Renate Griffith

Therapeutic targeting of c-KIT in cancer

Introduction: Mutated forms of the receptor tyrosine kinase c-KIT are “drivers” in several cancers and are attractive targets for therapy. While benefits have been obtained from use of inhibitors of KIT kinase activity such as imatinib, especially in gastrointestinal stromal tumours (GIST), primary resistance occurs with certain oncogenic mutations. Furthermore, resistance frequently develops due to secondary mutations. Approaches to addressing both of these issues as well as combination therapies to optimise use of KIT kinase inhibitors are discussed. Areas covered: This review covers the occurrence of oncogenic KIT mutations in different cancers and the molecular basis of their action. The action of KIT kinase inhibitors, especially imatinib, sunitinib, dasatinib and PKC412, on different primary and secondary mutants is discussed. Outcomes of clinical trials in GIST, acute myeloid leukaemia (AML), systemic mastocytosis and melanoma and their implications for future directions are considered. Expert opinion: Analysis of KIT mutations in individual patients is an essential prerequisite to the use of kinase inhibitors for therapy, and monitoring for development of secondary mutations that confer drug resistance is necessary. However, it is unlikely that KIT inhibitors alone can lead to cure. KIT mutations alone do not seem to be sufficient for transformation; thus identification and co-targeting of synergistic oncogenic pathways should lead to improved outcomes.

Combination of ligand- and structure-based methods in virtual screening

The combination of ligand- and structure-based molecular modelling methods has become a common approach in virtual screening. This review describes different strategies for integration of ligand- and structure-based methods which can be divided into sequential, parallel or hybrid approaches. Although no thorough performance comparisons between combined approaches are available, examples of successful applications in prospective and retrospective virtual screening are discussed. Most published studies use a sequential approach, utilising well-documented single methods successfully.

Identification of Glutathionyl-3-hydroxykynurenine Glucoside as a Novel Fluorophore Associated with Aging of the Human Lens

A novel fluorophore was isolated from human lenses using high performance liquid chromatography (HPLC). The new fluorophore was well separated from 3-hydroxykynurenine glucoside (3-OHKG) and its deaminated isoform, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside, which are known UV filter compounds. The new compound exhibited UV absorbance maxima at 260 and 365 nm, was fluorescent (Ex360 nm/Em500 nm), and increased in concentration with age. Further analysis of the purified compound by microbore HPLC with in-line electrospray ionization mass spectrometry revealed a molecular mass of 676 Da. This mass corresponds to that of an adduct of GSH with a deaminated form of 3-OHKG. This adduct was synthesized using 3-OHKG and GSH as starting materials. The synthetic glutathionyl-3-hydroxykynurenine glucoside (GSH-3-OHKG) adduct had the same HPLC elution time, thin-layer chromatographyR F value, UV absorbance maxima, fluorescence characteristics, and mass spectrum as the lens-derived fluorophore. Furthermore, the 1H and 13C NMR spectra of the synthetic adduct were entirely consistent with the proposed structure of GSH-3-OHKG. These data indicate that GSH-3-OHKG is present as a novel fluorophore in aged human lenses. The GSH-3-OHKG adduct was found to be less reactive with β-glucosidase compared with 3-OHKG, and this could be due to a folded conformation of the adduct that was suggested by molecular modeling.

Ligand design for α1 adrenoceptor subtype selective antagonists

Abstract α1 Adrenoceptors have three subtypes and drugs interacting selectively with these subtypes could be useful in the treatment of a variety of diseases. In order to gain an insight into the structural principles governing subtype selectivity, ligand based drug design (pharmacophore development) methods have been used to design a novel 1,2,3-thiadiazole ring d analogue of the aporphine system. Synthesis and testing of this compound as a ligand on cloned and expressed human α1 adrenoceptors is described. Low binding affinity was found, possibly due to an unfavourable electrostatic potential distribution. Pharmacophore models for antagonists at the three adrenoceptor sites (α1A, α1B, α1D) were generated from a number of different training sets and their value for the design of new selective antagonists discussed. The first preliminary antagonist pharmacophore model for the α1D adrenoceptor subtype is also reported.

Resistance to c-KIT kinase inhibitors conferred by V654A mutation

Certain mutations within c-KIT cause constitutive activation of the receptor and have been associated with several human malignancies. These include gastrointestinal stromal tumors (GIST), mastocytosis, acute myelogenous leukemia, and germ cell tumors. The kinase inhibitor imatinib potently inhibits c-KIT and is approved for treatment of GIST. However, secondary point mutations can develop within the kinase domain to confer resistance to imatinib and cause drug-resistant relapse. A common mutation, which results in a V654A substitution, has been documented in imatinib-resistant GIST patients. We expressed c-KIT cDNA constructs encoding the V654A substitution alone and in combination with a typical activating exon 11 mutation characteristic of GIST, V560G, in factor-dependent FDC-P1 cells. The V654A substitution alone resulted in enhanced proliferation in c-KIT ligand (stem cell factor) but not factor independence. Cells expressing the double mutant were, like those expressing single V560G mutant c-KIT, factor independent. Analysis of cellular proliferation in the presence of imatinib showed that the V654A substitution alone conferred resistance. The difference in sensitivity was especially pronounced for cells expressing single mutant V560G c-KIT compared with double mutant V560G/V654A c-KIT. The findings were supported by studies of c-KIT phosphorylation. Analysis of the crystal structure of imatinib in complex with the kinase domain of c-KIT predicts that the V654A substitution directly affects the binding of imatinib to the receptor. Alternative c-KIT inhibitors, nilotinib (AMN107) and PKC412, were also less active on V560G/V654A c-KIT than on the V560G single mutant; however, nilotinib, like imatinib, potently inhibited the V560G mutant. PKC412 strongly inhibited imatinib-resistant D816V c-KIT. [Mol Cancer Ther 2007;6(3):1159–66]

Association of paediatric mastocytosis with a polymorphism resulting in an amino acid substitution (M541L) in the transmembrane domain of c-KIT

Background  The receptor tyrosine kinase c‐KIT plays a key role in normal mast cell development. Point mutations in c‐KIT have been associated with sporadic or familial mastocytosis.

The Search for a Topical Dual Action Spermicide/Microbicide

There is an urgent clinical need to research novel methods of fertility control that are also protective against sexually transmitted diseases (STDs) such as the human immunodeficiency virus (HIV) or Chlamydia. The most obvious way to generate such a dual-purpose contraceptive method would be to develop safe, effective spermicides that were also active against a wide range of pathogenic organisms. The currently available formulations such as nonoxynol-9, gramicidin and benzalkonium chloride are effective spermicides but are toxic to the vaginal epithelium and do not provide protection against STDs. Over 60 agents are in clinical trials as potentially safer topical spermicides and/or microbicides. Compounds that have reached this stage of development include acid buffers, detergents, dendrimers, non-nucleoside reverse transcriptase inhibitors and anionic polymers. In addition, a number of potential spermicides/microbicides are the subject of preclinical investigation, including beta-cyclodextrin, cyanovirin, porphyrins, cyclotriazadisulfonamides, dermaseptins, short-interfering RNA (siRNA) and HIV antibodies. The chemical principles underlying these disparate approaches and potential avenues for future investigation are discussed.

Novel Pharmacophore Based Methods Reveal Gossypol as a Reverse Transcriptase Inhibitor

In a program to identify new structural entities for the inhibition of the HIV-1 reverse transcriptase (RT) enzyme via database searching, a series of RT pharmacophores were developed. By utilising a novel filtering technique, the National Cancer Institute database of compounds was scanned producing 15 compounds to be screened for activity. A notable inclusion was a series of gossypol derivatives. The testing of a series of compounds revealed the parent compound gossypol to be an HIV-1 reverse transcriptase inhibitor. These results suggest that at least a part of its anti-HIV activity is due to gossypol targeting the non-nucleoside inhibitor binding pocket of RT.

Glycosylation and Sialylation of Macrophage-derived Human Apolipoprotein E Analyzed by SDS-PAGE and Mass Spectrometry EVIDENCE FOR A NOVEL SITE OF GLYCOSYLATION ON SER290

Apolipoprotein E (apoE) is a 34-kDa glycoprotein secreted from various cells including hepatocytes and macrophages and plays an important role in remnant lipoprotein clearance, immune responses, Alzheimer disease, and atherosclerosis. Cellular apoE and plasma apoE exist as multiple glycosylated and sialylated glycoforms with plasma apoE being less glycosylated/sialylated than cell-derived apoE. Some of the glycan structures on plasma apoE are characterized; however, the more complicated structures on plasma and cellular/secreted apoE remain unidentified. We investigated glycosylation and sialylation of cellular and secreted apoE from primary human macrophages by one- and two-dimensional gel electrophoresis and mass spectrometry. Our results identify eight different glycoforms with (HexNAc)2-Hex2-(NeuAc)2 being the most complex glycan detected on Thr194 in both cellular and secreted apoE. Four additional glycans were identified on apoE(283–299), and using β-elimination/alkylation by methylamine in vitro, we identified Ser290 as a novel site of glycan attachment. Comparison of plasma and cellular/secreted apoE from the same donor confirmed that cell-derived apoE is more extensively sialylated than plasma apoE. Given the importance of the C terminus of apoE in regulating apoE solubility, stability, and lipid binding, these results may have important implications for our understanding of apoE biochemistry.

Stereoselective Fluorination Alters the Geometry of a Cyclic Peptide: Exploration of Backbone‐Fluorinated Analogues of Unguisin A

New methods for enhancing the efficiency of peptide cyclization, and for fine-tuning the conformations of cyclic peptides, are valuable from a drug development perspective. Herein stereoselective fluorination is investigated as a new strategy for achieving these goals. Four vicinal difluorinated analogues of the natural cyclic heptapeptide unguisin A have been efficiently synthesized. The analogues are found to adopt dramatically different secondary structures, controlled by the fluorine stereochemistry.