Discovery of a new series of PI3K-δ inhibitors from Virtual Screening.

PI3K-δ mediates key immune cell signaling pathways and is a target of interest for treatment of oncological and immunological disorders. Here we describe the discovery and optimization of a novel series of PI3K-δ selective inhibitors. We first identified hits containing an isoindolinone scaffold using a combined ligand- and receptor-based virtual screening workflow, and then improved potency and selectivity guided by structural data and modeling. Careful optimization of molecular properties led to compounds with improved permeability and pharmacokinetic profile, and high potency in a whole blood assay.

1-Isoindolinone scaffold-based natural products with a promising diverse bioactivity.

Isoindolin-1-one or 1-isoindolinone framework is referred to phthalimidines or benzo fused γ-lactams of the corresponding γ-amino carboxylic acids and has been of prime interest for scientists for last several decades. 1-Isoindolinone framework is found in a wide range of naturally occurring compounds with diverse biological activities and therapeutic potential for various chronic diseases. Recent developments in synthetic methods for their procurement have opened a new era of 1-isoindolinone chemistry. This review aims to provide an alphabetical quick reference guide to only 1-isoindolinone based natural products and its variable fused, oxidized and reduced state skeleton with information for advanced chemotaxonomic analyses, cellular targets/pathways and diverse biological activities and future use for medicinal chemistry.

Synthesis and evaluation of novel S-benzyl- and S-alkylphthalimide- oxadiazole -benzenesulfonamide hybrids as inhibitors of dengue virus protease.

Direct acting antiviral drugs (DAADs) are becoming therapeutics of choice for the treatment of viral infections. Successful development of anti HIV and HCV drugs by targeting the viral proteases has provided impetus for discovering newer DAADs. Dengue virus (DENV) protease, which is composed of two nonstructural proteins, NS2B and NS3pro, can be likewise exploited for discovering new anti-dengue therapeutics. In this study, we have linked together two pharmaceutically interesting motifs, namely 1,3,4-oxadiazole and benzenesulfonamide in two alternative series to develop novel S-benzylated and S-alkylphthalimidated hybrids. For the first series of hybrids, 4-aminobenzoic acid (1) was reacted with substituted benzenesulfonyl chlorides via its amino group, whereas the carboxylic acid side was elaborated to sulfonamido-1,3,4-oxadiazole-2-thiols (6a/b) in three steps. At this stage, the intermediates 6a/b were bifurcated to either S-alkylphthalimidated (8a-j) or S-benzylated (9a-c) hybrids by reacting with corresponding halides. For the alternative series of hybrids, the carboxylic acid group of probenecid (10) was similarly elaborated to sulfonamido-1,3,4-oxadiazole-2-thiols (13), and diverged to S-alkylphthalimidated (14a-f) and S-benzylated hybrids (15a-e). Bioactivity assays demonstrated that 8g and 8h are the most potent inhibitors among the synthesized analogs, exhibiting the IC50 values of 13.9 µM and 15.1 µM, respectively. Computational assessment predicted the binding of the inhibitors at an allosteric site developed in the open conformation of DENV2 NS2B/NS3pro. Taken together these findings point out that the synthesized hybrid inhibitors possess a great potential for further antiviral drug development.

N-arylmethylideneaminophthalimide: Design, Synthesis and Evaluation as Analgesic and Anti-inflammatory Agents.

BACKGROUND AND OBJECTIVE: N-aryl derivatives of phthalimide and 4-nitro phthalimide have demonstrated cyclooxygenase inhibitory activity. Also, they possess excellent analgesic and antiinflammatory activity. In this work, a new series of N-arylmethylideneamino derivatives of phthalimide and 4-nitro phthalimide were designed and synthesized. METHODS: The designed compounds were synthesized by condensation of the appropriate aldehyde and N-aminophthalimide in ethanol at room temperature at PH around 3. Their analgesic and antiinflammatory activity were evaluated by acetic acid-induced pain test and carrageenan-induced paw edema test in mice and rats, respectively. RESULTS AND CONCLUSION: The details of the synthesis and chemical characterization of the analogs are described. In vivo screening showed compounds 3a, 3b, 3f and 3h were the most potent analgesic compounds. In addition, compounds 3a, 3c, 3d, 3e and 3j indicated comparable anti-inflammatory activity to indomethacin as a reference drug.

Glycine Protects H9C2 Cardiomyocytes from High Glucose- and Hypoxia/Reoxygenation-Induced Injury via Inhibiting PKCß2 Activation and Improving Mitochondrial Quality.

BACKGROUND: Patients with diabetes are more vulnerable to myocardial ischemia reperfusion injury (IRI), which is involved in PKCß2 activation and mitochondrial dysfunction. Glycine has been documented as a cytoprotective agent to attenuate diabetes-related abnormalities and reduce myocardial IRI, but the underlying mechanisms are still unclear. We determined whether glycine could attenuate high glucose- (HG-) and hypoxia/reoxygenation- (H/R-) induced injury by inhibiting PKCß2 activation and improving mitochondrial quality in cultured H9C2 cells. METHODS: H9C2 cells were either exposed to low glucose (LG) or HG conditions with or without treatment of glycine or CGP53353 (a selective inhibitor of PKCß2) for 48 h, then subjected to 4 h of hypoxia followed by 2 h of reoxygenation (H/R). Cell viability, lactate dehydrogenase (LDH) release, mitochondrial membrane potential (MMP), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration were detected using corresponding commercial kits. Mitochondrial quality control-related proteins (LC-3II, Mfn-2, and Cyt-C) and PKCß2 activation were detected by Western blot. RESULTS: HG stimulation significantly decreased cell viability and SOD activity and increased LDH release, MDA production, and PKCß2 activation as compared to LG group, all of which changes were further increased by H/R insult. Glycine or CGP53353 treatment significantly reduced the increase of LDH release, MDA production, PKCß2 activation, and Cyt-C expression and the decrease of cell viability, SOD activity, MMP, Mfn-2 expression, and LC-3II/LC-3I ratio induced by HG and H/R stimulation. CONCLUSIONS: Supplementary glycine protects H9C2 cells from HG- and H/R-induced cellular injury by suppressing PKCß2 activation and improving mitochondria quality.

Role of CovR phosphorylation in gene transcription in Streptococcus mutans.

Streptococcus mutans, the primary aetiological agent of dental caries, is one of the major bacteria of the human oral cavity. The pathogenicity of this bacterium is attributed not only to the expression of virulence factors, but also to its ability to respond and adapt rapidly to the ever-changing conditions of the oral cavity. The two-component signal transduction system (TCS) CovR/S plays a crucial role in virulence and stress response in many streptococci. Surprisingly, in S. mutans the response regulator CovR appears to be an orphan, as the cognate sensor kinase, CovS, is absent in all the strains. We found that acetyl phosphate, an intracellular phosphodonor molecule known to act in signalling, might play a role in CovR phosphorylation in vivo. We also found that in vitro, upon phosphorylation by potassium phosphoramide (a high-energy phophodonor) CovR formed a dimer and showed altered electrophoretic mobility. As expected, we found that the conserved aspartic acid residue at position 53 (D53) was the site of phosphorylation, since neither phosphorylation nor dimerization was seen when an alanine-substituted CovR mutant (D53A) was used. Surprisingly, we found that the ability of CovR to act as a transcriptional regulator does not depend upon its phosphorylation status, since the D53A mutant behaved similarly to the wild-type protein in both in vivo and in vitro DNA-binding assays. This unique phosphorylation-mediated inhibition of CovR function in S. mutans sheds light on an unconventional mechanism of the signal transduction pathway.

Comparison of the Effects of Curcumin and RG108 on NGF-Induced PC-12 Adh Cell Differentiation and Neurite Outgrowth.

DNA methyltransferases (DNMTs) are promising epigenetic targets for the development of novel drugs, especially for neurodegenerative disorders. In recent years, there has been increased interest in small molecules that can cross the blood-brain barrier for the treatment of neurodegenerative diseases. Therefore, comparing the neuronal differentiative effects of a natural compound curcumin and a synthetic small molecule RG108 was the aim of this study. The effects of curcumin and RG108 on neuronal differentiation and neurite outgrowth were investigated in the PC-12 Adh cell line. First, a nontoxic concentration was determined to be 100 nM with WST-1 assay. Subsequently, cells were treated with 100 nM curcumin and RG108 alone or in combination with 50 nM nerve growth factor (NGF). Cell differentiations were evaluated by a real-time cell analyzer system. Neurite outgrowth was determined and morphologically shown by immunofluorescence staining with anti-beta III tubulin antibody on PC-12 Adh cells. Also, growth-associated protein-43 (GAP-43) and ß-tubulin III mRNA expression levels, associated with neurite outgrowth promotion, were determined with real-time polymerase chain reaction (RT-PCR). According to our results, 100 nM curcumin and RG108 significantly induced neurite outgrowth of PC-12 Adh cells with 50 nM NGF. Curcumin + NGF combination further increased cell differentiations and total neurite lengths more than curcumin alone and RG108 + NGF combination groups. Strikingly, curcumin and NGF combination upregulated GAP-43 and ß-tubulin mRNA expression levels excessively. In conclusion, curcumin was found to be more effective than RG108 on neuronal differentiation and neurite outgrowth of PC-12 Adh cells in a combination with NGF. Therefore, natural DNMT1 inhibitors, such as curcumin, can be a novel approach for the neurodegenerative disorders treatment.

Auxin Biosynthesis, Accumulation, Action and Transport are Involved in Stress-Induced Microspore Embryogenesis Initiation and Progression in Brassica napus.

Isolated microspores are reprogrammed in vitro by stress, becoming totipotent cells and producing embryos and plants via a process known as microspore embryogenesis. Despite the abundance of data on auxin involvement in plant development and embryogenesis, no data are available regarding the dynamics of auxin concentration, cellular localization and the expression of biosynthesis genes during microspore embryogenesis. This work involved the analysis of auxin concentration and cellular accumulation; expression of TAA1 and NIT2 encoding enzymes of two auxin biosynthetic pathways; expression of the PIN1-like efflux carrier; and the effects of inhibition of auxin transport and action by N-1-naphthylphthalamic acid (NPA) and α-(p-chlorophenoxy) isobutyric acid (PCIB) during Brassica napus microspore embryogenesis. The results indicated de novo auxin synthesis after stress-induced microspore reprogramming and embryogenesis initiation, accompanying the first cell divisions. The progressive increase of auxin concentration during progression of embryogenesis correlated with the expression patterns of TAA1 and NIT2 genes of auxin biosynthetic pathways. Auxin was evenly distributed in early embryos, whereas in heart/torpedo embryos auxin was accumulated in apical and basal embryo regions. Auxin efflux carrier PIN1-like gene expression was induced in early multicellular embryos and increased at the globular/torpedo embryo stages. Inhibition of polar auxin transport (PAT) and action, by NPA and PCIB, impaired embryo development, indicating that PAT and auxin action are required for microspore embryo progression. NPA also modified auxin embryo accumulation patterns. These findings indicate that endogenous auxin biosynthesis, action and polar transport are required in stress-induced microspore reprogramming, embryogenesis initiation and progression.

A novel phthalimide derivative, TC11, has preclinical effects on high-risk myeloma cells and osteoclasts.

Despite the recent advances in the treatment of multiple myeloma (MM), MM patients with high-risk cytogenetic changes such as t(4;14) translocation or deletion of chromosome 17 still have extremely poor prognoses. With the goal of helping these high-risk MM patients, we previously developed a novel phthalimide derivative, TC11. Here we report the further characterization of TC11 including anti-myeloma effects in vitro and in vivo, a pharmacokinetic study in mice, and anti-osteoclastogenic activity. Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous tumors in human myeloma-bearing SCID mice. Immunohistochemical analyses showed that TC11 induced apoptosis of MM cells in vivo. In the pharmacokinetic analyses, the Cmax was 2.1 µM at 1 h after the injection of TC11, with 1.2 h as the half-life. TC11 significantly inhibited the differentiation and function of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts in mouse osteoclast cultures using M-CSF and RANKL. We also revealed that TC11 induced the apoptosis of myeloma cells accompanied by α-tubulin fragmentation. In addition, TC11 and lenalidomide, another phthalimide derivative, directly bound to nucleophosmin 1 (NPM1), whose role in MM is unknown. Thus, through multiple molecular interactions, TC11 is a potentially effective drug for high-risk MM patients with bone lesions. The present results suggest the possibility of the further development of novel thalidomide derivatives by drug designing.

Antimalarial activity of abietane ferruginol analogues possessing a phthalimide group.

The abietane-type diterpenoid (+)-ferruginol, a bioactive compound isolated from New Zealand's Miro tree (Podocarpus ferruginea), displays relevant pharmacological properties, including antimicrobial, cardioprotective, anti-oxidative, anti-plasmodial, leishmanicidal, anti-ulcerogenic, anti-inflammatory and anticancer. Herein, we demonstrate that ferruginol (1) and some phthalimide containing analogues 2-12 have potential antimalarial activity. The compounds were evaluated against malaria strains 3D7 and K1, and cytotoxicity was measured against a mammalian cell line panel. A promising lead, compound 3, showed potent activity with an EC50 = 86 nM (3D7 strain), 201 nM (K1 strain) and low cytotoxicity in mammalian cells (SI>290). Some structure-activity relationships have been identified for the antimalarial activity in these abietane analogues.

Genetic and phenotypic analyses of carpel development in Arabidopsis.

Carpels are the female reproductive organs of the flower, organized in a gynoecium, which is arguably the most complex organ of a plant. The gynoecium provides protection for the ovules, helps to discriminate between male gametophytes, and facilitates successful pollination. After fertilization, it develops into a fruit, a specialized organ for seed protection and dispersal. To carry out all these functions, coordinated patterning and tissue specification within the developing gynoecium have to be achieved. In this chapter, we describe different methods to characterize defects in carpel patterning and morphogenesis associated with developmental mutations as well as a list of reporter lines that can be used to facilitate genetic analyses.

Root-derived auxin contributes to the phosphorus-deficiency-induced cluster-root formation in white lupin (Lupinus albus).

Formation of cluster roots is a typical morphological response to phosphorus (P) deficiency in white lupin (Lupinus albus), but its physiological and molecular mechanisms are still unclear. We investigated the role of auxin in the initiation of cluster roots by distinguishing the sources of auxin, measuring the longitudinal distribution patterns of free indole-3-acetic acid (IAA) along the root and the related gene expressions responsible for polar auxin transport (PAT) in different developmental stages of cluster roots. We found that removal of shoot apex or primary root apex and application of auxin-influx or -efflux transport inhibitors, 3-chloro-4-hydroxyphenylacetic acid, N-1-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid, to the stem did not affect the number of cluster roots and the free-IAA concentration in the roots of P-deficient plants, but when these inhibitors were applied directly to the growth media, the cluster-root formation was greatly suppressed, suggesting the fundamental role of root-derived IAA in cluster-root formation. The concentration of free IAA in the roots was higher in P-deficient plants than in P-adequate ones, and the highest in the lateral-root apex and the lowest in the mature cluster roots. Meanwhile the expression patterns of LaAUX1, LaPIN1 and LaPIN3 transcripts related to PAT was consistent with concentrations of free IAA along the lateral root, indicating the contribution of IAA redistribution in the cluster-root development. We proposed that root-derived IAA plays a direct and important role in the P-deficiency-induced formation of cluster roots.

Inhibition of monoamine oxidase by C5-substituted phthalimide analogues.

Literature reports that isatin as well as C5- and C6-substituted isatin analogues are reversible inhibitors of human monoamine oxidase (MAO) A and B. In general, C5- and C6-substitution of isatin leads to enhanced binding affinity to both MAO isozymes compared to isatin and in most instances result in selective binding to the MAO-B isoform. Crystallographic and modeling studies suggest that the isatin ring binds to the substrate cavities of MAO-A and -B and is stabilized by hydrogen bond interactions between the NH and the C2 carbonyl oxygen of the dioxoindolyl moiety and water molecules present in the substrate cavities of MAO-A and -B. Based on these observations and the close structural resemblances between isatin and its phthalimide isomer, a series of phthalimide analogues were synthesized and evaluated as MAO inhibitors. While phthalimide and N-aryl-substituted phthalimides were found to be weak MAO inhibitors, phthalimide homologues containing C5 substituents were potent reversible inhibitors of recombinant human MAO-B with IC(50) values ranging from 0.007 to 2.5 µM and moderately potent reversible inhibitors of recombinant human MAO-A with IC(50) values ranging from 0.22 to 9.0 µM. By employing molecular docking the importance of hydrogen bonding between the active sites of MAO-A and -B and the phthalimide inhibitors are highlighted.

Multidentate terephthalamidate and hydroxypyridonate ligands: towards new orally active chelators.

The limitations of current therapies for the treatment of iron overload or radioisotope contamination have stimulated efforts to develop new orally bioavailable iron and actinide chelators. Siderophore-inspired tetradentate, hexadentate and octadentate terephthalamidate and hydroxypyridonate ligands were evaluated in vivo as selective and efficacious iron or actinide chelating agents, with several metal loading and ligand assessment procedures, using (59)Fe, (238)Pu, and (241)Am as radioactive tracers. The compounds presented in this study were compared to commercially available therapeutic sequestering agents [deferoxamine (DFO) for iron and diethylenetriaminepentaacetic acid (DPTA) for actinides] and are unrivaled in terms of affinity, selectivity and decorporation efficacy, which attests to the fact that high metal affinity may overcome the low bioavailability properties commonly associated to multidenticity.

SIZ1 regulation of phosphate starvation-induced root architecture remodeling involves the control of auxin accumulation.

Phosphate (Pi) limitation causes plants to modulate the architecture of their root systems to facilitate the acquisition of Pi. Previously, we reported that the Arabidopsis (Arabidopsis thaliana) SUMO E3 ligase SIZ1 regulates root architecture remodeling in response to Pi limitation; namely, the siz1 mutations cause the inhibition of primary root (PR) elongation and the promotion of lateral root (LR) formation. Here, we present evidence that SIZ1 is involved in the negative regulation of auxin patterning to modulate root system architecture in response to Pi starvation. The siz1 mutations caused greater PR growth inhibition and LR development of seedlings in response to Pi limitation. Similar root phenotypes occurred if Pi-deficient wild-type seedlings were supplemented with auxin. N-1-Naphthylphthalamic acid, an inhibitor of auxin efflux activity, reduced the Pi starvation-induced LR root formation of siz1 seedlings to a level equivalent to that seen in the wild type. Monitoring of the auxin-responsive reporter DR5::uidA indicated that auxin accumulates in PR tips at early stages of the Pi starvation response. Subsequently, DR5::uidA expression was observed in the LR primordia, which was associated with LR elongation. The time-sequential patterning of DR5::uidA expression occurred earlier in the roots of siz1 as compared with the wild type. In addition, microarray analysis revealed that several other auxin-responsive genes, including genes involved in cell wall loosening and biosynthesis, were up-regulated in siz1 relative to wild-type seedlings in response to Pi starvation. Together, these results suggest that SIZ1 negatively regulates Pi starvation-induced root architecture remodeling through the control of auxin patterning.

Inhibitory effects of phthalimide derivatives on the activity of the hepatic cytochrome P450 monooxygenases CYP2C9 and CYP2C19.

Affecting hepatic cytochrome (CYP) activity is one of the major concerns in drug-drug interaction. Thus the testing of drug candidates on their impact on these enzymes is an essential step in early drug discovery. We tested a collection of 480 in-house phthalimide derivatives against different CYP450s using a high throughput inhibition assay. In initial tests with the isoform CYP2C19 about 57.5% of the tested phthalimide derivatives showed significantly enhanced inhibitory effects against this enzyme. In addition similar patterns of phthalimide inhibition for CYP2C9 and CYP2C19 were found, whereas the unrelated isoforms CYP2D6 and CYP3A4 were not specifically affected. Also less than 10% of randomly chosen substances inhibited CYP2C9. Analyses of structure-function relationships revealed that the substituent at the nitrogen atom in the isoindole ring is of crucial impact for the activity of CYP2C9/19.

Efficient reversal of Alzheimer's disease fibril formation and elimination of neurotoxicity by a small molecule.

The Abeta1-42 peptide that is overproduced in Alzheimer's disease (AD) from a large precursor protein has a normal amino acid sequence but, when liberated, misfolds at neutral pH to form "protofibrils" and fibrils that are rich in beta-sheets. We find that these protofibrils or fibrils are toxic to certain neuronal cells that carry Ca-permeant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Disrupting the structure of the Abeta1-42 fibrils and protofibrils might lead to the discovery of molecules that would be very useful in the treatment of AD. A high-throughput screen of a library of >3,000 small molecules with known "biological activity" was set up to find compounds that efficiently decrease the beta-sheet content of aggregating Abeta1-42. Lead compounds were characterized by using thioflavin T (ThT) as a beta-sheet assay. The most effective of six compounds found was 4,5-dianilinophthalimide (DAPH) under the following conditions: DAPH at low micromolar concentrations abolishes or greatly reduces previously existing fully formed Abeta1-42 fibrils, producing instead amorphous materials without fibrils but apparently containing some protofibrils and smaller forms. Coincubation of the Abeta1-42 peptide with DAPH produces either amorphous materials or empty fields. Coincubation of DAPH and Abeta1-42 greatly reduces the beta-sheet content, as measured with ThT fluorescence, and produces a novel fluorescent complex with ThT. When the Abeta1-42 peptide was coincubated with DAPH at very low micromolar concentrations, the neuronal toxicity mentioned above (Ca(2+) influx) was eliminated. Clearly, DAPH is a promising candidate for AD therapy.

Comparative anticonvulsant activity and neurotoxicity of 4-amino-N-(2,6-dimethylphenyl)phthalimide and prototype antiepileptic drugs in mice and rats.

We compared the anticonvulsant activity and neurotoxicity of 4-amino-N-(2,6-dimethylphenyl)phthalimide (ADD 213063) with those of phenytoin (PHT), carbamazepine (CBZ), phenobarbital (PB), ethosuximide (ESM), valproate (VPA), and felbamate (FBM). Evaluation of anticonvulsant properties performed according to well-established procedures in rats and mice showed that ADD 213063 is most effective in protecting animals against maximal electroshock seizures (MES). This anti-MES activity is achieved with nontoxic doses, with the optimal effect recorded in rats dosed orally with anti-MES ED50 and protective index (PI) values of 25.2 mumol/kg and > 75, respectively. ADD 213063 protects to a lesser extent against seizures induced by subcutaneous (s.c.) picrotoxin and subcutaneous pentylenetetrazol (PTZ) in mice dosed intraperitoneally and orally, respectively. The profile of anticonvulsant action of ADD 213063 closely parallels that of CBZ.

Characterization of [3H]acifluorfen binding to purified pea etioplasts, and evidence that protoporphyrinogen oxidase specifically binds acifluorfen.

It is now generally accepted that protoporphyrinogen oxidase is the target-enzyme for diphenyl-ether-type herbicides. Recent studies [Camadro, J-M., Matringe M., Scalla, R. & Labbe, P. (1991) Biochem. J. 277, 17-21] have revealed that in maize, diphenyl ethers competitively inhibit protoporphyrinogen oxidase with respect to its substrate, protoporphyrinogen IX. In this study, we show that, in purified pea etioplast, [3H]acifluorfen specifically binds to a single class of high-affinity binding sites with an apparent dissociation constant of 6.2 +/- 1.3 nM and a maximum density of 29 +/- 5 nmol/g protein. [3H]Acifluorfen binding reaches equilibrium in about 1 min at 30 degrees C. Half dissociation occurs in less than 30 s, indicating that the binding is fully reversible. The specificity of [3H]acifluorfen binding to protoporphyrinogen oxidase is examined. [3H]Acifluorfen binding is inhibited by all the peroxidizing molecules tested. The phthalimide derivative, N-(4-chloro-2-fluoro-5-isopropoxy)phenyl-3,4,5,6-tetra hydrophthalimide, exerts a mixed-competitive inhibition on this binding. The effects of all these molecules on the binding of [3H]acifluorfen are tightly linked to their capacity to inhibit pea etioplast protoporphyrinogen oxidase activity. Furthermore, protoporphyrinogen IX, the substrate of the reaction catalyzed by protoporphyrinogen oxidase, was able to competitively inhibit the binding of [3H]acifluorfen. In contrast, protoporphyrin IX, the product of the reaction, did not inhibit this binding. All these results provide clear evidence that in pea etioplasts, [3H]acifluorfen exclusively binds to protoporphyrinogen oxidase, that the protoporphyrinogen oxidase inhibitors tested so far bind to the same region of the enzyme and that this region overlaps the catalytic site of the enzyme.