New TRAP1 and Hsp90 chaperone inhibitors with cationic components: Preliminary studies on mitochondrial targeting.

TRAP1 (Hsp75) is the mitochondrial paralog of the Hsp90 molecular chaperone family. Due to structural similarity among Hsp90 chaperones, a potential strategy to induce apoptosis through mitochondrial TRAP1 ATPase inhibition has been envisaged and a series of compounds has been developed by binding the simple pharmacophoric core of known Hsp90 inhibitors with various appendages bearing a permanent cationic head, or a basic group highly ionizable at physiologic pH. Cationic appendages were selected as vehicles to deliver drugs to mitochondria. Indeed, masses of new derivatives were evidenced to accumulate in the mitochondrial fraction from colon carcinoma cells and a compound in the series, with a guanidine appendage, demonstrated good activity in inhibiting recombinant TRAP1 ATPase and cell growth and in inducing apoptotic cell death in colon carcinoma cells.

Homodimeric enzymes as drug targets.

Many enzymes and proteins are regulated by their quaternary structure and/or by their association in homo- and/or hetero-oligomer complexes. Thus, these protein-protein interactions can be good targets for blocking or modulating protein function therapeutically. The large number of oligomeric structures in the Protein Data Bank (http://www.rcsb.org/) reflects growing interest in proteins that function as multimeric complexes. In this review, we consider the particular case of homodimeric enzymes as drug targets. There is intense interest in drugs that inhibit dimerization of a functionally obligate homodimeric enzyme. Because amino acid conservation within enzyme interfaces is often low compared to conservation in active sites, it may be easier to achieve drugs that target protein interfaces selectively and specifically. Two main types of dimerization inhibitors have been developed: peptides or peptidomimetics based on sequences involved in protein-protein interactions, and small molecules that act at hot spots in protein-protein interfaces. Examples include inhibitors of HIV protease and HIV integrase. Studying the mechanisms of action and locating the binding sites of such inhibitors requires different techniques for different proteins. For some enzymes, ligand binding is only detectable in vivo or after unfolding of the complexes. Here, we review the structural features of dimeric enzymes and give examples of inhibition through interference in dimer stability. Several techniques for studying these complex phenomena will be presented.

Design and characterization of a mutation outside the active site of human thymidylate synthase that affects ligand binding.

Owing to its central role in DNA synthesis, human thymidylate synthase (hTS) is a well-established target for chemotherapeutic agents, such as fluoropyrimidines. The use of hTS inhibitors in cancer therapy is limited by their toxicity and the development of cellular drug resistance. Here, with the aim of shedding light on the structural role of the A-helix in fluoropyrimidine resistance, we have created a fluoropyrimidine-resistant mutant by making a single point mutation, Glu30Trp. We postulated that residue 30, which is located in the A-helix, close to but outside the enzyme active site, could have a long-range effect on inhibitor binding. The mutant shows 100 times lower specific activity with respect to the wild-type hTS and is resistant to the classical inhibitor, FdUMP, as shown by a 6-fold higher inhibition constant. Circular dichroism experiments show that the mutant is folded. The results of molecular modeling and simulation suggest that the Glu30Trp mutation gives rise to resistance by altering the hydrogen-bond network between residue 30 and the active site.

New thymidylate synthase inhibitors induce apoptosis in melanoma cell lines.

Malignant melanoma is particularly resistant to conventional chemotherapy and radiotherapy. For this reason in the past years a huge variety of new compounds has been developed with potential chemotherapeutic activity which needs to be tested in vitro and in vivo. We investigated the in vitro action of three new experimental antifolate substances (MR7, MR21 and MR36) with a critical target for thymidylate synthase (TS), an essential enzyme for DNA synthesis. The response of two melanoma cell lines (SK-MEL-2 derived from malignant melanoma metastasis and SK-MEL-28 derived from primary malignant melanoma) was examined after treatment with these substances. The antifolate agents induced apoptosis in SK-MEL-2 and SK-MEL-28 cells as confirmed by the TUNEL technique and Comet Assay. Western-blot analysis showed a down-regulation of Bcl-2 protein level and PARP cleavage, otherwise p53 and Bax expressions were not modulated. Moreover, these antifolate-induced apoptosis was accompanied by both pro-caspase-9 and -8 activations. These results were supported by the use of the pan-caspases inhibitor Z-VAD-FMK that almost completely decreased the amount of apoptosis in both the melanoma cell lines treated with antifolate. In conclusion our results show that TS inhibitors are able to induce apoptosis through a caspase-mediated pathway, but without the involvement of the p53/Bax signalling.

Thymidylate synthase structure, function and implication in drug discovery.

Recent methodologies applied to the drug discovery process, such as genomics and proteomics, have greatly implemented our basic understanding of drug action and are giving more input to medicinal chemists, in finding genuinely new targets and opportunities for the development of drugs with original mechanisms of action. In this paper, an example of the successful application of some new techniques to the target enzymes with the Thymidylate Synthase (TS) function is given. The improved knowledge of the complex mechanism of the biological pathways in which thymidylate synthase is involved represents a unique chance to find new mechanism-based inhibitors, aimed to treat not only cancerous diseases, but also infectious pathologies. Thymidylate synthase (TS or ThyA) has long been considered as one of the best-known drug targets in the anti-cancer area, after which old and new drugs, such as 5-fluoro uracil and the anti-folate ZD1694, have been introduced into chemotherapy to treat solid tumours. Only a few attempts have been made to find non-classical anti-folate inhibitors that are dissimilar to the folate co-factor, with the aim of finding unshared protein target domains on the enzyme structure, in order to specifically inhibit TS enzymes from pathogens. Only recently from omic studies, a new Thymidylate Synthase Complementing Protein (TSCP or ThyX) has been identified in a number of pathogens, showing a different structure with respect to human TS, thus opening new avenues to specific inhibitions. A depiction of the most recent progress in the study of Thymidylate Synthase enzymes is presented in the following sections.

Predicting and harnessing protein flexibility in the design of species-specific inhibitors of thymidylate synthase.

BACKGROUND: Protein plasticity in response to ligand binding abrogates the notion of a rigid receptor site. Thus, computational docking alone misses important prospective drug design leads. Bacterial-specific inhibitors of an essential enzyme, thymidylate synthase (TS), were developed using a combination of computer-based screening followed by in-parallel synthetic elaboration and enzyme assay [Tondi et al. (1999) Chem. Biol. 6, 319-331]. Specificity was achieved through protein plasticity and despite the very high sequence conservation of the enzyme between species. RESULTS: The most potent of the inhibitors synthesized, N,O-didansyl-L-tyrosine (DDT), binds to Lactobacillus casei TS (LcTS) with 35-fold higher affinity and to Escherichia coli TS (EcTS) with 24-fold higher affinity than to human TS (hTS). To reveal the molecular basis for this specificity, we have determined the crystal structure of EcTS complexed with DDT and 2'-deoxyuridine-5'-monophosphate (dUMP). The 2.0 A structure shows that DDT binds to EcTS in a conformation not predicted by molecular docking studies and substantially differently than other TS inhibitors. Binding of DDT is accompanied by large rearrangements of the protein both near and distal to the enzyme's active site with movement of C alpha carbons up to 6 A relative to other ternary complexes. This protein plasticity results in novel interactions with DDT including the formation of hydrogen bonds and van der Waals interactions to residues conserved in bacterial TS but not hTS and which are hypothesized to account for DDT's specificity. The conformation DDT adopts when bound to EcTS explains the activity of several other LcTS inhibitors synthesized in-parallel with DDT suggesting that DDT binds to the two enzymes in similar orientations. CONCLUSIONS: Dramatic protein rearrangements involving both main and side chain atoms play an important role in the recognition of DDT by EcTS and highlight the importance of incorporating protein plasticity in drug design. The crystal structure of the EcTS/dUMP/DDT complex is a model system to develop more selective TS inhibitors aimed at pathogenic bacterial species. The crystal structure also suggests a general formula for identifying regions of TS and other enzymes that may be treated as flexible to aid in computational methods of drug discovery.

Update on antifolate drugs targets.

Antifolate drugs are molecules directed to interfere with the folate metabolic pathway at some level. They can be recognized among the first rationally designed compounds applying the principle of structural analogy with the substrate developing the antimetabolite strategy. This strategy has taken advantage of the basic different features of the microbial and human folate metabolism and therefore allows targeting the pathway at different steps generating a specificity tools for Medicinal Chemists. Two main problems are giving renewed importance to such targets and therefore improving the efforts to discover new targets in the folate metabolism area. The first one is the increasing resistance to the present drugs due to different mechanisms such as the enzyme modification and the increased production of enzymes with not well recognized importance. The second one is the development of techniques directed to highlight the interference at genetic level of molecular probes as antifolate drug to develop new enzymes previously unknown. This approach is defined as genetic approach to drug discovery, from gene to drugs. The present article describes the importance in drug design and discovery of some antifolate targets among the best known at the present status of research such as thymidylate synthase (TS), dhydrofolate reductases, (DHFR) serine hydroxymethyltransferase (SHMT), folyilpolyglutamyl synthetase (FPGS), gamma-glutamyl hydrolase (gamma-GH), glycinamide-ribonucleotide transformylase (GARTfase), amino-imidazole-carboxamide-ribonucleotide transformylase (AICARTfase) and Folate transporters. Discovery, known functions, structure/function studies and inhibition will be described.

Structure-based design and in-parallel synthesis of inhibitors of AmpC beta-lactamase.

BACKGROUND: Group I beta-lactamases are a major cause of antibiotic resistance to beta-lactams such as penicillins and cephalosporins. These enzymes are only modestly affected by classic beta-lactam-based inhibitors, such as clavulanic acid. Conversely, small arylboronic acids inhibit these enzymes at sub-micromolar concentrations. Structural studies suggest these inhibitors bind to a well-defined cleft in the group I beta-lactamase AmpC; this cleft binds the ubiquitous R1 side chain of beta-lactams. Intriguingly, much of this cleft is left unoccupied by the small arylboronic acids. RESULTS: To investigate if larger boronic acids might take advantage of this cleft, structure-guided in-parallel synthesis was used to explore new inhibitors of AmpC. Twenty-eight derivatives of the lead compound, 3-aminophenylboronic acid, led to an inhibitor with 80-fold better binding (2; K(i) 83 nM). Molecular docking suggested orientations for this compound in the R1 cleft. Based on the docking results, 12 derivatives of 2 were synthesized, leading to inhibitors with K(i) values of 60 nM and with improved solubility. Several of these inhibitors reversed the resistance of nosocomial Gram-positive bacteria, though they showed little activity against Gram-negative bacteria. The X-ray crystal structure of compound 2 in complex with AmpC was subsequently determined to 2.1 A resolution. The placement of the proximal two-thirds of the inhibitor in the experimental structure corresponds with the docked structure, but a bond rotation leads to a distinctly different placement of the distal part of the inhibitor. In the experimental structure, the inhibitor interacts with conserved residues in the R1 cleft whose role in recognition has not been previously explored. CONCLUSIONS: Combining structure-based design with in-parallel synthesis allowed for the rapid exploration of inhibitor functionality in the R1 cleft of AmpC. The resulting inhibitors differ considerably from beta-lactams but nevertheless inhibit the enzyme well. The crystal structure of 2 (K(i) 83 nM) in complex with AmpC may guide exploration of a highly conserved, largely unexplored cleft, providing a template for further design against AmpC beta-lactamase.

Structure-based discovery and in-parallel optimization of novel competitive inhibitors of thymidylate synthase.

BACKGROUND: The substrate sites of enzymes are attractive targets for structure-based inhibitor design. Two difficulties hinder efforts to discover and elaborate new (nonsubstrate-like) inhibitors for these sites. First, novel inhibitors often bind at nonsubstrate sites. Second, a novel scaffold introduces chemistry that is frequently unfamiliar, making synthetic elaboration challenging. RESULTS: In an effort to discover and elaborate a novel scaffold for a substrate site, we combined structure-based screening with in-parallel synthetic elaboration. These techniques were used to find new inhibitors that bound to the folate site of Lactobacillus casei thymidylate synthase (LcTS), an enzyme that is a potential target for proliferative diseases, and is highly studied. The available chemicals directory was screened, using a molecular-docking computer program, for molecules that complemented the three-dimensional structure of this site. Five high-ranking compounds were selected for testing. Activity and docking studies led to a derivative of one of these, dansyltyrosine (Ki 65 microM). Using solid-phase in-parallel techniques 33 derivatives of this lead were synthesized and tested. These analogs are dissimilar to the substrate but bind competitively with it. The most active analog had a Ki of 1.3 microM. The tighter binding inhibitors were also the most specific for LcTS versus related enzymes. CONCLUSIONS: TS can recognize inhibitors that are dissimilar to, but that bind competitively with, the folate substrate. Combining structure-based discovery with in-parallel synthetic techniques allowed the rapid elaboration of this series of compounds. More automated versions of this approach can be envisaged.

Structure-based design of inhibitors specific for bacterial thymidylate synthase.

Thymidylate synthase is an attractive target for antiproliferative drug design because of its key role in the synthesis of DNA. As such, the enzyme has been widely targeted for anticancer applications. In principle, TS should also be a good target for drugs used to fight infectious disease. In practice, TS is highly conserved across species, and it has proven to be difficult to develop inhibitors that are selective for microbial TS enzymes over the human enzyme. Using the structure of TS from Lactobacillus casei in complex with the nonsubstrate analogue phenolphthalein, inhibitors were designed to take advantage of features of the bacterial enzyme that differ from those of the human enzyme. Upon synthesis and testing, these inhibitors were found to be up to 40-fold selective for the bacterial enzyme over the human enzyme. The crystal structures of two of these inhibitors in complex with TS suggested the design of further compounds. Subsequent synthesis and testing showed that these second-round compounds inhibit the bacterial enzyme at sub-micromolar concentrations, while the human enzyme was not inhibited at detectable levels (selectivities of 100-1000-fold or greater). Although these inhibitors share chemical similarities, X-ray crystal structures reveal that the analogues bind to the enzyme in substantially different orientations. Site-directed mutagenesis experiments suggest that the individual inhibitors may adopt multiple configurations in their complexes with TS.

Quinoxaline chemistry. Part 11. 3-Phenyl-2[phenoxy- and phenoxymethyl]-6(7) or 6,8-substituted quinoxalines and N-[4-(6(7)-substituted or 6,8-disubstituted-3-phenylquinoxalin-2-yl)hydroxy or hydroxymethyl] benzoylglutamates. Synthesis and evaluation of in vitro anticancer activity and enzymatic inhibitory activity against dihydrofolate reductase and thymidylate synthase.

Twenty-four out of twenty-nine quinoxalines were selected at the National Cancer Institute, Bethesda, Md, USA, for in vitro anticancer screening. Among these, 10 derivatives exhibited high values of percent tumor growth inhibition at a concentration of 10(-4) M in all cancer cell lines. Four of these compounds maintained these values at 10(-5) M, whereas a certain number exhibited significant values of percent inhibition at the most diluted concentrations (10(-8)-10(-6) M). Inhibitory activity against dihydrofolate reductase (DHFR) (bovine and rat liver) was determined for the most active compounds. This test showed that this type of quinoxaline exhibited an appreciable activity in comparison with the previously described aza analogues. In the other test (Lactobacillus casei, thymidylate synthase (TS), human HTS) no or poor activity was detected in both series of compounds.

Thymidylate synthase inhibition: a structure-based rationale for drug design.

Thymidylate synthase (TS) is a very interesting target in antiproliferative diseases. Its inhibition causes thimineless death of the cells and compounds inhibiting TS are widely used in anticancer therapy. The classical antifolate TS inhibitors are structural analogs of the folate cofactor; they often share the same metabolic pathways and this causes the development of resistance inside the cells. A detailed analysis of the available x-ray crystal structures of the complexes of the enzyme with different substrates and inhibitors support the finding of a structural basis of their biological activity. TS inhibitors nonstructural analog of folate, non-analog antifolate inhibitors (NAAI), are welcome as a new interesting research topic. Among the most recent and interesting ones, compounds from Agouron related to the indole structure, are independent on the folate metabolism, highly active and specific for human TS. Other compounds, phthalein derivatives, can inhibit TS enzymes from various sources and show an interesting biological activity profile: they inhibit better bacterial and fungal TS than human TS. The x-ray crystal structures of some of these inhibitors with TS show that they bind in a different binding site from that of the classical folate TS inhibitors. This indicates a potential allosteric binding site useful for future drug discovery studies.

Conformational analysis of phthalein derivatives acting as thymidylate synthase inhibitors by means of 1H NMR and quantum chemical calculations.

The conformations of a set of phthalein derivatives with bacterial thymidylate synthase (TS) inhibitory activity were investigated by 1H NMR spectra, performed at both room and low temperature, and by quantum chemical calculations. Since the crystal structure of the binary complex of phenolphthalein with the enzyme is known, we set out to study the conformation of various of its analogues in solution in order to observe the effects of the substituents on the phenolic rings, of the alpha-naphthol derivative and of the rigid analogue, fluorescein, and compare the results with the X-ray crystal structure studies. A relationship between the chemical shift of the proton on C4 (H4) of the phthalidic ring and the averaged angle formed by the phthalidic and the aromatic ring planes was found in which the most perpendicular conformations have the lowest H4 chemical shift values. At room temperature, the rotational freedom of all the studied compounds was similar, while at lower temperature the naphthol derivative assumed a partially blocked conformation. Finally, a qualitative relationship between the inhibitory properties of the compounds and their conformations is discussed.

Theoretical study of electronic spectra and photophysics of uracil derivatives.

The changes that the UV absorption spectrum and the photophysics of uracil undergo under hydrogen substitution or deprotonation, were studied theoretically within the CS-INDO/CI scheme. First of all this method was tested on uracil. It was then used for the calculation of the electronic structure of excited states (Sn, Tn) of a large number of uracil derivatives (1-, 3- and 5-methyluracil; 1,3-, 1,5- and 3,5-dimethyluracil; 5-fluoro- and 5-chlorouracil), including some anions (1- and 3-methyluracil anion). The excited states were obtained in the singly-excited configuration interaction approximation (S-CI) and the correlation effects on (pi pi*) states were studied by including the most important doubly- and triply-excited configurations in the CI. The S-CI wavefunctions were used for the calculation of the most important electronic matrix elements for spin-orbit coupling. The photophysics of these compounds is discussed using Jablonski diagrams.

Preparation and physicochemical properties of uracil derivatives with potential biological activity.

The characterization of mono- and dimethylated 5-substituted uracils was re-examined. Analysis of their physicochemical properties (pKa, U.V., delta 1H-N.M.R.) affords insight into the structural characteristics of 5-substituted uracil alkylated at the ring nitrogens.

Antimycotic action of alkyl derivatives of 5-(benzenesulfonamide)-1,2,3,4-tetrahydro-2-thioxo-4-pyrimidinon e.

Several series of mono-, di- and trimethyl derivatives of N-(6-amino-1,2,3,4-tetrahydro-4-oxo-2-thioxo-5-pyrimidinyl)benzene sulfonamide substituted at the benzene ring (Z), were synthesized and studied spectrophotometrically. The spectral and physical data enabled the structures of the methyl derivatives obtained by methylating (Z) to be identified. When assayed biologically as antimycotics, a small percentage of the substances exhibited mild fungicide activity.