Synthesis and evaluation of novel aromatic substrates and competitive inhibitors of GABA aminotransferase

The design, synthesis, and evaluation of novel gamma-aminobutyric acid aminotransferase (GABA-AT) inhibitors and inactivators can lead to the discovery of new GABA-related therapeutics. To this end, a series of aromatic amino acid compounds was synthesized to aid in the design of new inhibitors and inactivators of GABA-AT. All compounds were tested as competitive inhibitors of GABA-AT. The amino acids with benzylic amines were also tested as substrates for GABA-AT. It was found that these compounds were all poor competitive inhibitors of GABA-AT, but some were substrates of the enzyme, suggesting their utility as scaffolds for potential GABA-AT mechanism-based inactivators. Computer modeling was used to rationalize the substrate activity of the various compounds.     

S-phase Inhibition of Cell Cycle Progression by a Novel Class of Pyridopyrimidine Tyrosine Kinase Inhibitors

Increased activity of the src family of oncogenic tyrosine kinases is seen in many human tumors and pharmacologic inhibitors of these kinases are investigated as potential anti-tumor agents. A family of pyrido [2, 3-d] pyrimidine compounds (PD) has been characterized as selective inhibitors of Src kinases. We studied the effects of this class of compounds on cancer cell lines and found that they were highly specific inhibitors of cell cycle progression. These compounds inhibit cells either in the mitotic phase or in mid S-phase; these two activities are mutually exclusive: no compound exerts both activities. We undertook experiments to determine the mechanistic basis for these differences and found additional biochemical activities associated with the S-phase inhibitors. Treatment of cells with the S-phase blocker PD179483 causes abnormal and persistent hyperactivation of Cdk2 and Cdc2 due to Tyr-15 dephosphorylation. These effects were associated with hyperphosphorylation of the upstream regulatory kinase Myt1 and Wee1. They were not observed with the anti-mitotic compounds. Furthermore, the S-phase inhibitors PD179483 and PD166326, but not the anti-mitotic compounds, inhibit Wee1 in vitro at concentrations that cause S-phase block in vivo. These data identify a novel subset of pyridopyrimidine compounds which are inhibitors of src and Wee1 kinases and which inhibit tumor cell growth through cell cycle arrest in mid S-phase.     

A scaffold hopping approach to identify novel monoamine oxidase B inhibitors

Monoamine oxidase B (MAO-B) inhibitors are used to treat Parkinson's disease. In this study, we searched for novel MAO-B inhibitors using a scaffold hopping approach based on our experience with the thiazolidinedione (TZD) class of compounds as MAO-B inhibitors. Several novel compounds were identified, with potencies in the low nanomolar and low micromolar range. We also found that derivatives of the natural product sulfuretin are potent MAO-A and MAO-B inhibitors.     

Computer-assisted search and optimization of new human immunodeficiency virus integrase inhibitors

Finding and optimization of new HIV-1 integrase inhibitors is an important task due to the drawbacks of the existing HIV/AIDS therapy caused by the significant adverse effects, high cost of the treatment, and emerging resistance. In this study we have demonstrated potential of computer-assisted methods for finding and optimization of new HIV-1 integrase inhibitors blocking the 3′-processing reaction. Applicability of the specialized version of the computer program PASS for searching original inhibitors of 3′-processing reaction among commercially available samples of chemical compounds, belonging to a new chemical class, is shown. We compared the general pharmacological profiles calculated by PASS for the sets of original integrase inhibitors found in the study with those for known inhibitors published in literature. It was shown that the predicted average selectivity is close for the compounds from both sets, while the probability of predicted adverse/toxic effects is lower for the original compounds. Differences between the pharmacological profiles of original compounds and inhibitors of 3′-processing known in the literature pointed out to the essential novelty of our substances of the investigated chemical class. Significantly less deviations from the recommended values for QikProp parameters, which characterized the ADME properties, have been observed for the original compounds than for the integrase inhibitors published in literature. Thus, one can conclude that the original compounds look more prospective comparing to the earlier known inhibitors of 3′-processing.     

Triazine-based tyrosinase inhibitors identified by chemical genetic screening

As most of the available depigmenting agents exhibit only modest activity and some exhibit toxicities that lead to adverse side effects after long-term usage, there remains a need for novel depigmenting agents. Chemical genetic screening was performed on cultured melanocytes to identify novel depigmenting compounds. By screening a tagged-triazine library, we identified four compounds, TGH11, TGD10, TGD39 and TGJ29, as potent pigmentation inhibitors with IC50 values in the range of 10 microM. These newly identified depigmenting compounds were found to function as reversible inhibitors of tyrosinase, the key enzyme involved in melanin synthesis. Tyrosinase was further confirmed as the cellular target of these compounds by affinity chromatography. Kinetic data suggest that all four compounds act as competitive inhibitors of tyrosinase, most likely competing with L-3,4-dihydroxyphenylalanine (L-DOPA) for binding to the DOPA-binding site of the enzyme. No effect on levels of tyrosinase protein, processing or trafficking was observed upon treatment of melanocytes with these compounds. Cytotoxicity was not observed with these compounds at concentrations up to 20 muM. Our data suggest that TGH11, TGD10, TGD39 and TGJ29 are novel potent tyrosinase inhibitors with potential beneficial effects in the treatment of cutaneous hyperpigmentation.     

Glycosidase inhibitors as antiviral and/or antitumor agents.

Glycoprotein processing inhibitors prevent the normal processing of N-linked glycoproteins by inhibiting specific glycosidases involved in these reactions. Thus, a number of compounds are now known that inhibit alpha-glucosidase I and alpha-glucosidase II and therefore prevent the removal of glucoses from the high-mannose chains. Some of these compounds are more potent inhibitors of one or the other of these glucosidases. There are also a number of inhibitors that affect one of the processing alpha-mannosidases (i.e. mannosidase I or mannosidase II). These compounds; especially the glucosidase inhibitors, have been valuable tools to help us understand the role of carbohydrate in viral envelope glycoprotein function. Such processing inhibitors have also been used with various tumorigenic cell lines to determine the function of N-linked glycoproteins in cancer.     

Detection of allosteric kinase inhibitors by displacement of active site probes

Non-adenosine triphosphate (ATP) competitive, allosteric inhibitors provide a promising avenue to develop highly selective small-molecule kinase inhibitors. Although this class of compounds is growing, detection of such inhibitors can be challenging as standard kinase activity assays preferentially detect compounds that bind to active kinases in an ATP competitive manner. We have previously described a time-resolved fluorescence resonance energy transfer (TR-FRET)-based kinase binding assay using the competitive displacement of ATP competitive active site fluorescent probes ("tracers"). Although this format has gained acceptance, published data with this and related formats are almost entirely without examples of non-ATP competitive compounds. Thus, this study addresses whether this format is useful for non-ATP competitive inhibitors. To this end, 15 commercially available non-ATP competitive inhibitors were tested for their ability to displace ATP competitive probes. Despite the diversity of both compound structures and their respective targets, 14 of the 15 compounds displaced the tracers with IC(50) values comparable to literature values. We conclude that such binding assays are well suited for the study of non-ATP competitive inhibitors. In addition, we demonstrate that allosteric inhibitors of BCR-Abl and MEK bind preferentially to the nonphosphorylated (i.e., inactive) form of the kinase, indicating that binding assays may be a preferred format in some cases.     

Recent syntheses of PI3K/Akt/mTOR signaling pathway inhibitors

This review focuses on the syntheses of PI3K/Akt/mTOR inhibitors that have been reported outside of the patent literature in the last 5 years but is largely centered on synthetic work reported in 2011 and 2012. While focused on syntheses of inhibitors, some information on in vitro and in vivo testing of compounds is also included. Many of these reported compounds are reversible, competitive adenosine triphosphate (ATP) binding inhibitors, so given the structural similarities of many of these compounds to the adenine core, this review presents recent work on inhibitors based on where the synthetic chemistry was started, that is, inhibitor syntheses which started with purines/pyrimidines are followed by inhibitor syntheses which began with pyridines, pyrazines, azoles, and triazines then moves to inhibitors which bear no structural resemblance to adenine: liphagal, wortmannin and quercetin analogs. The review then finishes with a short section on recent syntheses of phosphotidyl inositol (PI) analogs since competitive PI binding inhibitors represent an alternative to the competitive ATP binding inhibitors which have received the most attention.     

Screening of non-alkaloidal natural compounds as acetylcholinesterase inhibitors

Acetylcholinesterase (AChE) inhibitors are currently the only approved therapy for the treatment of Alzheimer's disease, only a limited number of drugs are commercially available. A library of non-alkaloidal natural compounds was investigated. To this end, a convenient microtitre plate method for assaying AChE inhibition, which allows a complete kinetic analysis of AChE inhibitors, was developed. Seven active compounds with Ki values in the micromolar range were identified, six of which were xanthones. This is the first report that a promising potential for AChE inhibition exists in such non-nitrogenous natural compounds. Furthermore, four xanthones among these xanthones had already been described as monoamine oxidase (MAO) inhibitors, making then dual AChE/MAO inhibitors of great interest.     

Immunoproteasome inhibition and bioactivity of thiasyrbactins

A family of macrodilactam natural products, the syrbactins, are known proteasome inhibitors. A small group of syrbactin analogs was prepared with a sulfur-for-carbon substitution to enhance synthetic accessibility and facilitate modulation of their solubility. Two of these compounds surprisingly proved to be inhibitors of the trypsin-like catalytic site, including of the immunoproteasome. Their bound and free conformations suggest special properties of the thiasyrbactin ring are responsible for this unusual preference, which may be exploited to develop drug-like immunoproteasome inhibitors. These compounds show greater selectivity than earlier compounds used to infer phenotypes of immunoproteasome inhibition, like ONX-0914.     

Characterization of the binding properties of SIRT2 inhibitors with a N-(3-phenylpropenoyl)-glycine tryptamide backbone

SIRT2 inhibitors with a N-(3-phenylpropenoyl)-glycine tryptamide backbone were studied. This backbone has been developed in our group, and it is derived from a compound originally found by virtual screening. In addition, compounds with a smaller 3-phenylpropenoic acid tryptamide backbone were also included in the study. Binding modes for the new compounds and the previously reported compounds were analyzed with molecular modelling methods. The approach, which included a combination of molecular dynamics, molecular docking and cluster analysis, showed that certain docking poses were favourable despite the conformational variation in the target protein. The N-(3-phenylpropenoyl)-glycine tryptamide backbone is also a good backbone for SIRT2 inhibitors, and the series of compounds includes several potent SIRT2 inhibitors.     

Evidence for the mechanism of the irreversible inhibition of oestrone sulphatase (ES) by aminosulphonate based compounds

In our search for the mechanism of the enzyme oestrone sulphatase (ES) we have synthesised and evaluated a number of compounds that were predicted to possess some inhibitory activity. Some of these compounds were indeed found to be inhibitors of ES, whilst other compounds were not. From a consideration of the structure–activity relationship (SAR) of the inhibitors and non-inhibitors of this enzyme, we discovered a factor which we now believe is the main inhibitory moiety within the aminosulphonated inhibitors. We therefore report the results of our study into a series of phenyl and alkyl sulphamated compounds as inhibitors of ES. The results of the study show that the substituted phenyl sulphamates are potent inhibitors, whereas the alkyl compounds are, in general, non-inhibitors. Using the results of our SAR study, we postulate the probable mechanism for the irreversible and reversible inhibition of ES, and rationalise the role of the different physicochemical factors in the inhibition of this crucial enzyme.     

Selective inhibitors of bacterial DNA adenine methyltransferases

The authors describe the discovery and characterization of several structural classes of small-molecule inhibitors of bacterial DNA adenine methyltransferases. These enzymes are essential for bacterial virulence (DNA adenine methyltransferase [DAM]) and cell viability (cell cycle-regulated methyltransferase [CcrM]). Using a novel high-throughput fluorescence-based assay and recombinant DAM and CcrM, the authors screened a diverse chemical library. They identified 5 major structural classes of inhibitors composed of more than 350 compounds: cyclopentaquinolines, phenyl vinyl furans, pyrimidine-diones, thiazolidine-4-ones, and phenyl-pyrroles. DNA binding assays were used to identify compounds that interact directly with DNA. Potent compounds selective for the bacterial target were identified, whereas other compounds showed greater selectivity for the mammalian DNA cytosine methyltransferase, Dnmt1. Enzyme inhibition analysis identified mechanistically distinct compounds that interfered with DNA or cofactor binding. Selected compounds demonstrated cell-based efficacy. These small-molecule DNA methyltransferase inhibitors provide useful reagents to probe the role of DNA methylation and may form the basis of developing novel antibiotics.     

Pseudoirreversible inhibition of prostate-specific membrane antigen by phosphoramidate peptidomimetics

The mode of inhibition for phosphoramidate peptidomimetic inhibitors of prostate-specific membrane antigen was determined by inhibition reversibility experiments. The results revealed that these inhibitors can be classified into three types: pseudoirreversible (compounds 1-3), moderately reversible (compounds 4-9), and rapidly reversible inhibitors (compounds 10 and 11). Representative compounds from each class were further evaluated for their ability to induce cellular internalization of PSMA. Results from these experiments revealed that the pseudoirreversible inhibitor 1 induced the greatest PSMA internalization. The discovery of pseudoirreversible PSMA inhibitors is expected to provide a new avenue of investigation and therapeutic applications for prostate cancer and neurological disorders.     

Triazine‐based tyrosinase inhibitors identified by chemical genetic screening

As most of the available depigmenting agents exhibit only modest activity and some exhibit toxicities that lead to adverse side effects after long‐term usage, there remains a need for novel depigmenting agents. Chemical genetic screening was performed on cultured melanocytes to identify novel depigmenting compounds. By screening a tagged‐triazine library, we identified four compounds, TGH11, TGD10, TGD39 and TGJ29, as potent pigmentation inhibitors with IC₅₀ values in the range of 10 μM. These newly identified depigmenting compounds were found to function as reversible inhibitors of tyrosinase, the key enzyme involved in melanin synthesis. Tyrosinase was further confirmed as the cellular target of these compounds by affinity chromatography. Kinetic data suggest that all four compounds act as competitive inhibitors of tyrosinase, most likely competing with l ‐3,4‐dihydroxyphenylalanine (l ‐DOPA) for binding to the DOPA‐binding site of the enzyme. No effect on levels of tyrosinase protein, processing or trafficking was observed upon treatment of melanocytes with these compounds. Cytotoxicity was not observed with these compounds at concentrations up to 20 μM. Our data suggest that TGH11, TGD10, TGD39 and TGJ29 are novel potent tyrosinase inhibitors with potential beneficial effects in the treatment of cutaneous hyperpigmentation.     

High throughput screening of potentially selective MMP-13 exosite inhibitors utilizing a triple-helical FRET substrate

The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Matrix metalloproteinase 13 (MMP-13) has been implicated as the protease responsible for collagen degradation in cartilage during osteoarthritis (OA). In the present study, a triple-helical FRET substrate has been utilized for high throughput screening (HTS) of MMP-13 with the MLSCN compound library (n approximately 65,000). Thirty-four compounds from the HTS produced pharmacological dose-response curves. A secondary screen using RP-HPLC validated 25 compounds as MMP-13 inhibitors. Twelve of these compounds were selected for counter-screening with 6 representative MMP family members. Five compounds were found to be broad-spectrum MMP inhibitors, 3 inhibited MMP-13 and one other MMP, and 4 were selective for MMP-13. One of the selective inhibitors was more active against MMP-13 triple-helical peptidase activity compared with single-stranded peptidase activity. Since the THP FRET substrate has distinct conformational features that may interact with MMP secondary binding sites (exosites), novel non-active site-binding inhibitors may be identified via HTS protocols utilizing such assays.     

Highly specific, bisubstrate-competitive Src inhibitors from DNA-templated macrocycles

Protein kinases are attractive therapeutic targets, but their high sequence and structural conservation complicates the development of specific inhibitors. We recently identified, in a DNA-templated macrocycle library, inhibitors with unusually high selectivity among Src-family kinases. Starting from these compounds, we developed and characterized in molecular detail potent macrocyclic inhibitors of Src kinase and its cancer-associated 'gatekeeper' mutant. We solved two cocrystal structures of macrocycles bound to Src kinase. These structures reveal the molecular basis of the combined ATP- and substrate peptide-competitive inhibitory mechanism and the remarkable kinase specificity of the compounds. The most potent compounds inhibit Src activity in cultured mammalian cells. Our work establishes that macrocycles can inhibit protein kinases through a bisubstrate-competitive mechanism with high potency and exceptional specificity, reveals the precise molecular basis for their desirable properties and provides new insights into the development of Src-specific inhibitors with potential therapeutic relevance.     

Design, synthesis and biochemical evaluation of AC ring mimics as novel inhibitors of the enzyme estrone sulfatase (ES)

We report the initial results of our study into a series of simple 4'-O-sulfamoyl-4-biphenyl based compounds as novel inhibitors of the enzyme estrone sulfatase (ES). The results of the study show that these compounds are potent inhibitors, possessing greater inhibitory activity than COUMATE, but weaker inhibitory activity than EMATE or the tricyclic derivative of COUMATE, namely 667-COUMATE. Furthermore, the compounds are observed to be irreversible inhibitors.     

Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity

Small-molecule protein kinase inhibitors are widely used to elucidate cellular signaling pathways and are promising therapeutic agents. Owing to evolutionary conservation of the ATP-binding site, most kinase inhibitors that target this site promiscuously inhibit multiple kinases. Interpretation of experiments that use these compounds is confounded by a lack of data on the comprehensive kinase selectivity of most inhibitors. Here we used functional assays to profile the activity of 178 commercially available kinase inhibitors against a panel of 300 recombinant protein kinases. Quantitative analysis revealed complex and often unexpected interactions between protein kinases and kinase inhibitors, with a wide spectrum of promiscuity. Many off-target interactions occur with seemingly unrelated kinases, revealing how large-scale profiling can identify multitargeted inhibitors of specific, diverse kinases. The results have implications for drug development and provide a resource for selecting compounds to elucidate kinase function and for interpreting the results of experiments involving kinase inhibitors.