Design, synthesis, and evaluation of acyclic C-nucleoside and N-methylated derivatives of the ribitylaminopyrimidine substrate of lumazine synthase as potential enzyme inhibitors and mechanistic probes

Lumazine synthase and riboflavin synthase catalyze the last two steps in the biosynthesis of riboflavin, a vitamin that is involved in many critical biochemical reactions that are essential for the maintenance of life. To obtain inhibitors and structural probes that could be useful in studying the structures of bound reaction intermediates, the ribitylamino N-H moiety of the lumazine synthase substrate was replaced by CH(2) and N-CH(3) groups. The CH(2) replacement unexpectedly and completely abolished the affinity for lumazine synthase, thus revealing a critical, yet unexplained, role of the ribitylamino N-H moiety in conferring affinity for the enzyme. In contrast, the N-CH(3) replacement resulted in an inhibitor of both lumazine synthase and riboflavin synthase. Replacement of the ribitylamino N-H moiety with epimeric C-F moieties led to inhibition of lumazine synthase and riboflavin synthase when combined with the replacement of the 5-amino group with a nitro substituent.     

Titration-based screening for evaluation of natural product extracts: identification of an aspulvinone family of luciferase inhibitors

The chemical diversity of nature has tremendous potential for the discovery of molecular probes and medicinal agents. However, sensitivity of HTS assays to interfering components of crude extracts derived from plants, and macro- and microorganisms has curtailed their use in lead discovery. Here, we describe a process for leveraging the concentration-response curves obtained from quantitative HTS to improve the initial selection of "actives" from a library of partially fractionated natural product extracts derived from marine actinomycetes and fungi. By using pharmacological activity, the first-pass CRC paradigm improves the probability that labor-intensive subsequent steps of reculturing, extraction, and bioassay-guided isolation of active component(s) target the most promising strains and growth conditions. We illustrate how this process identified a family of fungal metabolites as potent inhibitors of firefly luciferase, subsequently resolved in molecular detail by X-ray crystallography.     

Proficiency evaluation as a traceability link in chemical metrology

Traceability implies comparison of the results of measurements, or comparison to national or international measurement standards. One of several approaches that have been used in chemistry to provide for such comparisons is distribution of proficiency evaluation materials which have been measured by a reference laboratory. A newer approach is based on receipt and measurement at a reference laboratory of materials that have been produced and analyzed by other laboratories. Traceability concepts and approaches to realization will be described together with discussion of the relative merits of various approaches. Extension into metrological fields other than chemistry will also be explored. Received: 14 November 2000 Accepted: 11 December 2000     

Development of a dual luciferase reporter screening assay for the detection of synthetic glucocorticoids in animal tissues

Synthetic glucocorticoids belong to the most frequently administered drugs in livestock production. These synthetic hormones are employed for therapeutic purposes against inflammatory reactions, disorders of the musculoskeletal system, bovine ketosis and many other diseases of farm animals. A widespread illegal use of synthetic glucocorticoids to improve feed intake and weight gain has also been observed. To enforce the residue limits imposed on glucocorticoid drugs and preclude their illicit administration as growth promoters, it is necessary to establish high throughput analytical methods that can be applied to the screening of animal tissues. Here, we developed a dual luciferase reporter assay that detects residues or contaminants with glucocorticoid activity. This screening assay is performed by transfection of human cell lines with two reporter constructs followed by the measurement of two distinct luminescence signals, one of which serves as internal control to correct for assay variabilities and unspecific matrix effects. The limit of detection (1.25 microg for dexamethasone in liver) depends on the biological potency of each synthetic glucocorticoid but, with all drugs tested, the maximal response reaches a 20 to 30 fold induction of luciferase activity. In combination with an appropriate sample clean-up method (recovery of 82%), this luciferase assay has been applied to the analysis of liver samples from calves treated with a single therapeutic injection of either dexamethasone or flumethasone. Thus, the dual luciferase reporter assay provides a new screening tool to detect unwanted glucocorticoid activities in animal tissues or other crude biological samples without knowledge of the precise chemical entity of the parent compounds or their metabolites.     

All eight stereoisomeric D-glyconic-delta-lactams: synthesis, conformational analysis, and evaluation as glycosidase inhibitors

An efficient and general synthetic route to all eight stereoisomeric D-glycono-delta-lactams has been developed. The strategy involves, as a key step, a stereodivergent delta-lactam formation with configurational retention or inversion at C-4 of a starting gamma-lactone to lead to two epimers of delta-lactam from one parent gamma-lactone. Conformations of eight glycono-delta-lactams were examined by X-ray crystallographic analysis and molecular modeling. Analyses of conformation and glycosidase-inhibition provide useful information for the design of new glycosidase inhibitors.     

Reactivity of amino acid nucleoside phosphoramidates: a mechanistic quantum chemical study

Recent experimental evidence (Maiti et al. Chem.-Eur. J., submitted) indicates that hydrolysis of nucleoside phosphoramidates is subjected to anchimeric influence by carboxyl moieties in the leaving group but also by the base in the nucleotide. A quantum chemical analysis of these findings is presented. First the intrinsic hydrolysis mechanism is investigated for simplified model compounds, and then both amino acid and nucleoside substituents are included. It is found that hydrolysis is assisted by the α-carboxyl group via formation of a five-membered intermediate and that the barrier for the reaction of this intermediate toward the product state can be influenced by the nucleobase. The adenine base protonated on N3 interacts with the transition state and considerably lowers the barrier for hydrolysis. The influence of several base modifications is explained by calculating the pK(a) for protonation on N3.     

Enzyme inhibitors as chemical tools to study enzyme catalysis: rational design, synthesis, and applications

Carefully designed molecules that are intimately related to the reaction mechanism of enzymes are often highly selective and potent inhibitors that serve as extremely useful chemical probes for understanding the reaction mechanism and structure of enzymes. This article describes the design, synthesis, and applications of specific inhibitors of two mechanistically distinct groups of enzymes, ATP-dependent amide ligases and Ser- and Thr-hydrolases. Our strategy is based on the premise that stable analogues of the transition state (transition-state analogues) are highly potent inhibitors that serve as good mechanistic probes, and that a key structure of a good inhibitor of one enzyme is also utilized for the inhibitors of other enzymes that share the same chemistry in their catalyzed reactions, irrespective of the degree of structural similarity and evolutionary link between the enzymes. According to these principles, we designed and synthesized a series of phosphinate- and sulfoximine-based transition-state analogue inhibitors of glutathione synthetase, gamma-glutamylcysteine synthetase and asparagine synthetase. For the second group of enzymes, we synthesized a gamma-monofluorophosphono glutamate analogue for mechanism-based affinity labeling of gamma-glutamyltranspeptidase and fluorescent phosphonic acid esters for the active-site titration of lipase. These inhibitors were used successfully as ligands for detailed kinetic analyses, X-ray crystallography, and mass analysis of the enzymes to identify the key amino acid residues responsible for catalysis and substrate recognition in the transition state.     

Aromatization of 3,4-dihydro-β-carboline-3-qarboxylic acid and its derivatives through a carbanion intermediate : mechanistic study and use in chemical synthesis

3,4-dlhydro-β-carboline-3-carboxylic acid , its esters and amide derivatives (AH₂) undergo complete aromatization into corresponding β-carboline derivatives (A) by basic treatment under mild conditions. This, together with the easy obtention of 3,4-dihydro-β-carboline from the parent N^αformyltryptophan derivatives constitutes an attractive possibility to obtain complex molecules containing the β-carboline ring. The mechanism of the reaction was investigated by polarography, U.V.spectrometry and polarimetry. In alcaline media, (AH₂) undergoes two successive equilibria : the first yielding a C₃ carbanion occurs with suppression of the chirality while the second yields a thermodynamically preferred C₄ carbanion which undergoes aromatization through an oxidative pathway (K'_a = 2×10^-14 at 45°C).     

Small molecules,big impact: a history of chemical inhibitors and the cytoskeleton

Chemical inhibitors, whether natural products or synthetic, have had an enormous impact on the study of the eukaryotic cytoskeleton. Here we review the history of some of the most widely used cytoskeletal poisons and their influence on our understanding of cytoskeletal functions. We then highlight several new inhibitors and the targeted screens used to identify them and discuss why this approach has been successful.     

Strategy for discovering chemical inhibitors of human cyclophilin a: focused library design, virtual screening, chemical synthesis and bioassay

The discovery of cyclophilin A (CypA) inhibitor is now of special interest in the treatment of immunological disorders. In this work, using a strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and bioassay, a series of novel small molecular CypA inhibitors have been discovered. First, using the fragments taken from our previously discovered CypA inhibitors (Bioorg. Med. Chem. 2006, 14, 2209-2224) as building blocks, we designed a focused combinatorial library containing 255 molecules employing the LD1.0 program (J. Comb. Chem. 2005, 7, 398-406) developed by us. Sixteen compounds (1a-e, 2a-b, 3a-b, and 4a-g) were selected by using virtual screening against the X-ray crystal structure of CypA as well as druglike analysis for further synthesis and bioassay. All these sixteen molecules are CypA binders with binding affinities (K(D) values) ranging from 0.076 to 41.0 microM, and five of them (4a, 4c, and 4e-g) are potent CypA inhibitors with PPIase inhibitory activities (IC(50) values) of 0.25-6.43 microM. The hit rates for binders and inhibitors are as high as 100% and 31.25%, respectively. Remarkably, both the binding affinity and inhibitory activity of the most potent compound increase approximately 10 times than that of the most active compound discovered previously. The high hit rate and the high potency of the new CypA inhibitors demonstrated the efficiency of the strategy for focused library design and screening. In addition, the novel chemical entities reported in this study could be leads for discovering new therapies against the CypA pathway.     

Synthesis and evaluation of novel pyrimido-acridone, -phenoxadine, and -carbazole as topoisomerase II inhibitors

As part of a series of studies to discover new topoisomerase II inhibitors, novel pyrimidoacridones, pyrimidophenoxadines, and pyrimidocarbazoles were synthesized, and in vitro and in vivo antitumor activities and DNA-protein and/or DNA-topoisomerase II cross-linking activity as an indicator of topoisomerase II-DNA cleavable complex formation were evaluated. The pyrimidocarbazoles possessed high in vitro and in vivo potencies. Compound 26 (ER-37326), 8-acetyl-2-[2-(dimethylamino)ethyl]-1H-pyrimido[5,6,1-jk]carbazole-1,3(2H)-dione, showed in vitro growth inhibitory activity with respective IC(50) values of 0.049 microM and 0.35 microM against mouse leukemia P388 and human oral cancer KB. In vivo, this compound inhibited the tumor growth of mouse sarcoma M5076 implanted into mice with T/C values of 42% and 13% at 3.13 and 6.25 mg/kg/d respectively without significantly affecting the body weight. In addition, compound 26 (ER-37326) increased the formation of DNA-topoisomerase II cross-linking in P388 cells.     

Silicon-based metalloprotease inhibitors: synthesis and evaluation of silanol and silanediol peptide analogues as inhibitors of angiotensin-converting enzyme

Silanols are best known as unstable precursors of siloxane (silicone) polymers, substances generally considered stable and inert, but have the potential to mimic a hydrated carbonyl and inhibit protease enzymes. While previous testing of simple silanediol and silanetriol species as inhibitors of hydrolase enzymes found them ineffective, this study reports polypeptide mimics with a central methylsilanol [SiMeOH] or silanediol [Si(OH)(2)] group and their assessment as effective transition state analogue inhibitors of the well-studied metalloprotease angiotensin-converting enzyme (ACE). Central to the synthesis strategy, phenylsilanes were employed as acid-hydrolyzable precursors of the silanol group. The N-benzoyl Leu-[SiMeOH]-Gly benzyl amides proved to be stable and readily characterized. In contrast, the Leu-[Si(OH)(2)]-Gly structure was difficult to characterize, possibly because of self-association. Capping the silanediol with chlorotrimethylsilane gave a well-defined trisiloxane, demonstrating that the silanediol was monomeric. The Leu-[Si]-Gly structures were converted to Leu-[Si]-Ala analogues by enolate alkylation. Coupling of the silanol precursors with proline tert-butyl ester gave N-benzoyl Leu-[Si]-Gly-Pro and N-benzoyl Leu-[Si]-Ala-Pro tripeptide analogues. Treatment of these with triflic acid formed the corresponding methylsilanols and silanediols, all of which were monomeric. The silanediol tripeptide mimics inhibited ACE with IC(50) values as low as 14 nM. Methylsilanols, in contrast, were poor inhibitors, with IC(50) values above 3000 nM. These data, including comparisons with inhibition data from carbon analogues, are consistent with binding of the silanediols by chelation of the ACE active site zinc, whereas the methylsilanols ligate poorly.     

Design, synthesis, and biological evaluation of beta-ketosulfonamide adenylation inhibitors as potential antitubercular agents

[reaction: see text] The antitubercular nucleoside antibiotics 1 and 2 were recently described that inhibit the adenylate-forming enzyme MbtA and disrupt biosynthesis of the virulence-conferring siderophore known as mycobactin in Mycobacterium tuberculosis. Herein, we report efforts to refine this inhibitor scaffold by replacing the labile acylsulfamate linkage (highlighted) with the more chemically robust beta-ketosulfonamide linkage of 3 and 4.     

Challenges and perspectives of chemical biology, a successful multidisciplinary field of natural sciences

Objects, goals, and main methods as well as perspectives of chemical biology are discussed. This review is focused on the fundamental aspects of this emerging field of life sciences: chemical space, the small molecule library and chemical sensibilization (small molecule microassays).     

SMM-chemokines: a class of unnatural synthetic molecules as chemical probes of chemokine receptor biology and leads for therapeutic development

Chemokines and their receptors play important roles in numerous physiological and pathological processes. To develop natural chemokines into receptor probes and inhibitors of pathological processes, the lack of chemokine-receptor selectivity must be overcome. Here, we apply chemical synthesis and the concept of modular modifications to generate unnatural synthetically and modularly modified (SMM)-chemokines that have high receptor selectivity and affinity, and reduced toxicity. A proof of the concept was shown by transforming the nonselective viral macrophage inflammatory protein-II into new analogs with enhanced selectivity and potency for CXCR4 or CCR5, two principal coreceptors for human immunodeficiency virus (HIV)-1 entry. These new analogs provided insights into receptor binding and signaling mechanisms and acted as potent HIV-1 inhibitors. These results support the concept of SMM-chemokines for studying and controlling the function of other chemokine receptors.