting the importance of having a hydrogen bond donor in this pocket for inhibiting PIM2; however, this interaction is not important for inhibiting PIM1 or PIM3. In addition, we report the discovery of a new class of PIM inhibitors by using computational de novo design tool implemented in MOE software (Chemical Computing Group). These inhibitors have a different interaction profile.

Selection of fragments for kinase inhibitor design: decoration is key.

In fragment-based screening, the choice of the best suited fragment hit among the detected hits is crucial for success. In our study, a kinase lead compound was fragmented, the hinge-binding motif extracted as a core fragment, and a minilibrary of five similar compounds with fragment-like properties was selected from our proprietary compound database. The structures of five fragments in complex with transforming growth factor β receptor type 1 kinase domain were determined by X-ray crystallography. Three different binding modes of the fragments are observed that depend on the position and the type of the substitution at the core fragment. The influence of different substituents on the preferred fragment pose was analyzed by various computational approaches. We postulate that the replacement of water molecules leads to the different binding modes.

Specificity rendering 'hot-spots' for aurora kinase inhibitor design: the role of non-covalent interactions and conformational transitions.

The present study examines the conformational transitions occurring among the major structural motifs of Aurora kinase (AK) concomitant with the DFG-flip and deciphers the role of non-covalent interactions in rendering specificity. Multiple sequence alignment, docking and structural analysis of a repertoire of 56 crystal structures of AK from Protein Data Bank (PDB) has been carried out. The crystal structures were systematically categorized based on the conformational disposition of the DFG-loop [in (DI) 42, out (DO) 5 and out-up (DOU) 9], G-loop [extended (GE) 53 and folded (GF) 3] and αC-helix [in (CI) 42 and out (CO) 14]. The overlapping subsets on categorization show the inter-dependency among structural motifs. Therefore, the four distinct possibilities a) 2W1C (DI, CI, GE) b) 3E5A (DI, CI, GF) c) 3DJ6 (DI, CO, GF) d) 3UNZ (DOU, CO, GF) along with their co-crystals and apo-forms were subjected to molecular dynamics simulations of 40 ns each to evaluate the variations of individual residues and their impact on forming interactions. The non-covalent interactions formed by the 157 AK co-crystals with different regions of the binding site were initially studied with the docked complexes and structure interaction fingerprints. The frequency of the most prominent interactions was gauged in the AK inhibitors from PDB and the four representative conformations during 40 ns. Based on this study, seven major non-covalent interactions and their complementary sites in AK capable of rendering specificity have been prioritized for the design of different classes of inhibitors.