Matthew J. Sykes

Amino acid conjugation: contribution to the metabolism and toxicity of xenobiotic carboxylic acids

Despite being the first conjugation reaction demonstrated in humans, amino acid conjugation as a route of metabolism of xenobiotic carboxylic acids is not well characterised. This is principally due to the small number and limited structural diversity of xenobiotic substrates for amino acid conjugation. Unlike CYP and uridine 5′-diphosphate glucuronosyltransferase, which are localised in the endoplasmic reticulum, the enzymes of amino acid conjugation reside in mitochondria. Unique among drug metabolism pathways, amino acid conjugation involves initial formation of a xenobiotic acyl-CoA thioester that is then conjugated principally with glycine in humans. However, formation of the xenobiotic acyl-CoA thioester does not always infer subsequent amino acid conjugation. Evidence is presented that in the absence of glycine conjugation substrates that form acyl-CoA thioesters perturb mitochondrial function. This review discusses literature on the enzymes involved and the concept that xenobiotic substrate selectivity provides a barrier to protect the metabolic integrity of the mitochondria.

Prediction of metabolism by cytochrome P450 2C9: Alignment and docking studies of a validated database of substrates

A validated database of 70 molecules known to undergo biotransformation by CYP2C9 was collated. The molecular alignment program ROCS was used with the query molecule flurbiprofen as a basis for predicting the correct active site orientation of the CYP2C9 database molecules. The quality of the results obtained was excellent, with 39 of the first 44 molecules (89%) sorted by ROCS combination score having alignments that accounted for the experimentally observed site of oxidation. Transposition of the first 39 correctly aligned molecules into the CYP2C9 active site yielded an average site of metabolism to iron heme distance of 5.21 A, in good agreement with previous experimental observations. Molecular docking studies were also undertaken, but the results were less successful than the ROCS-based alignment method, indicating that ligand-based approaches with chemical typing are important in the prediction of metabolism by CYP2C9.

Discovery of 5‐(2‐(Phenylamino)pyrimidin‐4‐yl)thiazol‐2(3H)‐one Derivatives as Potent Mnk2 Inhibitors: Synthesis, SAR Analysis and Biological Evaluation

Phosphorylation of eIF4E by human mitogen‐activated protein kinase (MAPK)‐interacting kinases (Mnks) is crucial for human tumourigenesis and development. Targeting Mnks may provide a novel anticancer therapeutic strategy. However, the lack of selective Mnk inhibitors has so far hampered pharmacological target validation and clinical drug development. Herein, we report, for the first time, the discovery of a series of 5‐(2‐(phenylamino)pyrimidin‐4‐yl)thiazole‐2(3H)‐one derivatives as Mnk inhibitors. Several derivatives demonstrate very potent Mnk2 inhibitory activity. The most active and selective compounds were tested against a panel of cancer cell lines, and the results confirm the cell‐type‐specific effect of these Mnk inhibitors. Detailed cellular mechanistic studies reveal that Mnk inhibitors are capable of reducing the expression level of anti‐apoptotic protein Mcl‐1, and of promoting apoptosis in MV4‐11 acute myeloid leukaemia cells.

Pharmacologic co-inhibition of Mnks and mTORC1 synergistically suppresses proliferation and perturbs cell cycle progression in blast crisis-chronic myeloid leukemia cells

The Ras/Raf/MAPK and PI3K/Akt/mTORC1 cascades are two most aberrantly regulated pathways in cancers. As MAPK-interacting kinases (Mnks) are part of the convergent node of these two pathways, and play a pivotal role in cellular transformation, targeting Mnks has emerged as a potential therapeutic strategy. Herein, a dual-specific Mnk1/2 inhibitor MNKI-57 and a potent Mnk2-specific inhibitor MNKI-4 were selected for a panel screen against 28 human cancer cell lines. The study reveals that MNKI-57 and MNKI-4 are most potent against leukemia cells KYO-1 (i.e. BC-CML) and KG-1 (i.e. AML). Interestingly, we found that sensitivity of selected leukemia cells to Mnk inhibitors is correlated with the level of phosphorylated 4E-BP1 at Thr70. The anti-proliferative effects of Mnk inhibitors are cytostatic in the sensitive KYO-1 cells, inducing significant G1 arrest via down-regulation of cyclin D1 expression. In KYO-1 cells where Akt is not constitutively active, Mnk inhibitors increase the sensitivity of cells to rapamycin, resulting in a more pronounced anti-proliferative activity. Remarkably, the synergistic anti-proliferative effects are associated with a marked de-phosphorylation of 4E-BP1 at Thr70. Collectively, these data highlight the importance of 4E-BP1 as a key integrator in the MAPK and mTORC1 cascades, and suggest that a combined pharmacologic inhibition of mTORC1 and Mnk kinases offers an innovative therapeutic opportunity in BC-CML.

Synthesis and extended activity of triazole-containing macrocyclic protease inhibitors.

Peptide-derived protease inhibitors are an important class of compounds with the potential to treat a wide range of diseases. Herein, we describe the synthesis of a series of triazole-containing macrocyclic protease inhibitors pre-organized into a β-strand conformation and an evaluation of their activity against a panel of proteases. Acyclic azido-alkyne-based aldehydes are also evaluated for comparison. The macrocyclic peptidomimetics showed considerable activity towards calpain II, cathepsin L and S, and the 20S proteasome chymotrypsin-like activity. Some of the first examples of highly potent macrocyclic inhibitors of cathepsin S were identified. These adopt a well-defined β-strand geometry as shown by NMR spectroscopy, X-ray analysis, and molecular docking studies.

Identification of a Novel Oligomerization Disrupting Mutation in CRYΑA Associated with Congenital Cataract in a South Australian Family

Congenital cataract is a heterogeneous disorder causing severe visual impairment in affected children. We screened four South Australian families with autosomal dominant congenital cataract for mutations in 10 crystallin genes known to cause congenital cataract. We identified a novel segregating heterozygous mutation, c.62G>A (p.R21Q), in the CRYΑA gene in one family. Western blotting of proteins freshly extracted from cataractous lens material of the proband demonstrated a marked reduction in the amount of the high‐molecular‐weight oligomers seen in the lens material of an unaffected individual. We conclude that the p.R21Q mutation, which is located in the highly conserved and structurally significant N‐terminal region of the protein, is responsible for the cataract phenotype observed in the family as this mutation likely reduces the formation of the functional oligomeric alpha‐crystallin.

Characterisation of the Binding of Cationic Amphiphilic Drugs to Phospholipid Bilayers Using Surface Plasmon Resonance

The interactions of three cationic amphiphilic drugs (CPZ, AMI, PROP) with phospholipid vesicles comprising DOPC, DMPC, or DSPC were investigated using surface plasmon resonance (SPR). Responses for CAD concentrations in the range 15.625 to 1500 μM were measured. The greatest uptake by each phospholipid bilayer occurred with CPZ. Inclusion of CAD concentrations between 750 and 1500 μM provided evidence for a second nonsaturable binding process, which may arise from intercalation of the drugs within the lipid bilayer. CAD binding was additionally shown to be dependent on membrane fluidity. Responses were initially fitted over a concentration range of 15.625 to 500 μM using a model which incorporated terms for a saturable binding site. This yielded very poor values of KD and nonsensible values of saturation responses. Subsequently, responses were fit to the expression for a model which incorporated terms for both a saturable binding site and second nonsaturable site. Measurable binding affinities (KD values ranged from 170 to 814 μM) were obtained for DOPC and DMPC bilayers which are similar to values reported previously. This work demonstrates that SPR studies with synthetic phospholipid bilayers provide a potentially useful approach for characterising drug–membrane binding interactions and for providing insight into the processes that contribute to drug–membrane binding.

Quantum-Mechanical QSAR/QSPR Descriptors from Momentum-Space Wave Functions

It is shown that quantum-mechanical descriptors obtained as parameters from the one-dimensional radial distribution function of electron momentum can be used to predict molecular activities or properties to a precision that compares favorably with the more traditional QSAR/QSPR methods. The distribution function is derived from momentum space ab initio wave functions. The predictive value of the descriptors is illustrated by their application to the estimation of McGowans volume, gas-chromatographic retention time, gas-hexadecane partition coefficient, second hyperpolarizability, and tadpole narcotic activity.

AFM study of the interaction of cytochrome P450 2C9 with phospholipid bilayers.

Cytochromes P450 (CYP) are key enzymes involved in the metabolism of drugs and other lipophilic xenobiotics and endogenous compounds. In this study, atomic force microscopy was applied to characterise the association of CYP2C9 to dimyristoylphosphatidylcholine (DMPC) supported phospholipid bilayers. CYP2C9 was found to exclusively localise in the gel domains of partially melted DMPC bilayers. Despite lacking the N-terminus transmembrane spanning domain, the CYP2C9 protein appeared to partially embed into the membrane bilayer, as evidenced by an increase in melting temperature of surrounding phospholipids. Reversible binding of CYP2C9 via an engineered His tag to a phospholipid bilayer was facilitated using nickel-chelating lipids, presenting potential applications for biosensor technologies.

Lateral heterogeneities in supported bilayers from pure and mixed phosphatidylethanolamine demonstrating hydrogen bonding capacity.

The phase behavior and lateral organization of saturated phosphatidylethanolamine (PE) and phosphatidylcholine (PC) bilayers were investigated using atomic force microscopy (AFM) and force-volume (FV) imaging for both pure and two component mixed layers. The results demonstrated the existence of unexpected segregated domains in pure PE membranes at temperatures well below the transition temperature (Tm) of the component phospholipid. These domains were of low mechanical stability and lacked the capacity for hydrogen bonding between lipid headgroups. Temperature dependent studies for different PC/PE ratios using AFM also demonstrated the mixing of these phospholipid bilayers to exhibit only a single gel to liquid transition temperature. Further work performed using FV imaging and chemically modified probes established that no lipid segregation exists at the PC/PE ratios investigated.