Selective AKR1C3 inhibitors do not recapitulate the anti-leukaemic activities of the pan-AKR1C inhibitor medroxyprogesterone acetate

Background: We and others have identified the aldo-keto reductase AKR1C3 as a potential drug target in prostate cancer, breast cancer and leukaemia. As a consequence, significant effort is being invested in the development of AKR1C3-selective inhibitors. Methods: We report the screening of an in-house drug library to identify known drugs that selectively inhibit AKR1C3 over the closely related isoforms AKR1C1, 1C2 and 1C4. This screen initially identified tetracycline as a potential AKR1C3-selective inhibitor. However, mass spectrometry and nuclear magnetic resonance studies identified that the active agent was a novel breakdown product (4-methyl(de-dimethylamine)-tetracycline (4-MDDT)). Results: We demonstrate that, although 4-MDDT enters AML cells and inhibits their AKR1C3 activity, it does not recapitulate the anti-leukaemic actions of the pan-AKR1C inhibitor medroxyprogesterone acetate (MPA). Screens of the NCI diversity set and an independently curated small-molecule library identified several additional AKR1C3-selective inhibitors, none of which had the expected anti-leukaemic activity. However, a pan AKR1C, also identified in the NCI diversity set faithfully recapitulated the actions of MPA. Conclusions: In summary, we have identified a novel tetracycline-derived product that provides an excellent lead structure with proven drug-like qualities for the development of AKR1C3 inhibitors. However, our findings suggest that, at least in leukaemia, selective inhibition of AKR1C3 is insufficient to elicit an anticancer effect and that multiple AKR1C inhibition may be required. © 2014 Cancer Research UK. All rights reserved.

Recent advances in the development of tissue transglutaminase (TG2) inhibitors

Tissue transglutaminase (TG2) is a Ca2+-dependent enzyme and probably the most ubiquitously expressed member of the mammalian transglutaminase family. TG2 plays a number of important roles in a variety of biological processes. Via its transamidating function, it is responsible for the cross-linking of proteins by forming isopeptide bonds between glutamine and lysine residues. Intracellularly, Ca2+ activation of the enzyme is normally tightly regulated by the binding of GTP. However, upregulated levels of TG2 are associated with many disease states like celiac sprue, certain types of cancer, fibrosis, cystic fibrosis, multiple sclerosis, Alzheimer's, Huntington's and Parkinson's disease. Selective inhibitors for TG2 both cell penetrating and non-cell penetrating would therefore serve as novel therapeutic tools for the treatment of these disease states. Moreover, they would provide useful tools to fully elucidate the cellular mechanisms TG2 is involved in and help comprehend how the enzyme is regulated at the cellular level. The current paper is intended to give an update on the recently discovered classes of TG2 inhibitors along with their structure-activity relationships. The biological properties of these derivatives, in terms of both activity and selectivity, will also be reported in order to translate their potential for future therapeutic developments. © 2011 Springer-Verlag.

Renal glucose reabsorption inhibitors to treat diabetes

Current therapies to reduce hyperglycaemia in type 2 diabetes mellitus (T2DM) mostly involve insulin-dependent mechanisms and lose their effectiveness as pancreatic ß-cell function declines. In the kidney, filtered glucose is reabsorbed mainly via the high-capacity, low-affinity sodium glucose cotransporter-2 (SGLT2) at the luminal surface of cells lining the first segment of the proximal tubules. Selective inhibitors of SGLT2 reduce glucose reabsorption, causing excess glucose to be eliminated in the urine; this decreases plasma glucose. In T2DM, the glucosuria produced by SGLT2 inhibitors is associated with weight loss, and mild osmotic diuresis might assist a reduction in blood pressure. The mechanism is independent of insulin and carries a low risk of hypoglycaemia. This review examines the potential of SGLT2 inhibitors as a novel approach to the treatment of hyperglycaemia in T2DM.

The stabilisation of purified, reconstituted P-glycoprotein by freeze drying with disaccharides

The drug efflux pump P-glycoprotein (P-gp) (ABCB1) confers multidrug resistance, a major cause of failure in the chemotherapy of tumours, exacerbated by a shortage of potent and selective inhibitors. A high throughput assay using purified P-gp to screen and characterise potential inhibitors would greatly accelerate their development. However, long-term stability of purified reconstituted ABCB1 can only be reliably achieved with storage at -80 °C. For example, at 20 °C, the activity of ABCB1 was abrogated with a half-life of <1 day. The aim of this investigation was to stabilise purified, reconstituted ABCB1 to enable storage at higher temperatures and thereby enable design of a high throughput assay system. The ABCB1 purification procedure was optimised to allow successful freeze drying by substitution of glycerol with the disaccharides trehalose or maltose. Addition of disaccharides resulted in ATPase activity being retained immediately following lyophilisation with no significant difference between the two disaccharides. However, during storage trehalose preserved ATPase activity for several months regardless of the temperature (e.g. 60% retention at 150 days), whereas ATPase activity in maltose purified P-gp was affected by both storage time and temperature. The data provide an effective mechanism for the production of resilient purified, reconstituted ABCB1.

The release of nitric oxide from S-nitrosothiols promotes angiogenesis

Free nitric oxide (NO) reacts with sulphydryl residues to form S-nitrosothiols, which act as NO reservoirs. We sought to determine whether thiol-preserving agents and antioxidants, such as dithiothreitol (DTT) and vitamin C, induce NO release from S-nitrosylated proteins in endothelial cell cultures to promote angiogenesis. NO release was measured directly in cell supernatants using a Sievers NO Analyser, and in vitro angiogenesis was assessed by quantifying capillary-like tube network formation of porcine aortic endothelial cells (PAEC) on growth factor-reduced Matrigel. Incubation of PAEC with DTT or vitamin C significantly increased NO release in a concentration-dependent manner. However, the nitric oxide synthase (NOS) inhibitors, L-NNA and L-NIO, had no effect on DTT- or vitamin C-induced NO release, and there was no concomitant increase in the phosphorylation of endothelial NOS at serine-1177 following DTT or vitamin C treatment. DTT and vitamin C increased capillary-like tube network formation by nine- and two-fold, respectively, and the addition of copper ions doubled the effect of vitamin C. Surprisingly, DTT maintained endothelial tube networks for up to one month under serum-free conditions, and selective inhibitors of guanylyl cyclase (ODQ) and PKG (KT-5823) blocked this, demonstrating the requirement of cyclic GMP and PKG in this process. Both DTT and vitamin C are capable of releasing sufficient NO from S-nitrosothiols to induce capillary morphogenesis. This study provides the first evidence that increased denitrosylation leads to increased bioavailability of NO, independent of NOS activity, to promote sustained angiogenesis.

Development of potent and selective tissue transglutaminase inhibitors:their effect on TG2 function and application in pathological conditions

Potent-selective peptidomimetic inhibitors of tissue transglutaminase (TG2) were developed through a combination of protein-ligand docking and molecular dynamic techniques. Derivatives of these inhibitors were made with the aim of specific TG2 targeting to the intra- and extracellular space. A cell-permeable fluorescently labeled derivative enabled detection of in situ cellular TG2 activity in human umbilical cord endothelial cells and TG2-transduced NIH3T3 cells, which could be enhanced by treatment of cells with ionomycin. Reaction of TG2 with this fluorescent inhibitor in NIH3T3 cells resulted in loss of binding of TG2 to cell surface syndecan-4 and inhibition of translocation of the enzyme into the extracellular matrix, with a parallel reduction in fibronectin deposition. In human umbilical cord endothelial cells, this same fluorescent inhibitor also demonstrated a reduction in fibronectin deposition, cell motility, and cord formation in Matrigel. Use of the same inhibitor in a mouse model of hypertensive nephrosclerosis showed over a 40% reduction in collagen deposition.

Design, synthesis and evaluation of cyclothialidine analogues as DNA gyrase inhibitors

Cyclothialidine, a natural product isolated from Streptomyces .filipinensis NR0484, has been proven to be a potent and selective inhibitor of the bacterial enzyme DNA gyrase. Gyrase inhibition results in cell death, the enzyme being the target of several currently used antibiotics. Cyclothialidine showed poor activity against whole bacterial cells, highlighting scope for improvement regarding cell membrane pemeability in order for the full potential of this new class of antibiotics to be realised, Structurally, cyclothialidine contains a 12-membered lactone ring which is partly integrated into a pentapeptide chain, with a substituted aromatic moiety bordering the lactone, Retrosynthetically it can be traced back to cis-3-hydroxyproline, 3,5-dihydroxy-2,6-dimethylbenzoic acid and four commercially available amino acids; two serine, one cysteine and one alanine. In this work, a model of cyclothialidine was synthesised in order to establish the methodology for more complex compounds. Analogues with hydroxy, dihydroxy and dihydroxymethyl substituted aromatic moieties were then prepared to ensure successful protection methods could be performed and the pharmacophore synthesised. The key aromatic moiety, 2,6-dimethyl-3,5-dihydroxybenzoic acid was produced via two successive Mannich reaction/reduction steps. Acid protection using 4-nitrobenzyl bromide and TBDMS hydroxyl protection followed by bromination of one methyl afforded the desired intermediate. Reaction with a serine/cysteine dipeptide, followed by deprotection and cyclisation under Mitsunobu conditions lead to the 12-membered lactone. An amine substituted aromatic analogue and also replacement of the cysteine sulphur by oxygen were attempted but without success. In an effort to improve cell permeability, a conjugate was synthesised between the pharmacophore and a cholesterol moiety. It was hoped the steroid fragment would serve to increase potency by escorting the molecule through the lipid environment of the cell membrane. The pharmacophore and conjugate were tested against a variety of bacterial strains but the conjugate failed to improve activity.

Flavones and related compounds as inhibitors of protein tyrosine kinases

Aberrant tyrosine protein kinase activity has been implicated in the formation and maintenance of malignancy and so presents a potential target for cancer chemotherapy. Quercetin, a naturally occuring flavonoid, inhibits the tyrosine protein kinase encoded by the Rous sarcoma virus but also exhibits many other effects. Analogues of this compound were synthesised by the acylation of suitable 2-hydroxyacetophenones with appropriately substituted aromatic (or alicyclic) acid chlorides, followed by base catalysed rearrangement to the 1-(2-hydroxyphenyl)-3-phenylpropan-1,3-diones. Acid catalysed ring closure furnished flavones. The majority of the 1-(2-hydroxyphenyl)-3-phenylpropan-1,3-diones were shown by NMR to exist in the enol form. This was supported by the crystal structure of 1-(2-hydroxy-4-methoxyphenyl)-3-phenylpropan-1,3-dione. In contrast, 1.(4,6-dimethoxy-2-hydroxyphenyl)-3-phenylpropan-1,3-dione did not exhibit keto-enol tautomerism in the NMR spectrum and was shown in its crystal structure to assume a twisted conformation. Assessment of the biological activity of the analogues of quercetin was carried out using whole cells and the kinase domain of the tyrosine protein kinase encoded by the Abelson murine leukaemia virus, ptab150 kinase. Single cell suspension cultures and clonogenic potential of murine fibroblasts transformed by the Abelson Murine leukaemia virus (ANN-1 cells) did not indicate the existence of any structure activity relationship required for cytotoxicity or cytostasis. No selective toxicity was apparent when the `normal' parent cell line, (3T3), was used to assess the cytotoxic potential of quercetin. The ICS50 for these compounds were generally in the region of 1-100M. The potential for these compounds to inhibit ptab150 kinase was determined. A definite substitution requirement emerged from these experiments indicating a necessity for substituents in the A ring or in the 3-position of the flavone nucleus. Kinetic data showed these inhibitors to be competitive for ATP.

The neurotoxic effects of enzyme inhibitors at the neuromuscular junction

Current knowledge of the long-term, low dose effects of carbamate (CB) anti-cholinesterases on skeletal muscle or on the metabolism and regulation of the molecular forms of acetylcholinesterase (AChE) is limited. This is largely due to the reversible nature of these inhibitors and the subtle effects they induce which has generally made their study difficult and preliminary investigations were conducted to determine suitable study methods. A sequential extraction technique was used to rapidly analyse AChE molecular form activity at the mouse neuromuscular junction and also in peripheral parts of muscle fibres. AChE in the synaptic cleft involved in the termination of cholinergic transmission was successfully assessed by the assay method and by an alternative method using a correlation equation which represented the relationship between synaptic AChE and the prolongation of extra-cellular miniature endplate potentials. It was found that inhibition after in vivo Carbamate (CB) dosing could not be maintained during tissue analysis because CB-inhibited enzyme complexes decarbamoylated vary rapidly and could not be prevented even when maintained on ice. The methods employed did not therefore give a measure of inhibition but presented a profile of metabolic responses to continual, low dose CB treatment. Repetitive and continual infusion with low doses of the CBs: pyridostigmine and physostigmine induced a variety of effects on mouse skeletal muscle. Both compounds induced a mild myopathy in the mouse diaphragm during continual infusion which was characterised by endplate deformation without necrosis; such deformation persisted on termination of treatment but had recovered slightly 14 days later. Endplate and non-endplate AChE molecular forms displayed selective responses to CB treatment. During treatment endplate AChE was reduced whereas non-endplate AChE was largely unaffected, and after treatment, endplate AChE recovered, whereas non-endplate AChE was up-regulated. The mechanisms by which these responses become manifest are unclear but may be due to CB-induced effects on nerve-mediated muscle activity, neurotrophic factors or morphological and physiological changes which arise at the neuromuscular junction. It was concluded that, as well as inhibiting AChE, CBs also influence the metabolism and regulation of the enzyme and induce persistent endplate deformation; possible detrimental effects of long-term, low-dose determination requires further investigation.