The role of protein kinase C isoenzymes in the pathogenesis of human autoimmune diseases.

The physiological role of protein kinase C (PKC) enzymes in the immune system is presented briefly. From earlier publications of others data were collected how the defects of one/two isoenzymes of PKC system suggested their involvement in the pathogenesis of human autoimmune diseases. Our observations on the defects of seven PKC isoenzymes in the peripheral blood mononuclear cells (PBMC) demonstrate that these molecular impairments are not prerequisits of the pathogenesis of systemic lupus erythematosus (SLE), mixed connective tissue disease and Sjögren's syndrome. However, these defects can modulate the disease activity and symptoms especially in SLE by several pathways. The role of PKC system in other forms of autoimmune diseases is also very small. It was of note that we detected decreased expression of PKC isoenzymes in PBMC of a European white family with an X-linked genetic background showing seasonal undulations in the lupus patient and also in her healthy mother.

Glycogen phosphorylase inhibitor, 2,3-bis[(2E)-3-(4-hydroxyphenyl)prop-2-enamido] butanedioic acid (BF142), improves baseline insulin secretion of MIN6 insulinoma cells.

Type 2 diabetes mellitus (T2DM), one of the most common metabolic diseases, is characterized by insulin resistance and inadequate insulin secretion of ß cells. Glycogen phosphorylase (GP) is the key enzyme in glycogen breakdown, and contributes to hepatic glucose production during fasting or during insulin resistance. Pharmacological GP inhibitors are potential glucose lowering agents, which may be used in T2DM therapy. A natural product isolated from the cultured broth of the fungal strain No. 138354, called 2,3-bis(4-hydroxycinnamoyloxy)glutaric acid (FR258900), was discovered a decade ago. In vivo studies showed that FR258900 significantly reduced blood glucose levels in diabetic mice. We previously showed that GP inhibitors can potently enhance the function of ß cells. The purpose of this study was to assess whether an analogue of FR258900 can influence ß cell function. BF142 (Meso-Dimethyl 2,3-bis[(E)-3-(4-acetoxyphenyl)prop-2-enamido]butanedioate) treatment activated the glucose-stimulated insulin secretion pathway, as indicated by enhanced glycolysis, increased mitochondrial oxidation, significantly increased ATP production, and elevated calcium influx in MIN6 cells. Furthermore, BF142 induced mTORC1-specific phosphorylation of S6K, increased levels of PDX1 and insulin protein, and increased insulin secretion. Our data suggest that BF142 can influence ß cell function and can support the insulin producing ability of ß cells.

Glucose-based spiro-oxathiazoles as in vivo anti-hyperglycemic agents through glycogen phosphorylase inhibition.

The design of glycogen phosphorylase (GP) inhibitors targeting the catalytic site of the enzyme is a promising strategy for a better control of hyperglycaemia in the context of type 2 diabetes. Glucopyranosylidene-spiro-heterocycles have been demonstrated as potent GP inhibitors, and more specifically spiro-oxathiazoles. A new synthetic route has now been elaborated through 1,3-dipolar cycloaddition of an aryl nitrile oxide to a glucono-thionolactone affording in one step the spiro-oxathiazole moiety. The thionolactone was obtained from the thermal rearrangement of a thiosulfinate precursor according to Fairbanks' protocols, although with a revisited outcome and also rationalised with DFT calculations. The 2-naphthyl substituted glucose-based spiro-oxathiazole 5h, identified as one of the most potent GP inhibitors (Ki = 160 nM against RMGPb) could be produced on the gram-scale from this strategy. Further evaluation in vitro using rat and human hepatocytes demonstrated that compound 5h is a anti-hyperglycaemic drug candidates performing slightly better than DAB used as a positive control. Investigation in Zucker fa/fa rat model in acute and subchronic assays further confirmed the potency of compound 5h since it lowered blood glucose levels by ∼36% at 30 mg kg-1 and ∼43% at 60 mg kg-1. The present study is one of the few in vivo investigations for glucose-based GP inhibitors and provides data in animal models for such drug candidates.

A multidisciplinary study of 3-(ß-d-glucopyranosyl)-5-substituted-1,2,4-triazole derivatives as glycogen phosphorylase inhibitors: Computation, synthesis, crystallography and kinetics reveal new potent inhibitors.

3-(ß-d-Glucopyranosyl)-5-substituted-1,2,4-triazoles have been revealed as an effective scaffold for the development of potent glycogen phosphorylase (GP) inhibitors but with the potency very sensitive to the nature of the alkyl/aryl 5-substituent (Kun et al., Eur. J. Med. Chem. 2014, 76, 567). For a training set of these ligands, quantum mechanics-polarized ligand docking (QM-PLD) demonstrated good potential to identify larger differences in potencies (predictive index PI = 0.82) and potent inhibitors with Ki's < 10 µM (AU-ROC = 0.86). Accordingly, in silico screening of 2335 new analogues exploiting the ZINC docking database was performed and nine predicted candidates selected for synthesis. The compounds were prepared in O-perbenzoylated forms by either ring transformation of 5-ß-d-glucopyranosyl tetrazole by N-benzyl-arenecarboximidoyl chlorides, ring closure of C-(ß-d-glucopyranosyl)formamidrazone with aroyl chlorides, or that of N-(ß-d-glucopyranosylcarbonyl)arenethiocarboxamides by hydrazine, followed by deprotections. Kinetics experiments against rabbit muscle GPb (rmGPb) and human liver GPa (hlGPa) revealed five compounds as potent low µM inhibitors with three of these on the submicromolar range for rmGPa. X-ray crystallographic analysis sourced the potency to a combination of favorable interactions from the 1,2,4-triazole and suitable aryl substituents in the GP catalytic site. The compounds also revealed promising calculated pharmacokinetic profiles.

Glycogen phosphorylase inhibition improves beta cell function.

BACKGROUND AND PURPOSE: Glycogen phosphorylase (GP) is the key enzyme for glycogen degradation. GP inhibitors (GPi-s) are glucose lowering agents that cause the accumulation of glucose in the liver as glycogen. Glycogen metabolism has implications in beta cell function. Glycogen degradation can maintain cellular glucose levels, which feeds into catabolism to maintain insulin secretion, and elevated glycogen degradation levels contribute to glucotoxicity. The purpose of this study was to assess whether influencing glycogen metabolism in beta cells by GPi-s affects the function of these cells. EXPERIMENTAL APPROACH: The effects of structurally different GPi-s were investigated on MIN6 insulinoma cells and in a mouse model of diabetes. KEY RESULTS: GPi treatment increased glycogen content and, consequently, the surface area of glycogen in MIN6 cells. Furthermore, GPi treatment induced insulin receptor ß (InsRß), Akt and p70S6K phosphorylation, as well as pancreatic and duodenal homeobox 1(PDX1) and insulin expression. In line with these findings, GPi-s enhanced non-stimulated and glucose-stimulated insulin secretion in MIN6 cells. The InsRß was shown to co-localize with glycogen particles as confirmed by in silico screening, where components of InsR signalling were identified as glycogen-bound proteins. GPi-s also activated the pathway of insulin secretion, indicated by enhanced glycolysis, mitochondrial oxidation and calcium signalling. Finally, GPi-s increased the size of islets of Langerhans and improved glucose-induced insulin release in mice. CONCLUSION AND IMPLICATIONS: These data suggest that GPi-s also target beta cells and can be repurposed as agents to preserve beta cell function or even ameliorate beta cell dysfunction in different forms of diabetes. LINKED ARTICLES: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Nanomolar Inhibitors of Glycogen Phosphorylase Based on ß-d-Glucosaminyl Heterocycles: A Combined Synthetic, Enzyme Kinetic, and Protein Crystallography Study.

Aryl substituted 1-(ß-d-glucosaminyl)-1,2,3-triazoles as well as C-ß-d-glucosaminyl 1,2,4-triazoles and imidazoles were synthesized and tested as inhibitors against muscle and liver isoforms of glycogen phosphorylase (GP). While the N-ß-d-glucosaminyl 1,2,3-triazoles showed weak or no inhibition, the C-ß-d-glucosaminyl derivatives had potent activity, and the best inhibitor was the 2-(ß-d-glucosaminyl)-4(5)-(2-naphthyl)-imidazole with a Ki value of 143 nM against human liver GPa. An X-ray crystallography study of the rabbit muscle GPb inhibitor complexes revealed structural features of the strong binding and offered an explanation for the differences in inhibitory potency between glucosyl and glucosaminyl derivatives and also for the differences between imidazole and 1,2,4-triazole analogues.

Synthetic, enzyme kinetic, and protein crystallographic studies of C-ß-d-glucopyranosyl pyrroles and imidazoles reveal and explain low nanomolar inhibition of human liver glycogen phosphorylase.

C-ß-d-Glucopyranosyl pyrrole derivatives were prepared in the reactions of pyrrole, 2-, and 3-aryl-pyrroles with O-peracetylated ß-d-glucopyranosyl trichloroacetimidate, while 2-(ß-d-glucopyranosyl) indole was obtained by a cross coupling of O-perbenzylated ß-d-glucopyranosyl acetylene with N-tosyl-2-iodoaniline followed by spontaneous ring closure. An improved synthesis of O-perbenzoylated 2-(ß-d-glucopyranosyl) imidazoles was achieved by reacting C-glucopyranosyl formimidates with α-aminoketones. The deprotected compounds were assayed with isoforms of glycogen phosphorylase (GP) to show no activity of the pyrroles against rabbit muscle GPb. The imidazoles proved to be the best known glucose derived inhibitors of not only the muscle enzymes (both a and b) but also of the pharmacologically relevant human liver GPa (Ki = 156 and 26 nM for the 4(5)-phenyl and -(2-naphthyl) derivatives, respectively). An X-ray crystallographic study of the rmGPb-imidazole complexes revealed structural features of the strong binding, and also allowed to explain the absence of inhibition for the pyrrole derivatives.

C-Glucopyranosyl-1,2,4-triazol-5-ones: synthesis and inhibition of glycogen phosphorylase.

Various C-glucopyranosyl-1,2,4-triazolones were designed as potential inhibitors of glycogen phosphorylase. Syntheses of these compounds were performed with O-perbenzoylated glucose derivatives as precursors. High temperature ring closure of N(1)-carbamoyl-C-ß-D-glucopyranosyl formamidrazone gave 3-ß-D-glucopyranosyl-1,2,4-triazol-5-one. Reaction of N(1)-tosyl-C-ß-D-glucopyranosyl formamidrazone with ClCOOEt furnished 3-ß-D-glucopyranosyl-1-tosyl-1,2,4-triazol-5-one. In situ prepared ß-D-glucopyranosylcarbonyl isocyanate was transformed by PhNHNHBoc into 3-ß-D-glucopyranosyl-1-phenyl-1,2,4-triazol-5-one, while the analogous 1-(2-naphthyl) derivative was obtained from the unsubstituted triazolone by naphthalene-2-boronic acid in a Cu(II) catalyzed N-arylation. Test compounds were prepared by Zemplén deacylation. The new glucose derivatives had weak or no inhibition of rabbit muscle glycogen phosphorylase b: the best inhibitor was 3-ß-D-glucopyranosyl-1-(2-naphthyl)-1,2,4-triazol-5-one (Ki = 80 µM).

PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines.

Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment.

Glucose-derived spiro-isoxazolines are anti-hyperglycemic agents against type 2 diabetes through glycogen phosphorylase inhibition.

Glycogen phosphorylase (GP) is a target for the treatment of hyperglycaemia in the context of type 2 diabetes. This enzyme is responsible for the depolymerization of glycogen into glucose thereby affecting the levels of glucose in the blood stream. Twelve new d-glucopyranosylidene-spiro-isoxazolines have been prepared from O-peracylated exo-D-glucals by regio- and stereoselective 1,3-dipolar cycloaddition of nitrile oxides generated in situ by treatment of the corresponding oximes with bleach. This mild and direct procedure appeared to be applicable to a broad range of substrates. The corresponding O-unprotected spiro-isoxazolines were evaluated as glycogen phosphorylase (GP) inhibitors and exhibited IC50 values ranging from 1 to 800 µM. Selected inhibitors were further evaluated in vitro using rat and human hepatocytes and exhibited significant inhibitory properties in the primary cell culture. Interestingly, when tested with human hepatocytes, the tetra-O-acetylated spiro-isoxazoline bearing a 2-naphthyl residue showed a much lower IC50 value (2.5 µM), compared to that of the O-unprotected analog (19.95 µM). The most promising compounds were investigated in Zucker fa/fa rat model in acute and sub-chronic assays and decreased hepatic glucose production, which is known to be elevated in type 2 diabetes. This indicates that glucose-based spiro-isoxazolines can be considered as anti-hyperglycemic agents in the context of type 2 diabetes.

4(5)-Aryl-2-C-glucopyranosyl-imidazoles as New Nanomolar Glucose Analogue Inhibitors of Glycogen Phosphorylase.

Inhibition of glycogen phosphorylases may lead to pharmacological treatments of diseases in which glycogen metabolism plays an important role: first of all in diabetes, but also in cardiovascular and tumorous disorders. C-(ß-d-Glucopyranosyl) isoxazole, pyrazole, thiazole, and imidazole type compounds were synthesized, and the latter showed the strongest inhibition against rabbit muscle glycogen phosphorylase b. Most efficient was 2-(ß-d-glucopyranosyl)-4(5)-(2-naphthyl)-imidazole (11b, K i = 31 nM) representing the best nanomolar glucose derived inhibitor of the enzyme.

Polymodal Transient Receptor Potential Vanilloid (TRPV) Ion Channels in Chondrogenic Cells.

Mature and developing chondrocytes exist in a microenvironment where mechanical load, changes of temperature, osmolarity and acidic pH may influence cellular metabolism. Polymodal Transient Receptor Potential Vanilloid (TRPV) receptors are environmental sensors mediating responses through activation of linked intracellular signalling pathways. In chondrogenic high density cultures established from limb buds of chicken and mouse embryos, we identified TRPV1, TRPV2, TRPV3, TRPV4 and TRPV6 mRNA expression with RT-PCR. In both cultures, a switch in the expression pattern of TRPVs was observed during cartilage formation. The inhibition of TRPVs with the non-selective calcium channel blocker ruthenium red diminished chondrogenesis and caused significant inhibition of proliferation. Incubating cell cultures at 41 °C elevated the expression of TRPV1, and increased cartilage matrix production. When chondrogenic cells were exposed to mechanical load at the time of their differentiation into matrix producing chondrocytes, we detected increased mRNA levels of TRPV3. Our results demonstrate that developing chondrocytes express a full palette of TRPV channels and the switch in the expression pattern suggests differentiation stage-dependent roles of TRPVs during cartilage formation. As TRPV1 and TRPV3 expression was altered by thermal and mechanical stimuli, respectively, these are candidate channels that contribute to the transduction of environmental stimuli in chondrogenic cells.

Insulin sensitivity is modified by a glycogen phosphorylase inhibitor: glucopyranosylidene-spiro-thiohydantoin in streptozotocin-induced diabetic rats.

The major role of liver glycogen is to supply glucose to the circulation maintaining the normal blood glucose level. In muscle and liver the accumulation and breakdown of glycogen are regulated by the reciprocal activities of glycogen phosphorylase and glycogen synthase. Glycogen phosphorylase catalyses the key step of glycogen degradation and its activity can be inhibited by glucose and its analogues. Obviously, any readily accessible inhibitor of glycogen phosphorylase can be used as a potential therapy of non-insulin-dependent or type 2 diabetes. Hepatic glycogen phosphorylase has been identified as a new target for drugs that control blood glucose concentration. In our experiments glucopyranosylidene-spirothiohydantoin (TH) was tested on the insulin sensitivity and blood glucose level of control and streptozotocin-treated rats. The streptozotocin-treated rats failed to gain weight and exhibited stable hyperglycemia (4.7 ± 0.5 mmol/L glucose in control vs. 7.8 ± 0.5 mmol/L) and low plasma insulin levels (9.6 ± 1.9 µIU/mL in control vs. 3.2 ± 2.2 µIU/mL). When insulin supplementation with slow-release implants (2 IU/day) was started 8 weeks after streptozotocin injection, blood glucose concentration remained suppressed, plasma insulin level dramatically increased and the insulin sensitivity restored. TH administration significantly reduced the high blood glucose concentration and restored the insulin sensitivity of STZtreated rats.

C-(2-Deoxy-d-arabino-hex-1-enopyranosyl)-oxadiazoles: synthesis of possible isomers and their evaluation as glycogen phosphorylase inhibitors.

Synthetic methods were elaborated for d-glucals attached to oxadiazoles by a C-C bond. Introduction of the double bond was effected by either DBU induced elimination of PhCOOH from the O-perbenzoylated glucopyranosyl precursors or Zn/N-methylimidazole mediated reductive elimination from the 1-bromoglucopyranosyl starting compounds. Alternatively, heterocyclizations of 2-deoxy-d-arabino-hex-1-enopyranosyl cyanide were also carried out. Test compounds were obtained by Zemplén debenzoylation, however, none of them showed significant inhibition of rabbit muscle glycogen phosphorylase b.

3-Glucosylated 5-amino-1,2,4-oxadiazoles: synthesis and evaluation as glycogen phosphorylase inhibitors.

Glycogen phosporylase (GP) is a promising target for the control of glycaemia. The design of inhibitors binding at the catalytic site has been accomplished through various families of glucose-based derivatives such as oxadiazoles. Further elaboration of the oxadiazole aromatic aglycon moiety is now reported with 3-glucosyl-5-amino-1,2,4-oxadiazoles synthesized by condensation of a C-glucosyl amidoxime with N,N'-dialkylcarbodiimides or Vilsmeier salts. The 5-amino group introduced on the oxadiazole scaffold was expected to provide better inhibition of GP through potential additional interactions with the enzyme's catalytic site; however, no inhibition was observed at 625 µM.

Poly(ADP) ribose polymerase-1 ablation alters eicosanoid and docosanoid signaling and metabolism in a murine model of contact hypersensitivity.

Poly(ADP­ribose) polymerase (PARP)­1 is a pro­inflammatory protein. The inhibition of PARP­1 reduces the activity of numerous pro­inflammatory transcription factors, which results in the reduced production of pro­inflammatory cytokines, chemokines, matrix metalloproteinases and inducible nitric oxide synthase, culminating in reduced inflammation of the skin and other organs. The aim of the present study was to investigate the effects of the deletion of PARP­1 expression on polyunsaturated fatty acids (PUFA), and PUFA metabolite composition, in mice under control conditions or undergoing an oxazolone (OXA)­induced contact hypersensitivity reaction (CHS). CHS was elicited using OXA in both the PARP­1+/+ and PARP­1/ mice, and the concentration of PUFAs and PUFA metabolites in the diseased skin were assessed using lipidomics experiments. The levels of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were shown to be increased in the PARP­1/ mice, as compared with the control, unsensitized PARP­1+/+ mice. In addition, higher expression levels of fatty acid binding protein 7 (FABP7) were detected in the PARP­1/ mice. FABP7 is considered to be a specific carrier of DHA and EPA. Furthermore, the levels of the metabolites of DHA and EPA (considered mainly as anti­inflammatory or pro­resolving factors) were higher, as compared with the metabolites of arachidonic acid (considered mainly pro­inflammatory), both in the unsensitized control and OXA­sensitized PARP­1/ mice. The results of the present study suggest that the genetic deletion of PARP­1 may affect the PUFA­homeostasis of the skin, resulting in an anti­inflammatory milieu, including increased DHA and EPA levels, and DHA and EPA metabolite levels. This may be an important component of the anti­inflammatory action of PARP­1 inhibition.

Synthesis of 4-amidomethyl-1-glucosyl-1,2,3-triazoles and evaluation as glycogen phosphorylase inhibitors.

Glycogen phosphorylase (GP) appears as a key enzyme for the control of hyperglycemia in the context of type 2 diabetes. In order to gain additional data for structure-activity studies of the inhibition of this enzyme, a series of eight GP inhibitor candidates were prepared from peracetylglucopyranosyl azide 1 by click-chemistry. The need for a N-Boc-protected propargylamine was identified in the CuAAC with azide 1 under Meldal's conditions, while Sharpless' conditions were better adapted to the CuAAC of azide 1 with propargyl bromide. Cycloaddition of Boc-propargylamine with azide 1 afforded the N-Boc precursor of a 4-aminomethyl-1-glucosyl-1,2,3-triazole which gave access to a series of eight amide and sulfonamide derivatives. After deacetylation, enzymatic studies revealed poor to moderate inhibitions toward this enzyme. The N-Boc-protected amine was the best inhibitor (IC50=620 µM) unexpectedly slightly better than the 2-naphthylamido substituted analogue (IC50=650 µM).

Synthesis of C-xylopyranosyl- and xylopyranosylidene-spiro-heterocycles as potential inhibitors of glycogen phosphorylase.

New derivatives of d-xylose with aglycons of the most efficient glucose derived inhibitors of glycogen phosphorylase were synthesized to explore the specificity of the enzyme towards the structure of the sugar part of the molecules. Thus, 2-(ß-d-xylopyranosyl)benzimidazole and 3-substituted-5-(ß-d-xylopyranosyl)-1,2,4-triazoles were obtained in multistep procedures from O-perbenzoylated ß-d-xylopyranosyl cyanide. Cycloadditions of nitrile-oxides and O-peracetylated exo-xylal obtained from the corresponding ß-d-xylopyranosyl cyanide furnished xylopyranosylidene-spiro-isoxazoline derivatives. Oxidative ring closure of O-peracetylated ß-d-xylopyranosyl-thiohydroximates prepared from 1-thio-ß-d-xylopyranose and nitrile-oxides gave xylopyranosylidene-spiro-oxathiazoles. The fully deprotected test compounds were assayed against rabbit muscle glycogen phosphorylase b to show moderate inhibition for 3-(2-naphthyl)-5-(ß-d-xylopyranosyl)-1,2,4-triazole (IC50=0.9mM) only.

Glucopyranosylidene-spiro-iminothiazolidinone, a new bicyclic ring system: synthesis, derivatization, and evaluation for inhibition of glycogen phosphorylase by enzyme kinetic and crystallographic methods.

The reaction of thiourea with O-perbenzoylated C-(1-bromo-1-deoxy-ß-D-glucopyranosyl)formamide gave the new anomeric spirocycle 1R-1,5-anhydro-D-glucitol-spiro-[1,5]-2-imino-1,3-thiazolidin-4-one. Acylation and sulfonylation with the corresponding acyl chlorides (RCOCl or RSO2Cl where R=tBu, Ph, 4-Me-C6H4, 1- and 2-naphthyl) produced the corresponding 2-acylimino- and 2-sulfonylimino-thiazolidinones, respectively. Alkylation by MeI, allyl-bromide and BnBr produced mixtures of the respective N-alkylimino- and N,N'-dialkyl-imino-thiazolidinones, while reactions with 1,2-dibromoethane and 1,3-dibromopropane furnished spirocyclic 5,6-dihydro-imidazo[2,1-b]thiazolidin-3-one and 6,7-dihydro-5H-thiazolidino[3,2-a]pyrimidin-3-one, respectively. Removal of the O-benzoyl protecting groups by the Zemplén protocol led to test compounds most of which proved micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). Best inhibitors were the 2-benzoylimino- (Ki=9µM) and the 2-naphthoylimino-thiazolidinones (Ki=10 µM). Crystallographic studies of the unsubstituted spiro-thiazolidinone and the above most efficient inhibitors in complex with RMGPb confirmed the preference and inhibitory effect that aromatic (and especially 2-naphthyl) derivatives show for the catalytic site promoting the inactive conformation of the enzyme.

Ser/Thr-phosphoprotein phosphatases in chondrogenesis: neglected components of a two-player game.

Protein phosphorylation plays a determining role in the regulation of chondrogenesis in vitro. While signalling pathways governed by protein kinases including PKA, PKC, and mitogen-activated protein kinases (MAPK) have been mapped in great details, published data relating to the specific role of phosphoprotein phosphatases (PPs) in differentiating chondroprogenitor cells or in mature chondrocytes is relatively sparse. This review discusses the known functions of Ser/Thr-specific PPs in the molecular signalling pathways of chondrogenesis. PPs are clearly equally important as protein kinases to counterbalance the effect of reversible protein phosphorylation. Of the main Ser/Thr PPs, some of the functions of PP1, PP2A and PP2B have been characterised in the context of chondrogenesis. While PP1 and PP2A appear to negatively regulate chondrogenic differentiation and maintenance of chondrocyte phenotype, calcineurin is an important stimulatory mediator during chondrogenesis but becomes inhibitory in mature chondrocytes. Furthermore, PPs are implicated to be mediators during the pathogenesis of osteoarthritis that makes them potential therapeutic targets to be exploited in the close future. Among the many yet unexplored targets of PPs, modulation of plasma membrane ion channel function and participation in mechanotransduction pathways are emerging novel aspects of signalling during chondrogenesis that should be further elucidated. Besides the regulation of cellular ion homeostasis, other potentially significant novel roles for PPs during the regulation of in vitro chondrogenesis are discussed.