Characterization of PARP1 binding to c-KIT1 G-quadruplex DNA: Insights into domain-specific interactions.

Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme involved in catalyzing Poly-(ADP-ribosyl)ation. PARP1 binds to different forms of DNA and DNA breaks and thus plays important roles in several cellular processes, including DNA damage repair, cell cycle regulation, chromatin remodeling, and maintaining genomic stability. In this study, we conducted biochemical and biophysical characterization of PARP1 binding to G-quadruplex DNA (G4-DNA). Our investigation identified ZnF1, ZnF3, and WGR as the critical domains to mediate PARP1 binding to G4-c-KIT1. Also, our results show that these domains together show cooperativity for G4-c-KIT1 recognition. Further, we establish that the presence of an oxidized (5-carboxylcytosine) base in the loop region of G4-c-KIT1 (G4-5caC-cKIT1) does not affect its recognition by PARP1. Both G4-c-KIT1 and G4-5caC-cKIT1 are potent stimulators of PARP1's catalytic activity. Our study advances the understanding of PARP1's versatile DNA binding capabilities for G4-c-KIT1 DNA irrespective of the oxidation/ modification in the DNA base. These insights into PARP1's domain-specific contributions to G4-c-KIT1 DNA recognition and catalysis expand our knowledge of its multifaceted roles in DNA repair and genome maintenance.

Heavy metal ions interactions with G-quadruplex-prone DNA sequences.

The industrial world exposes living organisms to a variety of metal pollutants. Here we investigated whether such elements affect G-rich sequences susceptible to fold into G-quadruplex (GQ) structures. Thermal stability and conformation of these oligoncleotides was studied at various molar ratios of a variety of heavy metal salts using thermal FRET, transition-FRET (t-FRET) and circular dichroism. Metal ions affected the thermal stability of the GQs to different extents; some metals had no effect on Tm while other metals caused small to moderate changes in Tm at 1:1 or 1:10 molar ratio. While most of the metals had no major effect, Al3+, Cd2+, Pb2+, Hg2+ and Zn2+ altered the thermal stability and structural features of the GQs. Some metals such as Pb2+ and Hg2+ exhibit differential interactions with telomere, c-myc and c-kit GQs. Overall, toxic heavy metals affect G-quadruplex stability in a sequence and topology dependent manner. This study provides new insight into how heavy metal exposure may affect gene expression and cellular responses.

The Inherent Coupling of Intrinsically Disordered Regions in the Multidomain Receptor Tyrosine Kinase KIT.

RTK KIT regulates a variety of crucial cellular processes via its cytoplasmic domain (CD), which is composed of the tyrosine kinase domain, crowned by the highly flexible domains-the juxtamembrane region, kinase insertion domain, and C-tail, which are key recruitment regions for downstream signalling proteins. To prepare a structural basis for the characterization of the interactions of KIT with its signalling proteins (KIT INTERACTOME), we generated the 3D model of the full-length CD attached to the transmembrane helix. This generic model of KIT in inactive state was studied by molecular dynamics simulation under conditions mimicking the natural environment of KIT. With the accurate atomistic description of the multidomain KIT dynamics, we explained its intrinsic (intra-domain) and extrinsic (inter-domain) disorder and represented the conformational assemble of KIT through free energy landscapes. Strongly coupled movements within each domain and between distant domains of KIT prove the functional interdependence of these regions, described as allosteric regulation, a phenomenon widely observed in many proteins. We suggested that KIT, in its inactive state, encodes all properties of the active protein and its post-transduction events.

Structural and functional analysis of target recognition by the lymphocyte adaptor protein LNK.

The SH2B family of adaptor proteins, SH2-B, APS, and LNK are key modulators of cellular signalling pathways. Whilst SH2-B and APS have been partially structurally and biochemically characterised, to date there has been no such characterisation of LNK. Here we present two crystal structures of the LNK substrate recognition domain, the SH2 domain, bound to phosphorylated motifs from JAK2 and EPOR, and biochemically define the basis for target recognition. The LNK SH2 domain adopts a canonical SH2 domain fold with an additional N-terminal helix. Targeted analysis of binding to phosphosites in signalling pathways indicated that specificity is conferred by amino acids one- and three-residues downstream of the phosphotyrosine. Several mutations in LNK showed impaired target binding in vitro and a reduced ability to inhibit signalling, allowing an understanding of the molecular basis of LNK dysfunction in variants identified in patients with myeloproliferative disease.

Evolution of KIPPIS as a versatile platform for evaluating intracellularly functional peptide aptamers.

Chimeric proteins have been widely used to evaluate intracellular protein-protein interactions (PPIs) in living cells with various readouts. By combining an interleukin-3-dependent murine cells and chimeric proteins containing a receptor tyrosine kinase c-kit, we previously established a c-kit-based PPI screening (KIPPIS) system to evaluate and select protein binders. In the KIPPIS components, proteins of interest are connected with a chemically inducible helper module and the intracellular domain of the growth-signaling receptor c-kit, which detects PPIs based on cell proliferation as a readout. In this system, proteins of interest can be incorporated into chimeric proteins without any scaffold proteins, which would be advantageous for evaluating interaction between small peptides/domains. To prove this superiority, we apply KIPPIS to 6 peptide aptamer-polypeptide pairs, which are derived from endogenous, synthetic, and viral proteins. Consequently, all of the 6 peptide aptamer-polypeptide interactions are successfully detected by cell proliferation. The detection sensitivity can be modulated in a helper ligand-dependent manner. The assay results of KIPPIS correlate with the activation levels of Src, which is located downstream of c-kit-mediated signal transduction. Control experiments reveal that KIPPIS clearly discriminates interacting aptamers from non-interacting ones. Thus, KIPPIS proves to be a versatile platform for evaluating the binding properties of peptide aptamers.

Nanomechanics of G-quadruplexes within the promoter of the KIT oncogene.

G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.

A new monoclonal antibody that blocks dimerisation and inhibits c-kit mutation-driven tumour growth.

PURPOSE: Imatinib, a small-molecule tyrosine kinase inhibitor, has shown good clinical activity by inhibiting adenosine triphosphate (ATP) binding to the receptor. Unfortunately, majority of patients eventually develop drug resistance, which limits the long-term benefits of the tyrosine kinase inhibitors and poses a significant challenge in the clinical management of GIST. The aim of our study was to explore the feasibility of blocking KIT dimerisation upstream of the phosphorylation in imatinib-resistant GIST. METHOD: KITMAb was prepared using hybridoma technique. The biological function of KITMAb was examined in KIT-dimer-expressing cells constructed by transfecting with liposomes using enzyme linked immunosorbent assay (ELISA), immunohistochemistry, western blot, MTT, Annexin V/FITC, and flow cytometry assay, respectively. RESULTS: KIT-dimer was expressed in 293 cells transfected with c-kit mutated-type pcDNA3.1. Treatment of KIT-dimer-expressing cells with the KITMAb significantly decreased the expression of both KIT-dimer and other phosphorylated proteins of KIT downstream signalling pathway. Furthermore, KITMAb slowed down cell growth and reduced the proportion of cells in the proliferative phase (S + G2-M). Finally, we also found that KITMAb treatment accelerated cell apoptosis. These results indicate that KITMAb strongly inhibits KIT receptor dimerisation-mediated signalling pathway and cell growth responses in vitro. CONCLUSIONS: We demonstrate c-kit mutation-driven KIT auto-dimerisation prior to tyrosine kinase phosphorylation as same as the procedure in ligand-dependent signalling pathway and describe a monoclonal antibody, KITMAb, with strong affinity to the dimerisation domain of KIT that blocks the important step in both the KIT signalling pathways. Further, the results suggest that treatment with KITMAb may be potentially therapeutic in imatinib-resistant GIST.

Molecular Modeling Study of c-KIT/PDGFRα Dual Inhibitors for the Treatment of Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumors (GISTs) are the most common Mesenchymal Neoplasm of the gastrointestinal tract. The tumorigenesis of GISTs has been associated with the gain-of-function mutation and abnormal activation of the stem cell factor receptor (c-KIT) and platelet-derived growth factor receptor alpha (PDGFRα) kinases. Hence, inhibitors that target c-KIT and PDGFRα could be a therapeutic option for the treatment of GISTs. The available approved c-KIT/PDGFRα inhibitors possessed low efficacy with off-target effects, which necessitated the development of potent inhibitors. We performed computational studies of 48 pyrazolopyridine derivatives that showed inhibitory activity against c-KIT and PDGFRα to study the structural properties important for inhibition of both the kinases. The derivative of phenylurea, which has high activities for both c-KIT (pIC50 = 8.6) and PDGFRα (pIC50 = 8.1), was used as the representative compound for the dataset. Molecular docking and molecular dynamics simulation (100 ns) of compound 14 was performed. Compound 14 showed the formation of hydrogen bonding with Cys673, Glu640, and Asp810 in c-KIT, and Cys677, Glu644, and Asp836 in PDGFRα. The results also suggested that Thr670/T674 substitution in c-KIT/PDGFRα induced conformational changes at the binding site of the receptors. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were developed based on the inhibitors. Contour map analysis showed that electropositive and bulky substituents at the para-position and the meta-position of the benzyl ring of compound 14 was favorable and may increase the inhibitory activity against both c-KIT and PDGFRα. Analysis of the results suggested that having bulky and hydrophobic substituents that extend into the hydrophobic pocket of the binding site increases the activity for both c-KIT and PDGFRα. Based on the contour map analysis, 50 compounds were designed, and the activities were predicted. An evaluation of binding free energy showed that eight of the designed compounds have potential binding affinity with c-KIT/PDGFRα. Absorption, distribution, metabolism, excretion and toxicity (ADMET) and synthetic feasibility tests showed that the designed compounds have reasonable pharmaceutical properties and synthetic feasibility. Further experimental study of the designed compounds is recommended. The structural information from this study could provide useful insight into the future development of c-KIT and PDGFRα inhibitors.

A shift of dynamic equilibrium between the KIT active and inactive states causes drug resistance.

Tyrosine phosphorylation, a highly regulated post-translational modification, is carried out by the enzyme tyrosine kinase (TK). TKs are important mediators in signaling cascades, facilitating diverse biological processes in response to stimuli. TKs may acquire mutations leading to malignancy and are viable targets for anti-cancer drugs. Mast/stem cell growth factor receptor KIT is a TK involved in cell differentiation, whose dysregulation leads to various types of cancer, including gastrointestinal stromal tumors, leukemia, and melanoma. KIT can be targeted by a range of inhibitors that predominantly bind to the inactive state of the enzyme. A mutation Y823D in the activation loop of KIT is known to be responsible for the loss of sensitivity to some drugs in metastatic tumors. We used all-atom molecular dynamics simulations to study the impact of Y823D on the KIT conformation and dynamics and compared it to the effect of phosphorylation of Y823. We simulated in total 6.4 µs of wild-type, mutant and phosphorylated KIT in the active- and inactive-state conformations. We found that Y823D affects the protein dynamics differently: in the active state, the mutation increases the protein stability, whereas in the inactive state it induces local destabilization, thus shifting the dynamic equilibrium towards the active state, altering the communication between distant regulatory regions. The observed dynamics of the Y823D mutant is similar to the dynamics of KIT phosphorylated at position Y823, thus we hypothesize that this mutation mimics a constitutively active kinase, which is not responsive to inhibitors that bind its inactive conformation.

Discovery of 8-Amino-Substituted 2-Phenyl-2,7-Naphthyridinone Derivatives as New c-Kit/VEGFR-2 Kinase Inhibitors.

The 2,7-naphthyridone scaffold has been proposed as a novel lead structure of MET inhibitors by our group. To broaden the application of this new scaffold, a series of 8-amino-substituted 2-phenyl-2,7-naphthyridin-1(2H)-one derivatives were designed and synthesized. Preliminary biological screening resulted in the discovery of a new lead of c-Kit and VEGFR-2 kinase inhibitors. Compound 9k exhibited excellent c-Kit inhibitory activity, with an IC50 value of 8.5 nM, i.e., it is 38.8-fold more potent than compound 3 (IC50 of 329.6 nM). Moreover, the compounds 10l and 10r exhibited good VEGFR-2 inhibitory activity, with IC50 values of 56.5 and 31.7 nM, respectively, i.e., they are 5.0-8.8-fold more potent than compound 3 (IC50 of 279.9 nM). Molecular docking experiments provided further insight into the binding interactions of the new lead compounds with c-Kit and VEGFR-2 kinase. In this study, an 8-amino-substituted 2-phenyl-2,7-naphthyridin-1(2H)-one scaffold was identified as the new lead structure of c-Kit and VEGFR-2 kinase inhibitors.

A non-synonymous SNP in exon 3 of the KIT gene is responsible for the classic grey phenotype in alpacas (Vicugna pacos).

The alpaca classic grey phenotype is of particular interest to the industry. Until now, there were only indirect data suggesting that the KIT gene was involved in the classic grey phenotype. All exons of KIT in three black and three classic silvergrey alpacas were sequenced. Five non-synonymous SNPs were observed. There was only one SNP found that was present only in the silvergrey alpacas, and this was also the only SNP predicted to be damaging. This variant results in a change of a glycine (Gly) to an arginine (Arg) at amino acid position 126 (c.376G>A), occurring in the second Ig-like domain of the extracellular domain of KIT. Basic protein modelling predicted that this variant is likely destabilising. Therefore, an additional 488 alpacas were genotyped for this SNP using the tetra-primer amplification refractory mutation system PCR (Tetra-primer ARMS-PCR). All classic grey alpacas were observed to be heterozygous, and 99.3% of non-grey dark base colour alpacas were found to be homozygous for the wildtype allele in this position. These results confirm that the classic grey phenotype in alpacas is the result of a c.376G>A (p.Gly126Arg) SNP in exon 3 of KIT. These data also support the hypothesis that the grey phenotype is autosomal dominant and that the mutation is most likely homozygous lethal.

Binding of quinazolinones to c-KIT G-quadruplex; an interplay between hydrogen bonding and π-π stacking.

Stabilization of G-quadruplex structures in the c-KIT promoter with the aid of ligands has become an area of great interest in potential cancer therapeutics. Understanding the binding process between ligands and G-quadruplex is essential for a discovery of selective ligands with high binding affinity to G-quadruplex. In the present work, binding mechanisms of 4-quinazolinones to c-KIT G-quadruplex were investigated theoretically by means of molecular dynamics (MD) simulations. To explore the binding affinity of ligands, binding free energy calculations were performed using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method. We demonstrate that the key interactions in G-quadruplex-ligand complexes are π-π stacking and hydrogen bond interactions. However, neither of these two interactions alone determines the stability of the G-quadruplex-ligand complexes; rather, it is the result of an intricate interplay between the two. To further examine the nature of the binding, a free energy decomposition analysis at residue level was carried out. The results clearly demonstrate the crucial roles of two hot spot residues (DG4 and DG8) for the binding of ligands to c-KIT G-quadruplex, and highlight the importance of the planar aromatic moiety of ligands in G-quadruplex stabilization via π-π stacking interactions. Our study can assist in the design of new derivatives of 4-quinazolinone with high binding affinity for c-KIT G-quadruplex.

ATP-Competitive Inhibitors Midostaurin and Avapritinib Have Distinct Resistance Profiles in Exon 17-Mutant KIT.

KIT is a type-3 receptor tyrosine kinase that is frequently mutated at exon 11 or 17 in a variety of cancers. First-generation KIT tyrosine kinase inhibitors (TKI) are ineffective against KIT exon 17 mutations, which favor an active conformation that prevents these TKIs from binding. The ATP-competitive inhibitors, midostaurin and avapritinib, which target the active kinase conformation, were developed to inhibit exon 17-mutant KIT. Because secondary kinase domain mutations are a common mechanism of TKI resistance and guide ensuing TKI design, we sought to define problematic KIT kinase domain mutations for these emerging therapeutics. Midostaurin and avapritinib displayed different vulnerabilities to secondary kinase domain substitutions, with the T670I gatekeeper mutation being selectively problematic for avapritinib. Although gatekeeper mutations often directly disrupt inhibitor binding, we provide evidence that T670I confers avapritinib resistance indirectly by inducing distant conformational changes in the phosphate-binding loop. These findings suggest combining midostaurin and avapritinib may forestall acquired resistance mediated by secondary kinase domain mutations. SIGNIFICANCE: This study identifies potential problematic kinase domain mutations for next-generation KIT inhibitors midostaurin and avapritinib.

A juxtacrine/paracrine loop between C-Kit and stem cell factor promotes cancer stem cell survival in epithelial ovarian cancer.

Receptors tyrosine kinase (RTK) enable normal and tumor cells to perceive and adapt to stimuli present in the microenvironment. These stimuli, also known as growth factors, are important molecular cues actively supporting cancer stem cell (CSC) self-renewal and viability. Since in epithelial ovarian cancer (EOC) the expression of c-Kit (CD117) has been identified as a CSC hallmark, we investigated the existence of a tumor growth-promoting loop between c-Kit and its ligand Stem Cell Factor (SCF). SCF exists as a soluble or transmembrane protein and through c-Kit interaction regulates cell viability, proliferation, and differentiation both in physiological and pathological conditions. High amounts of SCF were found in the ascitic effusions collected from EOC patients. While tumor cells and CSC only expressed the membrane-associated SCF isoform, both secreted and membrane-bound isoforms were expressed by tumor-associated macrophages (TAM, here shown to be M2-like) and fibroblasts (TAF). Circulating monocytes from EOC-bearing patients and healthy donors did not express both SCF isoforms. However, monocytes isolated from healthy donors produced SCF upon in vitro differentiation into macrophages, irrespectively of M1 or M2 polarization. In vitro, both SCF isoforms were able to activate the Akt pathway in c-Kit+ cells, and this effect was counteracted by the tyrosine kinase inhibitor imatinib. In addition, our results indicated that SCF could help c-Kit+ CSC survival in selective culture conditions and promote their canonical stemness properties, thus indicating the possible existence of a juxtacrine/paracrine circuit in EOC.

Two-quartet kit* G-quadruplex is formed via double-stranded pre-folded structure.

In the promoter of c-KIT proto-oncogene, whose deregulation has been implicated in many cancers, three G-rich regions (kit1, kit* and kit2) are able to fold into G-quadruplexes. While kit1 and kit2 have been studied in depth, little information is available on kit* folding behavior despite its key role in regulation of c-KIT transcription. Notably, kit* contains consensus sites for SP1 and AP2 transcription factors. Herein, a set of complementary spectroscopic and biophysical methods reveals that kit*, d[GGCGAGGAGGGGCGTGGCCGGC], adopts a chair type antiparallel G-quadruplex with two G-quartets at physiological relevant concentrations of KCl. Heterogeneous ensemble of structures is observed in the presence of Na+ and NH4+ ions, which however stabilize pre-folded structure. In the presence of K+ ions stacking interactions of adenine and thymine residues on the top G-quartet contribute to structural stability together with a G10•C18 base pair and a fold-back motif of the five residues at the 3'-terminal under the bottom G-quartet. The 3'-tail enables formation of a bimolecular pre-folded structure that drives folding of kit* into a single G-quadruplex. Intriguingly, kinetics of kit* G-quadruplex formation matches timescale of transcriptional processes and might demonstrate interplay of kinetic and thermodynamic factors for understanding regulation of c-KIT proto-oncogene expression.

Molecular Dynamics Simulations of the c-kit1 Promoter G-Quadruplex: Importance of Electronic Polarization on Stability and Cooperative Ion Binding.

G-quadruplexes (GQs) are guanine-rich, noncanonical nucleic acid structures that play fundamental roles in genomic stability and the regulation of gene expression. GQs are enriched in promoter sequences of growth regulatory genes and proto-oncogenes such as c-kit, which is linked to gastrointestinal stromal tumors, mast cell disease, and leukemia. While GQs have become a popular subject for experimental and computational research, the forces governing GQ dynamics are not fully understood. To gain insights into cation interactions and base-dipole moments of these highly ordered nucleic acid structures, we performed molecular dynamics simulations on the c-kit1 GQ using the CHARMM36 additive and Drude-2017 polarizable force fields. These simulations are the first of their kind to investigate the role of electronic polarization on interactions dictating GQ conformational sampling and cation interactions. Use of a polarizable model revealed differences in base dipole moments between GQs and B-form duplex DNA, force field-dependent ion binding pathways, and allowed for quantification of multibody contributions of water to ion-GQ interactions. These results emphasize the importance of electronic polarization as a contribution to the forces underlying nucleic acid dynamics.

KIT as a therapeutic target for non-oncological diseases.

KIT is a receptor tyrosine kinase that after binding to its ligand stem cell factor activates signaling cascades linked to biological processes such as proliferation, differentiation, migration and cell survival. Based on studies performed on SCF and/or KIT mutant animals that presented anemia, sterility, and/or pigmentation disorders, KIT signaling was mainly considered to be involved in the regulation of hematopoiesis, gametogenesis, and melanogenesis. More recently, novel animal models and ameliorated cellular and molecular techniques have led to the discovery of a widen repertoire of tissue compartments and functions that are being modulated by KIT. This is the case for the lung, heart, nervous system, gastrointestinal tract, pancreas, kidney, liver, and bone. For this reason, the tyrosine kinase inhibitors that were originally developed for the treatment of hemato-oncological diseases are being currently investigated for the treatment of non-oncological disorders such as asthma, rheumatoid arthritis, and alzheimer's disease, among others. The beneficial effects of some of these tyrosine kinase inhibitors have been proven to depend on KIT inhibition. This review will focus on KIT expression and regulation in healthy and pathologic conditions other than cancer. Moreover, advances in the development of anti-KIT therapies, including tyrosine kinase inhibitors, and their application will be discussed.

Residue-level determinants of angiopoietin-2 interactions with its receptor Tie2.

We combined computational and experimental methods to interrogate the binding determinants of angiopoietin-2 (Ang2) to its receptor tyrosine kinase (RTK) Tie2-a central signaling system in angiogenesis, inflammation, and tumorigenesis. We used physics-based electrostatic and surface-area calculations to identify the subset of interfacial Ang2 and Tie2 residues that can affect binding directly. Using random and site-directed mutagenesis and yeast surface display (YSD), we validated these predictions and identified additional Ang2 positions that affected receptor binding. We then used burial-based calculations to classify the larger set of Ang2 residues that are buried in the Ang2 core, whose mutations can perturb the Ang2 structure and thereby affect interactions with Tie2 indirectly. Our analysis showed that the Ang2-Tie2 interface is dominated by nonpolar contributions, with only three Ang2 and two Tie2 residues that contribute electrostatically to intermolecular interactions. Individual interfacial residues contributed only moderately to binding, suggesting that engineering of this interface will require multiple mutations to reach major effects. Conversely, substitutions in substantially buried Ang2 residues were more prevalent in our experimental screen, reduced binding substantially, and are therefore more likely to have a deleterious effect that might contribute to oncogenesis. Computational analysis of additional RTK-ligand complexes, c-Kit-SCF and M-CSF-c-FMS, and comparison to previous YSD results, further show the utility of our combined methodology.

Molecular mechanism of D816X mutation-induced c-Kit activation and -mediated inhibitor resistance in gastrointestinal stromal tumor.

The D816X (X = V, H, Y or F) missense mutation constitutively activates c-Kit kinase in gastrointestinal stromal tumor (GIST) and has been observed to cause acquired resistance against first-line and second-line kinase inhibitors. In the present study, the allosteric mechanism of D816X-induced c-Kit conformational change is investigated at molecular level. The Asp816 residue is located at the activation loop (A-loop) of c-Kit and the mutation can eliminate a negative formal charge from the loop region by substituting the acidic asparagic acid residue with neutral valine, histidine, tyrosine or phenylalanine. Here, we classify the c-Kit kinase into four states in terms of its mutation (wild type or mutant) and conformation (DFG-in or DFG-out). The wild-type kinase is electrostatically stabilized in inactive DFG-out conformation, whereas the D816X mutation can promote the conformational conversion to active DFG-in and then activate the kinase. Structural analysis reveals that the Asp816 residue in DFG-out is surrounded by a number of polar and positively charged residues within its first and second shells of protein context, and kinase conformational change to DFG-in brings this residue into a negative electrostatic potential environment. Dynamics simulation characterizes that the c-Kit conformational conversion from DFG-out to DFG-in can cause local unfavorable effect to type-II inhibitor, while the mutation-induced global structural rearrangement would participate in the favorable interaction of c-Kit with type-I inhibitor.

The role of small molecule Kit protein-tyrosine kinase inhibitors in the treatment of neoplastic disorders.

The Kit proto-oncogene was found as a consequence of the discovery of the feline v-kit sarcoma oncogene. Stem cell factor (SCF) is the Kit ligand and it mediates Kit dimerization and activation. The Kit receptor contains an extracellular segment that is made up of five immunoglobulin-like domains (D1/2/3/4/5), a transmembrane segment, a juxtamembrane segment, a protein-tyrosine kinase domain that contains an insert of 77 amino acid residues, and a carboxyterminal tail. Activating somatic mutations in Kit have been documented in various neoplasms including gastrointestinal stromal tumors (GIST), mast cell overexpression (systemic mastocytosis), core-binding factor acute myeloid leukemias (AML), melanomas, and seminomas. In the case of gastrointestinal stromal tumors, most activating mutations occur in the juxtamembrane segment and these mutants are initially sensitive to imatinib. As with many targeted anticancer drugs, resistance to Kit antagonists occurs in about two years and is the result of secondary KIT mutations. An activation segment exon 17 D816V mutation is one of the more common resistance mutations in Kit and this mutant is resistant to imatinib and sorafenib. Type I protein kinase inhibitors interact with the active enzyme form with DFG-D of the proximal activation segment directed inward toward the active site (DFG-Din). In contrast, type II inhibitors bind to their target with the DFG-D pointing away from the active site (DFG-Dout). Based upon the X-ray crystallographic structures, imatinib, sunitinib, and ponatinib are Type II Kit inhibitors. We used the Schrödinger induced fit docking protocol to model the interaction of midostaurin with Kit and the result indicates that it binds to the DFG-Din conformation of the receptor and is thus classified as type I inhibitor. This medication inhibits the notoriously resistant Kit D816V mutant and is approved for the treatment of systemic mastocytosis and is effective against tumors bearing the D816V activation/resistance mutation.