Gatekeeper Residue Research Articles

Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation.

Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril’s morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4′-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54–63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54–63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although with different efficiency, all formed fibrils morphologically similar to wild-type MccE492. The physiological implication of MccE492 intracellular amyloid formation is probably similar to the inactivation process observed for extracellular amyloids, and could be used as a mean of sequestering potentially toxic species inside the cell when this bacteriocin is produced in large amounts.

Exploration of Novel Inhibitors for Bruton's Tyrosine Kinase by 3D QSAR Modeling and Molecular Dynamics Simulation.

Bruton’s tyrosine kinase (BTK) is a cytoplasmic, non-receptor tyrosine kinase which is expressed in most of the hematopoietic cells and plays an important role in many cellular signaling pathways. B cell malignancies are dependent on BCR signaling, thus making BTK an efficient therapeutic target. Over the last few years, significant efforts have been made in order to develop BTK inhibitors to treat B-cell malignancies, and autoimmunity or allergy/hypersensitivity but limited success has been achieved. Here in this study, 3D QSAR pharmacophore models were generated for Btk based on known IC50 values and experimental energy scores with extensive validations. The five features pharmacophore model, Hypo1, includes one hydrogen bond acceptor lipid, one hydrogen bond donor, and three hydrophobic features, which has the highest correlation coefficient (0.98), cost difference (112.87), and low RMS (1.68). It was further validated by the Fisher’s randomization method and test set. The well validated Hypo1 was used as a 3D query to search novel Btk inhibitors with different chemical scaffold using high throughput virtual screening technique. The screened compounds were further sorted by applying ADMET properties, Lipinski’s rule of five and molecular docking studies to refine the retrieved hits. Furthermore, molecular dynamic simulation was employed to study the stability of docked conformation and to investigate the binding interactions in detail. Several important hydrogen bonds with Btk were revealed, which includes the gatekeeper residues Glu475 and Met 477 at the hinge region. Overall, this study suggests that the proposed hits may be more effective inhibitors for cancer and autoimmune therapy.

A systematic molecular dynamics approach to the structural characterization of amyloid aggregation propensity of β2-microglobulin mutant D76N.

Beta-2 microglobulin (β2m) is an amyloidogenic protein belongs to the immunoglobulin superfamily, responsible for the dialysis-related amyloidosis (DRA). Misfolding of β2m is a prerequisite to the formation of systemic amyloidosis that has an effect on the structure and function of the affected organ. The aim of our present study is to intensively explore the structural characterization of amyloid aggregation propensity of recently identified natural mutation D76N by applying the classical molecular dynamics (MD) approach. The MD result revealed that mutant D76N exhibited a wide variation in the evolutionarily conserved secondary structure profile. Due to an unsatisfied position of main chain donor/acceptor atoms that unable to form essential hydrogen bonds resulted to cause misfolding of mutant D76N by disrupting the local folding of β-strands and turn region. Analysis of time evolution of various structural properties, especially those of the functionally important residues: aggregation determining, initiating, and gatekeeper residues gave some possible insights into the structural characteristics of the disease mutant D76N. In a nutshell, compared to the wild-type β2m, aggregation promoting propensity of mutant D76N has established a long β-strand D owing to an inward movement of residue, Asp(53). Besides, aggregation forming characteristic of the DE loop in mutant D76N showed greater flexibility along the first principal eigenvector that favored to enhance an unusual conformational dynamics may lead toward self-aggregation and amyloid fibrils.

Imidazopyridazine Inhibitors of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 Also Target Cyclic GMP-Dependent Protein Kinase and Heat Shock Protein 90 To Kill the Parasite at Different Stages of Intracellular Development.

Imidazopyridazine compounds are potent, ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1) and of Plasmodium falciparum parasite growth in vitro. Here, we show that these compounds can be divided into two classes depending on the nature of the aromatic linker between the core and the R2 substituent group. Class 1 compounds have a pyrimidine linker and inhibit parasite growth at late schizogony, whereas class 2 compounds have a nonpyrimidine linker and inhibit growth in the trophozoite stage, indicating different modes of action for the two classes. The compounds also inhibited cyclic GMP (cGMP)-dependent protein kinase (PKG), and their potency against this enzyme was greatly reduced by substitution of the enzyme's gatekeeper residue at the ATP binding site. The effectiveness of the class 1 compounds against a parasite line expressing the modified PKG was also substantially reduced, suggesting that these compounds kill the parasite primarily through inhibition of PKG rather than CDPK1. HSP90 was identified as a binding partner of class 2 compounds, and a representative compound bound to the ATP binding site in the N-terminal domain of HSP90. Reducing the size of the gatekeeper residue of CDPK1 enabled inhibition of the enzyme by bumped kinase inhibitors; however, a parasite line expressing the modified enzyme showed no change in sensitivity to these compounds. Taken together, these findings suggest that CDPK1 may not be a suitable target for further inhibitor development and that the primary mechanism through which the imidazopyridazines kill parasites is by inhibition of PKG or HSP90.

Molecular modeling studies on series of Btk inhibitors using docking, structure-based 3D-QSAR and molecular dynamics simulation: a combined approach.

Bruton tyrosine kinase (Btk) is a non-receptor tyrosine kinase. It is a crucial component in BCR pathway and expressed only in hematopoietic cells except T cells and Natural killer cells. BTK is a promising target because of its involvement in signaling pathways and B cell diseases such as autoimmune disorders and lymphoma. In this work, a combined molecular modeling study of molecular docking, 3D-QSAR and molecular dynamic (MD) simulation were performed on a series of 2,5-diaminopyrimidine compounds as inhibitors targeting Btk kinase to understand the interaction and key residues involved in the inhibition. A structure based CoMFA (q (2) = 0.675, NOC = 5, r (2) = 0.961) and COMSIA (q (2) = 0.704, NOC = 6, r (2) = 0.962) models were developed from the conformation obtained by docking. The developed models were subjected to various validation techniques such as leave-five-out, external test set, bootstrapping, progressive sampling and rm (2) metrics and found to have a good predictive ability in both internal and external validation. Our docking results showed the important residues that interacts in the active site residues in inhibition of Btk kinase. Furthermore, molecular dynamics simulation was employed to study the stability of the docked conformation and to investigate the binding interactions in detail. The MD simulation analyses identified several important hydrogen bonds with Btk, including the gatekeeper residue Thr474 and Met477 at the hinge region. Hydrogen bond with active site residues Leu408 and Arg525 were also recognized. A good correlation between the MD results, docking studies and the contour map analysis are observed. This indicates that the developed models are reliable. Our results from this study can provide insights in the designing and development of more potent Btk kinase inhibitors.

Exome Sequencing Informs Mechanisms of Clinical Resistance to the FLT3-D835 Inhibitor Crenolanib

Introduction: Variation of the D835 residue of FLT3 occurs in ~5% of de novo AML cases and is a prominent feature of drug resistance in the setting of FLT3-ITD. Crenolanib treatment of AML patients exhibiting FLT3 D835 and/or FLT3-ITD positive tumors has yielded significant clinical responses. However, these clinical responses to single-agent crenolanib were transient, so we performed whole exome sequencing to determine the mechanisms of crenolanib resistance. Mutation of the FLT3 F691 "gatekeeper" residue is a known resistance mechanism to crenolanib and other FLT3 inhibitors in vitro. Hence, secondary mutation of FLT3 at the gatekeeper or other sites was hypothesized to be a predominant mechanism of resistance. We report that a minority of patients acquired FLT3 gatekeeper or other secondary FLT3 mutations, however, the majority of cases relapsed in the absence of additional FLT3 mutations indicating alternative mechanisms of drug resistance.Methods: Genomic DNA was isolated from bone marrow aspirates from 44 AML patients treated with crenolanib. We performed whole exome sequencing using Illumina Nextera exome capture and paired end sequencing on an Illumina 2500 HiSeq. Paired specimens (pre/post-treatment) were available for 24 of these patients, allowing for analysis of acquired mutations in the context of crenolanib therapy.Results: Although secondary mutation of FLT3 at the gatekeeper residue, FLT3 F691, was predicted to be a prominent mechanism of resistance, we did not observe FLT3 gatekeeper mutations in most patients. In total, only 3/44 (7%) of cases exhibited a FLT3 gatekeeper mutation despite good coverage of the FLT3 locus (average of 134 fold read depth; range of 77-177 fold average read depth across all 44 cases). In addition, we observed secondary FLT3 mutations at alternative residues, D200N, K429E, and L601F, in 3 other patients. Hence, only 6/44 (14%) of patients exhibited evidence of relapse due to secondary mutation of FLT3, and the majority of patients (38/44; 86%) presented with crenolanib resistance in the absence of secondary FLT3 mutations. Exome analysis revealed that many of these cases acquired mutations involving transcriptional regulators, suggesting alternate pathways of escape as the predominant mechanism to crenolanib therapy.Conclusions: Crenolanib is broadly effective against FLT3-ITD and FLT3-D835 mutant AML. Despite predictions of FLT3 gatekeeper mutations (or other secondary FLT3 mutations) as a primary mechanism of resistance, we only observe these events in a minority of patients. Instead, we observe a prominent signal of acquired mutations in transcriptional regulators, suggesting a more elaborate genetic/epi-genetic mechanism of resistance to crenolanib. DisclosuresHo:AROG Pharmaceuticals: Employment. Davis:AROG Pharmaceuticals: Employment. Ramachandran:AROG Pharmaceuticals: Employment. Cortes:ARIAD Pharmaceuticals Inc.: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Consultancy, Research Funding; BMS: Consultancy, Research Funding. Tyner:Aptose Biosciences: Research Funding; Constellation Pharmaceuticals: Research Funding; Array Biopharma: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding.

Abstract A168: Discovery of exquisitely selective MerTK inhibitors

Abstract MerTK is a receptor tyrosine kinase most prominently expressed in myeloid cells and known for its role in phagocytosis of early apoptotic cells and concomitant establishment of tolerogenic microenvironment. Hence, MerTK is thought to play a significant role in cancer immune evasion as, for example, a defining component of M2c-type tumor-associated macrophages (TAMs). Moreover, it has also been increasingly recognized that MerTK is overexpressed in a variety of malignant tumor cells, promoting the cancer cell survival, invasion, and treatment resistance. As such, it is anticipated that the cancer cell intrinsic and extrinsic effects might conspire to make MerTK a highly promising target for anticancer therapy. We describe herein an exquisitely selective series of MerTK inhibitors. A lead compound, XL1547, exhibited an IC50 of 1 nM against MerTK in an enzyme assay, with a greater than 300-fold selectivity over closely related AXL kinase (IC50 = 350 nM). In fact, MerTK was the only enzyme that was inhibited by XL1547 (> 99% at 100 nM) in the panel of 386 kinases (Reaction Biology Corp.; all others < 35%). X-ray crystal structure revealed a unique binding mode where the compound exploits a large back pocket next to the gatekeeper residue (known as a Type 1.5 binder). XL1547 potently inhibited autophosphorylation as well as the viability of engineered BaF3(CD8:MerTK) cells (GI50 = 230 nM) with little toxicity. Extensive medicinal chemistry efforts yielded a more advanced compound SA3686 with at least 10-fold improvement in potency. More importantly, it displayed a pharmacokinetic profile suitable for oral dosing. Indeed, SA3686 was highly efficacious in vivo in an allograft tumor model of subcutaneously implanted Ba/F3(CD8:MerTK) cells. [SA3686 is being tested in multiple other in vivo models at the time of abstract submission]. In summary, we have identified an essentially mono-selective MerTK inhibitor series that will help delineate the biology of MerTK-specific inhibition and hopefully validate the translational potential of MerTK as a therapeutic target for human diseases including cancer. Citation Format: Taeyoung Yoon, Joon-Ho Sheen, Seong Jin Park, Jin Kwan Kim, Hadong Kim, Eun-Jung Kim, Munjin Kwon, Hyung Ki Lee, Okyung Lee, Youngjee Jeong, Ki Moon Ryu, Miyeon Jang, Jae-Sung Hwang. Discovery of exquisitely selective MerTK inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A168.

Noncovalent Mutant Selective Epidermal Growth Factor Receptor Inhibitors: A Lead Optimization Case Study.

Because of their increased activity against activating mutants, first-generation epidermal growth factor receptor (EGFR) kinase inhibitors have had remarkable success in treating non-small-cell lung cancer (NSCLC) patients, but acquired resistance, through a secondary mutation of the gatekeeper residue, means that clinical responses only last for 8-14 months. Addressing this unmet medical need requires agents that can target both of the most common double mutants: T790M/L858R (TMLR) and T790M/del(746-750) (TMdel). Herein we describe how a noncovalent double mutant selective lead compound was optimized using a strategy focused on the structure-guided increase in potency without added lipophilicity or reduction of three-dimensional character. Following successive rounds of design and synthesis it was discovered that cis-fluoro substitution on 4-hydroxy- and 4-methoxypiperidinyl groups provided synergistic, substantial, and specific potency gain through direct interaction with the enzyme and/or effects on the proximal ligand oxygen atom. Further development of the fluorohydroxypiperidine series resulted in the identification of a pair of diastereomers that showed 50-fold enzyme and cell based selectivity for T790M mutants over wild-type EGFR (wtEGFR) in vitro and pathway knock-down in an in vivo xenograft model.

Targeting the Gatekeeper MET146 of C-Jun N-Terminal Kinase 3 Induces a Bivalent Halogen/Chalcogen Bond.

We target the gatekeeper MET146 of c-Jun N-terminal kinase 3 (JNK3) to exemplify the applicability of X···S halogen bonds in molecular design using computational, synthetic, structural and biophysical techniques. In a designed series of aminopyrimidine-based inhibitors, we unexpectedly encounter a plateau of affinity. Compared to their QM-calculated interaction energies, particularly bromine and iodine fail to reach the full potential according to the size of their σ-hole. Instead, mutation of the gatekeeper residue into leucine, alanine, or threonine reveals that the heavier halides can significantly influence selectivity in the human kinome. Thus, we demonstrate that, although the choice of halogen may not always increase affinity, it can still be relevant for inducing selectivity. Determining the crystal structure of the iodine derivative in complex with JNK3 (4X21) reveals an unusual bivalent halogen/chalcogen bond donated by the ligand and the back-pocket residue MET115. Incipient repulsion from the too short halogen bond increases the flexibility of Cε of MET146, whereas the rest of the residue fails to adapt being fixed by the chalcogen bond. This effect can be useful to induce selectivity, as the necessary combination of methionine residues only occurs in 9.3% of human kinases, while methionine is the predominant gatekeeper (39%).

A Proteometric Analysis of Human Kinome: Insight into Discriminant Conformation-dependent Residues.

Because of the success of imatinib, the first type-II kinase inhibitor approved by the FDA in 2001, sustained efforts have been made by the pharmaceutical industry to discover novel compounds stabilizing the inactive conformation of protein kinases. On the seven type-II inhibitors having reached the market, four were released in 2012, suggesting an acceleration of the research of such a class of compounds. Still, they represent less than a third of the protein kinase inhibitors available to patients today. The identification of key residues involved in the binding of this type of ligands in the kinase active site might ease the design of potent and selective type-II inhibitors. In order to identify those discriminant residues, we have developed a proteometric approach combining residue descriptors of protein kinase sequences and biological activities of various type-II kinase inhibitors. We applied Partial Least Squares (PLS) regression to identify 29 key residues that influence the binding of four type-II inhibitors to most proteins of the kinome. The gatekeeper residue was found to be the most relevant, confirming an essential role in ligand binding as well as in protein kinase conformational changes. Using the newly developed proteometric model, we predicted the propensity of each protein kinase to be inhibited by type-II ligands. The model was further validated using an external data set of protein/ligand activity pairs. Other residues present in the kinase domain, and more specifically in the binding site, have been highlighted by this approach, but their role in biological mechanisms is still unknown.

Abstract LB-256: Optimal clinical dose-finding strategies: Translational preclinical pharmacokineitcs, pharmacodynamics, and efficacy analysis of HM61713, an orally selective EGFR mutant inhibitor

Abstract The first-generation of EGFR1 inhibitors (Gefitinib and Erlotinib) has significant clinical benefits in NSCLC caused by activating mutations, but the efficacy of these agents is often limited due to the emergence of drug resistance conferred by a gatekeeper residue, T790M. HM61713 is a third-generation EGFR tyrosine kinase inhibitor that has been evaluated as a novel therapeutic agent for the treatment of non-small cell lung cancer (NSCLC) with EGFR mutations. HM61713 is an orally active and a novel EGFR mutant selective inhibitor which is potent on resistance mutation (T790M) without affecting EGFR wild type at efficacious dose level. HM61713 showed an anti-cancer activity in several EGFR mutant lung cancer cell lines including T790M mutation harboring cell line. Integrated pharmacokinetic - pharmacodynamic - xenograft tumor model (PK-PD-XTG) was used to characterize the relationship between HM61713 plasma concentration and tumor growth inhibition (TGI) in H1975 (T790M mutation) xenograft model. Simple one-compartment model applied re-absorption compartment with first-order absorption/elimination was used to describe HM61713 plasma concentration-time profiles. Biophase distribution model with baseline inhibition Emax equation was applied to characterize the PD marker (p-EGFR) and tumor volume shrinkage was explained by michaelis-menten kinetics of p-EGFR. Estimated in vivo IC50 value of p-EGFR inhibition (%) based on plasma free concentration in xenograft mice was 1.14 ng/mL. The human PD marker response curve and the tumor growth inhibition plot were obtained by replacing the mice PK to human PK in our developed model based on the hypothesis thattranslation providesa good relationship of surrogate mice tumor PD to human tumor regression corresponding human PK. According to our simulated curves, we predicted appropriate human active dose from 300 to 800 mg/man and it would be an efficacious dose in patients with NSCLC harboring the EGFR activating and also with T790M resistant mutation. Currently, HM61713 is undergoing in clinical trial phase I/II in NSCLC (ClinicalTrials.gov, NCT01588145). Citation Format: Jooyun Byun, Taehun Song, Donghyun Kim, Junhyeng Son, Kwang-Ok Lee, Jaeho Lee, Yong Hoon Kim, Young-Mi Lee, Kwee Hyun Suh. Optimal clinical dose-finding strategies: Translational preclinical pharmacokineitcs, pharmacodynamics, and efficacy analysis of HM61713, an orally selective EGFR mutant inhibitor. [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 LB-256. doi:10.1158/1538-7445.AM2015-LB-256

Abstract 2436: Understanding the molecular mechanism of targeted kinase inhibitor resistance mediated by the FGFR1 gatekeeper mutation

Abstract Human fibroblast growth factor receptors (FGFRs) can serve as drivers of oncogenesis and are a target of several inhibitors in clinical trials. Unfortunately, resistance severely hampers the long-term success of tyrosine kinase inhibitors (TKIs), with mutations at the gatekeeper residue often serving as an early means of resistance to cancer therapy, resulting in tumor progression. Here we show the first crystal structures of the FGFR1 gatekeeper mutation, V561M FGFR1, and use kinetic and structural methods to explore the mechanisms of the catalytic activation we observe for this mutation. We show that while the FGFR1 gatekeeper mutation confers resistance to two inhibitors currently in clinical trials, the extent of change in efficacy differ, indicating two unique resistance profiles. We show that some affinity for one inhibitor is maintained by the V561M mutation due to a flexible linker that allows multiple binding conformations. This is the first example showing the same inhibitor binding in unique ways to the WT and gatekeeper mutant forms of FGFR, highlighting some regions of drug design flexibility both within the binding pocket and in the linker of the inhibitor. Identifying this flexibility in the context of the structural features of an active conformation of V561M FGFR1, the form most kinase inhibitors target, will provide critical insights for designing future inhibitors effective against the FGFR gatekeeper mutations. Citation Format: Christal D. Sohl, Molly Ryan, BeiBei Luo, Kathleen Frey, Karen S. Anderson. Understanding the molecular mechanism of targeted kinase inhibitor resistance mediated by the FGFR1 gatekeeper mutation. [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 2436. doi:10.1158/1538-7445.AM2015-2436

Abstract 5450: Naturally occurring mutations in the MPS1 gene predispose cells to kinase inhibitor drug-resistance

Abstract Acquired resistance is the greatest challenge to the effectiveness of targeted anti-cancer therapies in the clinic. With several MPS1 inhibitors under pre-clinical development, we aimed to investigate how cancer cells will develop resistance against these inhibitors; therefore we modeled acquired resistance using a range of MPS1 inhibitors. We identified and characterized five point mutations in the kinase domain of MPS1 that confer resistance against multiple inhibitors. Structural studies showed that several MPS1 mutants conferred resistance by causing steric hindrance to inhibitor binding. One mutation in particular, p.C604W, which is close to the gatekeeper residue, rendered MPS1 resistant to all the inhibitors we tested. However, we were able to design new compounds to specifically overcome this mutation, which in fact targeted the mutant with more potency than the wild-type MPS1 protein. Importantly, we show that these mutations are present in untreated cancer cell lines and primary tumour samples, and also pre-exist in normal lymphoblast and breast tissues. Furthermore, to confirm this is not specific to MPS1, we show that the EGFR p.T790M mutation is also pre-existing in cancer cell lines and normal tissue. Our data therefore suggest that mutations conferring resistance to targeted therapy are naturally occurring mutations in normal and cancer cells that are not introduced due to cancer cells being more mutagenic. Citation Format: Mark D. Gurden, Isaac Westwood, Amir Faisal, Sébastien Naud, Jack Cheung, Craig McAndrew, Amy Wood, Jessica Schmitt, Kathy Boxall, Grace Mak, Paul Workman, Rosemary Burke, Swen Hoelder, Julian Blagg, Rob Van Montfort, Spiros Linardopoulos. Naturally occurring mutations in the MPS1 gene predispose cells to kinase inhibitor drug-resistance. [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 5450. doi:10.1158/1538-7445.AM2015-5450

Abstract 2450: Identifying novel substrates of PLK2 using a chemical genetics approach

Abstract Polo like kinase 2 (PLK2/SNK) is a Ser/Thr kinase with roles identified in G1-S phase transition and in centriole duplication prior to entry into mitosis. PLK2 null embryonic fibroblasts have a slower proliferative rate and delayed entry into S-phase than their wild-type counterparts validating its role in cell cycle. Furthermore, wild-type p53, in response to DNA damage, induces PLK2 expression and checkpoint arrest. While these studies suggest that PLK2 is a tumor suppressor, studies have also shown that PLK2 binds to and phosphorylates mutant p53, but not wild-type p53, thereby promoting an oncogenic, auto-regulatory feedback loop, suggesting a potential role for PLK2 in proliferation. These observations suggest that PLK2 can function as an oncogene as well as a tumor suppressor. It is likely that cellular context and the nature of substrates are key to our understanding of the role that PLK2 plays in tumorigenesis. Although these reports highlight the importance of PLK2 in a variety of cellular processes, there is limited knowledge on the various proteins that are regulated by PLK2. We have optimized a chemical genetics system to identify novel substrates of PLK2. To that end, we mutated the conserved gatekeeper residue in the kinase domain of PLK2 (Leu159) to a smaller amino acid (Gly) to enlarge the ATP binding pocket. This engineered kinase, L159G-PLK2 (Plk2-as) is now able to utilize ATP analogs that carry a N6 modified bulky substituent of nucleoside residue such as N6-Phenylethyl ATP to phosphorylate substrates. We took advantage of this system to tag and isolate PLK2 substrates in vitro. Wild type and analog sensitive-PLK2 kinase domains were purified from bacteria and conditions were optimized for specific thio-phosphorylation of cell lysates by PLK2-as kinase. Labeling of cell lysates and subsequent mass spectrometry identified Signal transducer and activator of transcription 1 (STAT1) as a novel substrate of PLK2. In vitro PLK2 kinase assays in the presence or absence of specific PLK2 inhibitor, ON1231320, validated STAT1 to be a novel substrate of PLK2. Since mass spectrometry identified Ser708 as the potential phosphorylation site, we designed WT and mutant S708A peptides that span 16 amino acids of the phosphorylation site and used them as substrates for PLK1 and PLK2 in vitro kinase assays. While PLK2 readily phosphorylated the WT peptide, phosphorylation of the S708A mutant peptide was significantly reduced suggesting that PLK2 phosphorylates STAT1 at Ser708. This was further confirmed by our observation that ON1231320, a PLK2- specific inhibitor, inhibited PLK2-mediated phosphorylation of WT peptide. In addition, neither the WT nor the S708A peptide was phosphorylated by PLK1, indicating that Ser708 is a PLK2-specific phosphorylation site. Together, these studies validate that STAT1 is a bona fide target of PLK2. Citation Format: Poornima Ramkumar, Rebecca S. Levin, Miriam Sansó, Shashidhar Jatiani, Arvin C. Dar, Robert P. Fisher, Kevan M. Shokat, E Premkumar Reddy. Identifying novel substrates of PLK2 using a chemical genetics approach. [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 2450. doi:10.1158/1538-7445.AM2015-2450

Abstract 2585: Discovery of a potent covalent mutant-selective EGFR inhibitor - the journey from high throughput screening to EGF816

Abstract Epidermal growth factor receptor (EGFR) is a validated therapeutic target for lung cancer. First and second generation EGFR inhibitors (e.g., gefitinib, erlotinib and afatinib) have revolutionized treatment paradigms of non-small cell lung cancer (NSCLC) patients with oncogenic EGFR mutations. The use of EGFR tyrosine kinase inhibitors (TKI) provides superior efficacy compared to chemotherapy in patients with EGFR L858R or exon 19 deletion tumors. However, resistance inevitably develops after 8-12 months of treatment; most commonly via a secondary T790M point mutation at the gatekeeper residue of EGFR. Furthermore, responses are hindered due to treatment intolerance in the form of rash and diarrhea that are mediated by simultaneous inhibition of wild-type (WT) EGFR at doses required for mutant EGFR suppression. To overcome these limitations, we initiated a project to identify mutant-selective EGFR inhibitors that potently inhibit both activating and T790M resistance EGFR mutations while sparing WT EGFR. In this presentation, we report our medicinal chemistry approach and optimization that led to the discovery of EGF816, a selective and potent covalent mutant-selective EGFR inhibitor with single digit nanomolar cellular target modulation on both activating and T790M resistance mutations. In addition, we will also report validated clinical efficacy data from the first patient treated with EGF816. Citation Format: Gerald Lelais, Robert Epple, Pierre-Yves Michellys, Thomas H. Marsilje, Yun Long, Matthew McNeill, Bei Chen, Wenshuo Lu, Badry Bursulaya, Michael DiDonato, Yong Jia, Shailaja Kasibhatla, Chun Li, Igor Matushansky, Steven Bender. Discovery of a potent covalent mutant-selective EGFR inhibitor - the journey from high throughput screening to EGF816. [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 2585. doi:10.1158/1538-7445.AM2015-2585

In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum.

We report on the in vitro effects of the bumped kinase inhibitor 1294 (BKI-1294) in cultures of virulent Neospora caninum isolates Nc-Liverpool (Nc-Liv) and Nc-Spain7 and in two strains of Toxoplasma gondii (RH and ME49), all grown in human foreskin fibroblasts. In these parasites, BKI-1294 acted with 50% inhibitory concentrations (IC50s) ranging from 20 nM (T. gondii RH) to 360 nM (N. caninum Nc-Liv), and exposure of intracellular stages to 1294 led to the nondisjunction of newly formed tachyzoites, resulting in the formation of multinucleated complexes similar to complexes previously observed in BKI-1294-treated N. caninum beta-galactosidase-expressing parasites. However, such complexes were not seen in a transgenic T. gondii strain that expressed CDPK1 harboring a mutation (G to M) in the gatekeeper residue. In T. gondii ME49 and N. caninum Nc-Liv, exposure of cultures to BKI-1294 resulted in the elevated expression of mRNA coding for the bradyzoite marker BAG1. Unlike in bradyzoites, SAG1 expression was not repressed. Immunofluorescence also showed that these multinucleated complexes expressed SAG1 and BAG1 and the monoclonal antibody CC2, which binds to a yet unidentified bradyzoite antigen, also exhibited increased labeling. In a pregnant mouse model, BKI-1294 efficiently inhibited vertical transmission in BALB/c mice experimentally infected with one of the two virulent isolates Nc-Liv or Nc-Spain7, demonstrating proof of concept that this compound protected offspring from vertical transmission and disease. The observed deregulated antigen expression effect may enhance the immune response during BKI-1294 therapy and will be the subject of future studies.

Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution.

Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib) and promiscuous (Bosutinib, Dasatinib) kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations of key mediating residues. This study has outlined mechanisms by which inhibitor binding could modulate resilience and efficiency of allosteric interactions in the kinase structures, while preserving structural topology required for catalytic activity and regulation.

Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase

Dethiobiotin synthetase (DTBS) plays a crucial role in biotin biosynthesis in microorganisms, fungi, and plants. Due to its importance in bacterial pathogenesis, and the absence of a human homologue, DTBS is a promising target for the development of new antibacterials desperately needed to combat antibiotic resistance. Here we report the first X-ray structure of DTBS from Mycobacterium tuberculosis (MtDTBS) bound to a nucleotide triphosphate (CTP). The nucleoside base is stabilized in its pocket through hydrogen-bonding interactions with the protein backbone, rather than amino acid side chains. This resulted in the unexpected finding that MtDTBS could utilise ATP, CTP, GTP, ITP, TTP, or UTP with similar Km and kcat values, although the enzyme had the highest affinity for CTP in competitive binding and surface plasmon resonance assays. This is in contrast to other DTBS homologues that preferentially bind ATP primarily through hydrogen-bonds between the purine base and the carboxamide side chain of a key asparagine. Mutational analysis performed alongside in silico experiments revealed a gate-keeper role for Asn175 in Escherichia coli DTBS that excludes binding of other nucleotide triphosphates. Here we provide evidence to show that MtDTBS has a broad nucleotide specificity due to the absence of the gate-keeper residue.

Rationally engineered flavin-dependent oxidase reveals steric control of dioxygen reduction.

The ability of flavoenzymes to reduce dioxygen varies greatly, and is controlled by the protein environment, which may cause either a rapid reaction (oxidases) or a sluggish reaction (dehydrogenases). Previously, a 'gatekeeper' amino acid residue was identified that controls the reactivity to dioxygen in proteins from the vanillyl alcohol oxidase superfamily of flavoenzymes. We have identified an alternative gatekeeper residue that similarly controls dioxygen reactivity in the grass pollen allergen Phl p 4, a member of this superfamily that has glucose dehydrogenase activity and the highest redox potential measured in a flavoenzyme. A substitution at the alternative gatekeeper site (I153V) transformed the enzyme into an efficient oxidase by increasing dioxygen reactivity by a factor of 60,000. An inverse exchange (V169I) in the structurally related berberine bridge enzyme (BBE) decreased its dioxygen reactivity by a factor of 500. Structural and biochemical characterization of these and additional variants showed that our model enzymes possess a cavity that binds an anion and resembles the 'oxyanion hole' in the proximity of the flavin ring. We showed also that steric control of access to this site is the most important parameter affecting dioxygen reactivity in BBE-like enzymes. Analysis of flavin-dependent oxidases from other superfamilies revealed similar structural features, suggesting that dioxygen reactivity may be governed by a common mechanistic principle. Structural data are available in PDB database under the accession numbers 4PVE, 4PVH, 4PVJ, 4PVK, 4PWB, 4PWC and 4PZF.

Oncogene addiction: pathways of therapeutic response, resistance, and road maps toward a cure.

A key goal of cancer therapeutics is to selectively target the genetic lesions that initiate and maintain cancer cell proliferation and survival. While most cancers harbor multiple oncogenic mutations, a wealth of preclinical and clinical data supports that many cancers are sensitive to inhibition of single oncogenes, a concept referred to as 'oncogene addiction'. Herein, we describe the clinical evidence supporting oncogene addiction and discuss common mechanistic themes emerging from the response and acquired resistance to oncogene-targeted therapies. Finally, we suggest several opportunities toward exploiting oncogene addiction to achieve curative cancer therapies.