Glycogen Phosphorylase A Research Articles

Diarylheptanoid‐flavanone Hybrids as Multiple‐target Antidiabetic Agents from Alpinia katsumadai

Main observation and conclusionThe EtOAc fraction of Alpinia katsumadai seeds showed significant inhibition on glycogen phosphorylase a (GPa) with inhibitory ratios of 97.9% and 64.6% at concentrations of 200 and 100 μg/mL, respectively. Bioactivity‐guided isolation afforded 15 new diarylheptanoid‐flavanone hybrids, katsumadainols B1—B15 (1—15), together with eight known ones (16—23). Compounds 4—10 and 12—21 exhibited activity against GPa with IC50 values of 10.1—95.4 μmol/L; compounds 4, 5, 16, and 17 displayed inhibitory effects on α‐glucosidase with IC50 values of 7.1, 12.4, 7.2, and 8.3 μmol/L, obviously higher than acarbose (IC50, 209.1 μmol/L); compounds 4—6, 14, 16—20, 22, and 23 were PTP1B/TCPTP selective inhibitors with IC50 values of 40.7—95.8 μmol/L; compounds 4, 5, 16, and 17 showed DPP4 inhibitory effects with inhibitory ratios of 50.0%—54.2% (200 μmol/L). Diarylheptanoid‐flavanone hybrids (4, 5, 16, and 17) with a p‐hydroxybenzyl at C‐6 position represent a promising class of multiple‐target antidiabetic agents inhibiting GPa, α‐glucosidase, PTP1B, and DPP4.

Synthesis, in vitro ADME profiling and in vivo pharmacological evaluation of novel glycogen phosphorylase inhibitors

A small set of indole-2-carboxamide derivatives identified from a high-throughput screening campaign has been described as a novel, potent, and glucose-sensitive inhibitors of glycogen phosphorylase a (GPa). Among this series of compounds, compound 2 exhibited moderate GP inhibitory activity (IC50 = 0.29 μM), good cellular efficacy (IC50 = 3.24 μM for HepG2 cells and IC50 = 7.15 μM for isolated rat hepatocytes), together with good absorption, distribution, metabolism, and elimination (ADME) profiles. The in vivo animal study revealed that compound 2 significantly inhibited an increase of fasting blood glucose level in adrenaline-induced diabetic mice.

Exploring Non-obvious Hydrophobic Binding Pockets on Protein Surfaces: Increasing Affinities in Peptide-Protein Interactions.

A 42-residue polypeptide conjugated to a small-molecule organic ligand capable of targeting the phosphorylated side chain of Ser15 was shown to bind glycogen phosphorylase a (GPa) with a KD value of 280 nm. The replacement of hydrophobic amino acids by Ala reduced affinities, whereas the incorporation of l-2-aminooctanoic acid (Aoc) increased them. Replacing Nle5, Ile9 and Leu12 by Aoc reduced the KD value from 280 to 27 nm. "Downsizing" the 42-mer to an undecamer gave rise to an affinity for GPa an order of magnitude lower, but the undecamer in which Nle5, Ile9 and Leu12 were replaced by Aoc showed a KD value of 550 nm, comparable with that of the parent 42-mer. The use of Aoc residues offers a convenient route to increased affinity in protein recognition as well as a strategy for the "downsizing" of peptides essentially without loss of affinity. The results show that hydrophobic binding sites can be found on protein surfaces by comparing the affinities of polypeptide conjugates in which Aoc residues replace Nle, Ile, Leu or Phe with those of their unmodified counterparts. Polypeptide conjugates thus provide valuable opportunities for the optimization of peptides and small organic compounds in biotechnology and biomedicine.

Multiple pharmacological targets, cytotoxicity, and phytochemical profile of Aphloia theiformis (Vahl.) Benn.

Aphloia theiformis (Vahl.) Benn. (AT) is traditionally used in Sub-Saharan African countries including Mauritius as a biomedicine for the management of several diseases. However, there is a dearth of experimental studies to validate these claims. We endeavoured to evaluate the inhibitory effects of crude aqueous extract as traditionally used together with the crude methanol extracts of AT leaves on urease, angiotensin (I) converting enzyme (ACE), acetylcholinesterase (AChE), cholesterol esterase (CEase), glycogen phosphorylase a (GPa), and glycation in vitro. The crude extract showing potent activity against the studied enzymes was further partitioned using different solvents of increasing polarity. The enzyme inhibitory and antiglycation activities of each fraction was assessed. Kinetic of inhibition of the active crude extract/fractions on the aforementioned enzymes was consequently determined using Lineweaver-Burk plots. An ultra-high performance liquid chromatography (UHPLC-UV/MS) system was used to establish the phytochemical profile of AT. The real time cell analysis system (iCELLigence™) was used to monitor any cellular cytotoxicity of AT. Crude methanolextract (CME) was a potent inhibitor of the studied enzymes, with IC50 ranging from 696.22 to 19.73μg/mL. CME (82.5%) significantly (p<0.05) inhibited glycation and was comparable to aminoguanidine (81.5%). Ethyl acetate and n-butanol fractions of CME showed non-competitive, competitive, and uncompetitive mode of inhibition against ACE, CEase, and AChE respectively. Mangiferin, a xanthone glucoside was present in CME, ethyl acetate, and n-butanol fractions. Active extract/fractions were found to be non-cytotoxic (IC50>20μg/mL) according to the U.S National Cancer Institute plant screening program. This study has established baseline data that tend to justify the traditional use of AT and open new avenues for future biomedicine development.

Metformin improves hepatic IRS2/PI3K/Akt signaling in insulin-resistant rats of NASH and cirrhosis.

Nonalcoholic fatty liver disease and cirrhosis are strongly associated with insulin resistance and glucose intolerance. To date, the influence of metformin on glycogen synthesis in the liver is controversial. Limited studies have evaluated the effect of metformin on hepatic insulin signaling pathway in vivo In this study, an insulin-resistant rat model of nonalcoholic steatohepatitis and cirrhosis was developed by high-fat and high-sucrose diet feeding in combination with subcutaneous injection of carbon tetrachloride. Liver tissues of the model rats were featured with severe steatosis and cirrhosis, accompanied by impaired liver function and antioxidant capacity. The glucose tolerance was impaired, and the index of insulin resistance was increased significantly compared with the control. The content of hepatic glycogen was dramatically decreased. The expression of insulin receptor β (IRβ); phosphorylations of IRβ, insulin receptor substrate 2 (IRS2), and Akt; and activities of phosphatidylinositol 3-kinase (PI3K) and glycogen synthase (GS) in the liver were significantly decreased, whereas the activities of glycogen synthase kinase 3α (GSK3α) and glycogen phosphorylase a (GPa) were increased. Metformin treatment remarkably improved liver function, alleviated lipid peroxidation and histological damages of the liver, and ameliorated glucose intolerance and insulin resistance. Metfromin also significantly upregulated the expression of IRβ; increased the phosphorylations of IRβ, IRS2, and Akt; increased the activities of PI3K and GS; and decreased GSK3α and GPa activities. In conclusion, our study suggests that metformin upregulates IRβ expression and the downstream IRS2/PI3K/Akt signaling transduction, therefore, to increase hepatic glycogen storage and improve insulin resistance. These actions may be attributed to the improved liver histological alterations by metformin.

Maslinic acid modulates glycogen metabolism by enhancing the insulin signaling pathway and inhibiting glycogen phosphorylase

AimTo investigate the molecular signaling mechanism by which the plant-derived, pentacyclic triterpene maslinic acid (MA) exerts anti-diabetic effects. MethodHepG2 cells were stimulated with various concentrations of MA. The effects of MA on glycogen phosphorylase a (GPa) activity and the cellular glycogen content were measured. Western blot analyses were performed with anti-insulin receptor β (IRβ), protein kinase B (also known as Akt), and glycogen synthase kinase-3β (GSK3β) antibodies. Activation status of the insulin pathway was investigated using phospho-IRβ, as well as phospho-Akt, and phospho-GSK3β antibodies. The specific PI3-kinase inhibitor wortmannin was added to the cells to analyze the Akt expression. Enzyme-linked immunosorbent assay (ELISA) was used to measure the effect of MA on IRβ auto-phosphorylation. Furthermore, the effect of MA on glycogen metabolism was investigated in C57BL/6J mice fed with a high-fat diet (HFD). ResultsThe results showed that MA exerts anti-diabetic effects by increasing glycogen content and inhibiting glycogen phosphorylase activity in HepG2 cells. Furthermore, MA was shown to induce the phosphorylation level of IRβ-subunit, Akt, and GSK3β. The MA-induced activation of Akt appeared to be specific, since it could be blocked by wortmannin. Finally, MA treatment of mice fed with a high-fat diet reduced the model-associated adiposity and insulin resistance, and increased the accumulated hepatic glycogen content. ConclusionThe results suggested that maslinic acid modulates glycogen metabolism by enhancing the insulin signaling pathway and inhibiting glycogen phosphorylase.

The glycogen phosphorylase inhibitor 2-(3-benzylamino-2-oxo-1,2-dihydropyridin-1-yl)-N-(3,4-dichlorobenzyl)acetamide

The molecular structure of the title compound, C21H19Cl2N3O2, a potent glycogen phosphorylase a (GPa) inhibitor (IC50 of 6.3 µM), consists of four distinct conjugated π systems separated by rotatable C-C bonds at the methylene groups. Molecules are linked into dimers disposed about a crystallographic centre of symmetry through a cyclic N-H...O hydrogen-bonding motif [graph set R2(2)(10)]. These dimers are further connected along the crystallographic c axis by N-H...O hydrogen bonding between the amide groups [graph set C(4)]. A comparison of this structure with that of the monohydrate of the significantly less active analogue (S)-2-(3-benzylamino-2-oxo-1,2-dihydropyridin-1-yl)-N-(2-hydroxy-1-phenylethyl)acetamide (IC50 of 120 µM) is presented.

Glycogen Phosphorylase-a is a Common Target for Anti-Diabetic Effect of Iridoid and Secoiridoid Glycosides

Diabetes mellitus is characterized by hyperglycemia resulting from defects in insulin secretion, action or both. The use of medicinal plants for the treatment of diabetes mellitus dates back from the Ebers papyrus of about 1550 B.C. One of the major problems with herbal drugs is that the active ingredients are not well defined. It is important to know the active components and their molecular interactions which will help to analyze their therapeutic efficacy and also to standardize the product. There are a number of medicinal plants known for their anti-diabetic effect that possess similarities in their active chemical components, e.g. iridoid and secoiridoid glycosides. In this study, we have compared the structure of various iridoid and secoiridoid glycosides to design a novel pharmacophore. We further developed a structure-activity relationship for the inhibition of glycogen phosphorylase-a. By using docking studies, we are proposing, for the first time, that inhibition of glycogen phosphorylase-a activity is a common target for iridoids and secoiridoids to elicit anti-diabetic effects. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Heat acclimation-induced changes in heart glycogen/glucose metabolism in rats

Based on the observation that heat acclimation is a slowly developing response, evoked by continuous exposure to moderate heat, we investigated the time-dependent acclimatory changes of heart glycogen metabolism. Cardiac levels of key carbohydrate-related enzymes and substrates were studied in the function of the duration of short-term (STHA; 6, 12, 24 and 48 h) and long-term heat acclimation (LTHA; 7, 14, 21 and 30 days) to high environmental temperature (35 ± 1°C). The changes in heart glycogen metabolism during STHA could be separated in two phases: up to 12 h exposure, where significant decrease of the heart glycogen (Glk), glucose-6-phosphate (G6P), hexokinase (HK) activity as well as increase of heart glucose was observed; and from 24 to 48 h exposure, manifested with elevation of Glk, Glu, glycogen phosphorylase a (GPa), phosphofructokinase (PFK) and HK activities. The metabolic changes in the period of LTHA could also be seen as separate phases: in a period of 7-14 days of heat exposure there was an increase of heart Glk, Glu, G6P, HK, as well as a decrease of GPa and PFK, while in the period of 21-28 days there was more intensive rebound of Glk and G6P, increase of GPa activity and non-significant changes of Glu, HK and PFK. The results obtained have showed that acclimation to moderate hyperthermic environment has caused significant changes in examined parameters which differ depending on duration to the exposure: intensive stress-induced glycogenolytic and glycolytic processes in the period of STHA and intensive energy sparing, manifested by Glk deposition in the period of LTHA.

Novel therapeutics based on inhibiting the interaction of glycogen phosphorylase and GL-subunit of glycogen-associated protein phosphatase 1: WO2009127723

Diabetes mellitus is a major challenge for healthcare systems worldwide because it is strongly associated with several major health risk factors. Currently marketed drugs are insufficient to achieve long-term control of blood glucose levels and induce considerable side effects. The development of more effective drugs for metabolic diseases is still of urgent interest. Recently, inhibiting the interaction of hepatic glycogen-targeting subunit (GL) of protein phosphatase 1 and glycogen phosphorylase a (GPa) is emerging as a novel mechanism to combat diabetic hyperglycemia by dephosphorylation and activation of glycogen synthase, converting high blood glucose to liver glycogen. Patent WO2009127723 describes a variety of arylsulfonylaminomethylphosphonic acids as small molecule inhibitors of GL–GPa interaction and their potential use as pharmaceutical compositions to treat type 1 and type 2 diabetes mellitus. Although lacking detailed in vivo biological data, WO2009127723 represents the first evidence for the novel rationale in antidiabetic pursuit, meriting the attention of the antidiabetic community.

Synthesis of 3-Deoxypentacyclic Triterpene Derivatives as Inhibitors of Glycogen Phosphorylase

The 3-deoxy-2-keto derivatives 5 and 7 of oleanolic acid (1) and ursolic acid (2), respectively, served as precursors to the synthesis of 35 3-deoxy derivatives of pentacyclic triterpenes. The synthesized compounds were biologically assayed for their inhibitory activity against rabbit muscle glycogen phosphorylase a (GPa). Among this series of compounds, 2alpha-hydroxyurs-12-en-28-oic acid (18) (IC(50) = 1.2 microM) exhibited the most potent activity. Preliminary structure-activity relationship analysis for the 3-deoxy triterpene derivatives as GP inhibitors is also discussed.

Mechanism of Thiol-Supported Arsenate Reduction Mediated by Phosphorolytic-Arsenolytic Enzymes

Enzymes catalyzing the phosphorolytic cleavage of their substrates can reduce arsenate (AsV) to the more toxic arsenite (AsIII) via the arsenolytic substrate cleavage in presence of a reductant, as glutathione or dithiotreitol (DTT). We have shown this for purine nucleoside phosphorylase (PNP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycogen phosphorylase-a (GPa), and phosphotransacetylase (PTA). Using a multidisciplinary approach, we explored the mechanism whereby these enzymes mediate AsV reduction. It is known that PNP cleaves inosine with AsV into hypoxanthine and ribose-1-arsenate. In presence of inosine, AsV and DTT, PNP mediates AsIII formation. In this study, we incubated PNP first with inosine and AsV, allowing the arsenolytic reaction to run, then blocked this reaction with the PNP inhibitor BCX-1777, added DTT and continued the incubation. Despite inhibition of PNP, large amount of AsIII was formed in these incubations, indicating that PNP does not reduce AsV directly but forms a product (i.e., ribose-1-arsenate) that is reduced to AsIII by DTT. Similar studies with the other arsenolytic enzymes (GPa, GAPDH, and PTA) yielded similar results. Various thiols that differentially supported AsV reduction when present during PNP-catalyzed arsenolysis (DTT approximately dimercaptopropane-1-sulfonic acid > mercaptoethanol > DMSA > GSH) similarly supported AsV reduction when added only after a transient PNP-catalyzed arsenolysis, which preformed ribose-1-arsenate. Experiments with progressively delayed addition of DTT after BCX-1777 indicated that ribose-1-arsenate is short-lived with a half-life of 4 min. In conclusion, phosphorolytic enzymes, such as PNP, GAPDH, GPa, and PTA, promote thiol-dependent AsV reduction because they convert AsV into arsenylated products reducible by thiols more readily than AsV. In support of this view, reactivity studies using conceptual density functional theory reactivity descriptors (local softness, nucleofugality) indicate that reduction by thiols of the arsenylated metabolites is favored over AsV.

Mechanism of Thiol-Supported Arsenate Reduction Mediated by Phosphorolytic-Arsenolytic Enzymes

Several mammalian enzymes catalyzing the phosphorolytic-arsenolytic cleavage of their substrates (thus yielding arsenylated metabolites) have been shown to facilitate reduction of arsenate (AsV) to the more toxic arsenite (AsIII) in presence of their substrate and a thiol. These include purine nucleoside phosphorylase (PNP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glycogen phosphorylase-a (GPa). In this work, we tested further enzymes, the bacterial phosphotransacetylases (PTAs) and PNP, for AsV reduction. The PTAs, which arsenolytically cleave acetyl-CoA producing acetyl-arsenate, were compared with GAPDH, which can also form acetyl-arsenate by arsenolysis of its nonphysiological substrate, acetyl-phosphate. As these enzymes also mediated AsV reduction, we can assert that facilitation of thiol-dependent AsV reduction may be a general property of enzymes that catalyze phosphorolytic-arsenolytic reactions. Because with all such enzymes arsenolysis is obligatory for AsV reduction, we analyzed the relationship between these two processes in presence of various thiol compounds, using PNP. Although no thiol influenced the rate of PNP-catalyzed arsenolysis, all enhanced the PNP-mediated AsV reduction, albeit differentially. Furthermore, the relative capacity of thiols to support AsV reduction mediated by PNP, GPa, PTA, and GAPDH apparently depended on the type of arsenylated metabolites (i.e., arsenate ester or anhydride) produced by these enzymes. Importantly, AsV reduction by both acetyl-arsenate-producing enzymes (i.e., PTA and GAPDH) exhibited striking similarities in responsiveness to various thiols, thus highlighting the role of arsenylated metabolite formation. This observation, together with the finding that PNP-mediated AsV reduction lags behind the PNP-catalyzed arsenolysis lead to the hypothesis that arsenolytic enzymes promote reduction of AsV by forming arsenylated metabolites which are more reducible to AsIII by thiols than inorganic AsV. This hypothesis is evaluated in the adjoining paper.

Synthesis of new modified truncated peptides and inhibition of glycogen phosphorylase

The first solution state structural analysis (NMR) of the C-terminal sequence of human G(L) that binds to glycogen phosphorylase a (GPa), PEWPSYLGYEKLGPYY-NH(2) (1), showed it to be in a random coil conformation. This was supported by molecular dynamics simulation (modelled in solution) using NAMD 2.6. The conformational ambiguity of the peptide makes the structural arrangement of the peptide (and internal residues) strongly dependent on the environment. Thirteen tetra-peptide fragments of the C-terminal sequence, YEKLG-NH(2), and the corresponding tri- and di-peptide sequences were used in a fragment screen against GPa. Compound 2 (H-GPYY-NH(2)) did not give an IC(50) value, whereas PEWPSYLGYEKLGPYY-NH(2) (1) displayed an IC(50) of 34 microM against GPa. Truncated peptides derived from 1, (EKL-NH(2), EKLG-NH(2), and AcEKNH(2)) inhibited GPa (21%, 32%, 63%, respectively at 22 mM). These studies suggest key residues within the peptide chain have additional molecular interactions with GPa. The interaction of intra-sequence residues in combination with the terminal residues of PEWPSYLGYEKLGPYY with GPa may form the basis for the design of new inhibitors of GPa.

The Structure Of Phosphorylase Kinase Holoenzyme At Subnanometer Resolution, Location Of The Catalytic Subunit And The Substrate Glycogen Phosphorylase

Phosphorylase kinase (PhK) coordinates hormonal and neuronal signals to initiate the breakdown of glycogen. The enzyme catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a (GPa) and the stimulation of glycogenolysis. PhK is one of the largest of the protein kinases (MW 1.3 x 106) and contains four copies of four subunits: α,β,γ and δ. Here we present a 9.9Å resolution structure of PhK determined by electron cryo-microscopy single-particle reconstruction. The enzyme has a butterfly-like shape comprised of two lobes with 222 symmetry. This 3D structure has allowed us to dock the catalytic γ domain, whose crystal structure is known, to the PhK holoenzyme at a location that is towards the ends of the lobes. The kinase domain is not involved in homo-dimer interactions. We have also determined the structure of PhK decorated with GPb at 18-Å resolution, which shows GPb located at the end of the lobes. Comparison of PhK/GPb complex with the volume of density for the GPb dimer derived from the X-ray model indicates that only one subunit of GPb is localized. Careful examination of the electron microscope images revealed a mixture of large and small PhK particles. In addition to the large particles described above we have determined the reduced size particles at 9.8Å resolution. This structure was consistent with a proteolysed activated form of PhK that had lost the α subunit and possibly the γ subunit.

Glycogen Phosphorylase a Inhibitors with a Phenethylphenylphthalimide Skeleton Derived from Thalidomide-Related .ALPHA.-Glucosidase Inhibitors and Liver X Receptor Antagonists

Novel glycogen phosphorylase a (GPa) inhibitors with a phenethylphenylphthalimide skeleton were prepared based on alpha-glucosidase inhibitors and liver X receptor (LXR) antagonists derived from thalidomide. Their structure-activity relationships were analyzed. Some of the compounds thus prepared showed potent inhibitory activity against rabbit muscle GPa with more than 10-fold greater efficacy than a typical GPa inhibitor, 1,4-dideoxy-1,4-imino-D-arabinitol.

Anthranilimide based glycogen phosphorylase inhibitors for the treatment of type 2 diabetes. Part 3: X-ray crystallographic characterization, core and urea optimization and in vivo efficacy

Key binding interactions of the anthranilimide based glycogen phosphorylase a (GPa) inhibitor 2 from X-ray crystallography studies are described. This series of compounds bind to the AMP site of GP. Using the binding information the core and the phenyl urea moieties were optimized. This work culminated in the identification of compounds with single nanomolar potency as well as in vivo efficacy in a diabetic model.

Pentacyclic triterpenes, inhibitors of glycogen phosphorylase, as potential drugs for type 2 diabetes: X-ray crystallographic studies

Twenty-five naturally occurring pentacyclic tritepenes were identified as inhibitors for muscle glycogen phosphorylase a (GPa), a molecular target to control hyperglycaemia in type 2 diabetes. To elucidate the mechanism of inhibition for these compounds, the crystal structures of the GPb-asiatic acid and GPb-maslinic acid complexes were performed and refined to 2.4 Å, and 2.7 Å resolution, respectively [1]. The structures demonstrated that the inhibitors bind at the allosteric activator site. The position of the inhibitors was partially overlapping, with the position of the physiological activator AMP bound to the R-state of the human liver enzyme. The contacts from the inhibitors to RMGPb are dominated by ionic interactions from the carboxylate group to the Arg310 of the allosteric phosphate-subsite, and also by nonpolar van der Waals interactions to Tyr75, Phe196, and Val45' (from the symmetry related subunit). These interactions appear important in stabilizing the inactive quaternary T-state of the enzyme. The structural analyses of these compounds can be further exploited towards the development of better inhibitors.

Naturally Occurring Pentacyclic Triterpenes as Inhibitors of Glycogen Phosphorylase: Synthesis, Structure−Activity Relationships, and X-ray Crystallographic Studies

Twenty-five naturally occurring pentacyclic triterpenes, 15 of which were synthesized in this study, were biologically evaluated as inhibitors of rabbit muscle glycogen phosphorylase a (GPa). From SAR studies, the presence of a sugar moiety in triterpene saponins resulted in a markedly decreased activity ( 7, 18- 20) or no activity ( 21, 22). These saponins, however, might find their value as potential natural prodrugs which are much more water-soluble than their corresponding aglycones. To elucidate the mechanism of GP inhibition, we have determined the crystal structures of the GPb-asiatic acid and GPb-maslinic acid complexes. The X-ray analysis indicates that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Pentacyclic triterpenes represent a promising class of multiple-target antidiabetic agents that exert hypoglycemic effects, at least in part, through GP inhibition.

Evaluation of novel Hyphodermin derivatives as Glycogen Phosphorylase a inhibitors

The lipophilicity, permeability, solubility, polar surface area and ‘rule-of-five’ properties were assessed, using QikProp v2.5 (Schrödinger, Inc.) and ALOGPS 2.1 calculations, for 25 Hyphodermin derivatives. These compounds obeyed the ‘rule-of-five’, and the calculated physicochemical values were generally within desired limits. All compounds were tested against Glycogen Phosphorylase a (GPa). Four phenyl and benzyl substituted 2-oxo-hexahydro and tetrahydrobenzo[ cd]indole carboxylic acids were identified as novel inhibitors of GPa with estimated IC 50 values in the range 0.8–1.3 mM. Molecular modelling of these novel inhibitors was used to obtain the main structural features of this class of molecule for future structure–activity relationship studies.