Highly tunable thiosulfonates as a novel class of cysteine protease inhibitors with anti-parasitic activity against Schistosoma mansoni.

The development of a new class of cysteine protease inhibitors utilising the thiosulfonate moiety as an SH specific electrophile is described. This moiety has been introduced into suitable amino acid derived building blocks, which were incorporated into peptidic sequences leading to very potent i.e. sub micromolar inhibitors of the cysteine protease papain in the same range as the vinyl sulfone based inhibitor K11777. Therefore, their inhibitory effect on Schistosoma mansoni, a human blood parasite, that expresses several cysteine proteases, was evaluated. The homophenylalanine side chain containing compounds 27-30 and especially 36 showed promising activities compared with K11777 and warrant further investigations of these peptidic thiosulfonate inhibitors as new potential anti-parasitic compounds.

Molecular construction of HIV-gp120 discontinuous epitope mimics by assembly of cyclic peptides on an orthogonal alkyne functionalized TAC-scaffold.

Mimics of discontinuous epitopes of for example bacterial or viral proteins may have considerable potential for the development of synthetic vaccines, especially if conserved epitopes can be mimicked. However, due to the structural complexity and size of discontinuous epitopes molecular construction of these mimics remains challeging. We present here a convergent route for the assembly of discontinuous epitope mimics by successive azide alkyne cycloaddition on an orthogonal alkyne functionalized scaffold. Here the synthesis of mimics of the HIV gp120 discontinuous epitope that interacts with the CD4 receptor is described. The resulting protein mimics are capable of inhibition of the gp120-CD4 interaction. The route is convergent, robust and should be applicable to other discontinuous epitopes.

Scaffolded multiple cyclic peptide libraries for protein mimics by native chemical ligation.

The accessibility to collections, libraries and arrays of cyclic peptides is increasingly important since cyclic peptides may provide better mimics of the loop-like structures ubiquitously present in and - especially - on the surface of proteins. The next important step is the preparation of libraries of ensembles of scaffolded cyclic peptides, which upon screening may lead to promising protein mimics. Here we describe the synthesis of a tri-cysteine containing scaffold as well as the simultaneous native chemical ligation of three cyclic peptides thereby affording a clean library of multiple cyclic peptides on this scaffold, representing potential mimics of gp120. Members of this collection of protein mimics showed a decent inhibition of the gp120-CD4 interaction.

Thermodynamics of phosphotyrosine peptide-peptoid hybrids binding to the p56lck SH2 domain.

A frequently used approach to transform peptides into more drug-like compounds is preparation of the corresponding peptoids or peptide-peptoid hybrids. Although peptoids have advantages, there may also be some disadvantages such as their increased flexibility and the reduced ability for hydrogen bond formation due to alkylation of the backbone amide nitrogen, which might affect the free Gibbs energy (DeltaG). To obtain more insight into these contributions to DeltaG, we performed thermodynamic analyses on the interaction between peptide-peptoid hybrids, based on the sequence -pTyr-Glu-Glu-Ile-, and the p56(lck) (Lck) Src homology 2 domain. van't Hoff analysis was performed on binding data obtained from surface plasmon resonance competition experiments in a temperature range of 10-40 degrees C. It is observed that amino acid-peptoid substitutions do not have a systemic negative effect on the entropic contributions to DeltaG. However, loss in hydrogen-bonding capacity of the backbone may strongly reduce the binding enthalpy and contribute to the observed lower binding affinity.

Triphenylethylenes: a new class of protein kinase C inhibitors.

The Ca2+- and phospholipid-dependent phosphotransferase activity of protein kinase C was inhibited by the triphenylethylene compounds clomiphene [drug concentration causing 50% inhibition (IC50) = 25 microM], 4-hydroxytamoxifen (IC50 = 25 microM), and N-desmethyltamoxifen (IC50 = 8 microM). The Ca2+- and phospholipid-independent phosphorylation of protamine sulfate, which is catalyzed by protein kinase C, was not inhibited by the triphenylethylenes, suggesting that they do not interact directly with the active site of protein kinase C. The inhibitory potency of each triphenylethylene was reduced when the phospholipid concentration was increased, providing evidence that these drugs inhibited protein kinase C by interacting with phospholipids. The potencies of the effects of the triphenylethylenes on protein kinase C in the lipid environment of intact cells were evaluated by determining their efficacies in the inhibition of [3H]phorbol 12,13-dibutyrate (PDBu) binding to mouse embryo C3H/10T1/2 cells. Micromolar concentrations of each drug inhibited [3H]PDBu binding in these cells. N-Desmethyltamoxifen, 4-hydroxytamoxifen, and tamoxifen inhibited protein kinase C with the same order of potency as that which has been reported for their inhibition of MCF-7 cell growth by Reddel et al. (1983). N-Desmethyltamoxifen and 4-hydroxytamoxifen were also more potent than tamoxifen in the inhibition of the growth of mouse embryo fibroblast C3H/10T1/2 cells. These correlations suggest that the mechanism of growth inhibition by tamoxifen and its metabolites includes interactions with protein kinase C.

Inhibition of protein kinase C by tamoxifen.

The antiestrogen drug tamoxifen inhibits rat brain protein kinase C in vitro, whether the enzyme is activated by Ca2+ and phospholipid (50% inhibitory dose, 100 microM), 12-O-tetradecanoylphorbol-13-acetate and phospholipid (50% inhibitory dose, 40 microM), or teleocidin and phospholipid. Tamoxifen does not inhibit the Ca2+- and phospholipid-independent phosphorylation of protamine sulfate by protein kinase C, indicating that the drug does not interact with the active site of the enzyme. The binding of [3H]phorbol dibutyrate to high-affinity membrane receptors of cultured mouse fibroblast cells is inhibited by tamoxifen (50% inhibitory dose, 5 microM). Our findings suggest that the growth-inhibitory and cytotoxic effects of tamoxifen, which have been observed at microM concentrations of the drug, may be in part due to its effects on protein kinase C.

The presence of C/EBPα and its degradation are both required for TRIB2-mediated leukaemia.

C/EBPα (p42 and p30 isoforms) is commonly dysregulated in cancer via the action of oncogenes, and specifically in acute myeloid leukaemia (AML) by mutation. Elevated TRIB2 leads to the degradation of C/EBPα p42, leaving p30 intact in AML. Whether this relationship is a cooperative event in AML transformation is not known and the molecular mechanism involved remains elusive. Using mouse genetics, our data reveal that in the complete absence of C/EBPα, TRIB2 was unable to induce AML. Only in the presence of C/EBPα p42 and p30, were TRIB2 and p30 able to cooperate to decrease the latency of disease. We demonstrate that the molecular mechanism involved in the degradation of C/EBPα p42 requires site-specific direct interaction between TRIB2 and C/EBPα p42 for the K48-specific ubiquitin-dependent proteasomal degradation of C/EBPα p42. This interaction and ubiquitination is dependent on a critical C terminal lysine residue on C/EBPα. We show effective targeting of this pathway pharmacologically using proteasome inhibitors in TRIB2-positive AML cells. Together, our data show that excess p30 cooperated with TRIB2 only in the presence of p42 to accelerate AML, and the direct interaction and degradation of C/EBPα p42 is required for TRIB2-mediated AML.

Structure-activity relationships of sparsomycin and its analogues. Octylsparsomycin: the first analogue more active than sparsomycin.

Nine analogues of sparsomycin were synthesized, and their cytostatic activity was studied in an in vitro clonogenic L1210 assay by measuring the inhibition of colony formation. The activity of an analogue, expressed as an ID50 value, was compared to that of sparsomycin. Each possesses not more than two structural modifications of the sparsomycin molecule 1. Comparison of the activity of with that of the stereomers, having RCSS, SCSS, and RCRS chirality, respectively, shows that the S configuration of the chiral carbon atom is essential for an optimal activity, whereas the R chirality of the sulfoxide sulfur atom of sparsomycin is of importance. Study of the ID50 values of the S-deoxo analogues, as well as the compounds having the beta-sulfoxide function, indicate that the presence of an oxygen atom on the alpha-sulfur atom is essential. Isomerization of the trans double bond into the cis double bond yields isosparsomycin, (Scheme II), which has a low activity. The cytostatic activity of sparsomycin seems to be related to its lipophilicity: octylsparsomycin was shown to be three times as effective as sparsomycin.

Structure-activity relationships of sparsomycin and its analogues. Inhibition of peptide bond formation in cell-free systems and of L1210 and bacterial cell growth.

The biological activity of 14 analogues of sparsomycin (1) was studied in cell-free systems of Escherichia coli, Saccharomyces cerevisiae, and Sulfolobus solfataricus by measuring the inhibition of protein synthesis. The inhibition of L1210 colony formation in soft agar and bacterial cell growth in solid as well as in liquid medium was also examined. Each analogue possesses not more than two structural modifications of the sparsomycin molecule. This enabled us to determine unambiguously several structural and stereochemical features that are required for an optimal biological activity in these assays. Sparsomycin, having the SCRS chirality, is the most potent of the four possible stereoisomers. The results obtained with compounds 5-7 indicate that the presence of an oxygen atom on the S (alpha) atom is essential. Substitution of the bivalent sulfur atom by a CH2 group (10) or of the SCH3 moiety by a Cl atom (12) affects the activity of the molecule partially. Compound 12 is surprisingly active against intact cells. Substitution of the C(6)-CH3 group by a H(14) reduces the activity of the molecule. Isomerization of the trans double bond into the cis double bond yields cis-sparsomycin (15), which is inactive. The hydrophobic derivatives 8, 9, and 11 are considerably more active than sparsomycin; thus the ribosomal binding site for sparsomycin may have a hydrophobic character.

N-myristyl-Lys-Arg-Thr-Leu-Arg: a novel protein kinase C inhibitor.

In view of the critical role that the Ca2+- and phospholipid-dependent enzyme protein kinase C (PKC) plays in mediating proliferative responses to a number of growth factors, hormones, and tumor promoters, it is thought that selective PKC inhibitors may provide a new class of antiproliferative drugs. Established PKC inhibitors include three major classes of agents: agents that compete with the substrate ATP, agents that compete with the protein substrate, and agents that both compete with ATP and interact with the cofactor phosphatidylserine (PS). In this report, we have characterized the interactions between PKC and N-myristyl-Lys-Arg-Thr-Leu-Arg, a myristylated analogue of a synthetic peptide substrate of PKC. We determined that the myristylated peptide was a novel PKC inhibitor that interacted with PS as well as competed with the protein substrate of PKC. The inhibitory activity of the peptide was conferred by myristylation. We found that the myristylated peptide antagonized Ca2+- and PS-activated PKC with an IC50 of 75 microns, whereas the nonmyristylated peptide lacked this inhibitory activity. A fully active, Ca2+- and PS-independent catalytic fragment of PKC can be generated by limited proteolysis. Although the myristylated peptide was a very poor PKC substrate, this peptide inhibited the catalytic fragment of PKC by apparent competition with the phosphoacceptor substrate histone IIIS with an IC50 of 200 microM, whereas the nonmyristylated peptide showed no inhibitory activity against the catalytic fragment. Thus, the myristylated peptide may serve as a model for the development of selective PKC inhibitors, because its inhibitory mechanism exploits the substrate specificity of PKC, as well as the novel regulation of the enzyme. Furthermore, since endogenous PKC substrates include acylated proteins, the observations that we report here concerning a myristylated synthetic peptide suggest that acylation of proteins may be important in the regulation of PKC activity in vivo.

A selectively deprotectable triazacyclophane scaffold for the construction of artificial receptors.

[reaction: see text]. The synthesis of a triazacylclophane scaffold bearing a set of selectively removable protecting groups is described. This versatile scaffold, which can be linked to a solid support, allows the attachment of three different side chains and can therefore be used for the combinatorial synthesis of libraries of artificial receptor molecules of high structural diversity.

Computer-assisted molecular modeling of tumor promoters: rationale for the activity of phorbol esters, teleocidin B, and aplysiatoxin.

In the two-stage model of skin carcinogenesis, it is believed that initiators bind to DNA and that tumor promoters such as phorbol 12-tetradecanoate 13-acetate (TPA) bind noncovalently to membrane-associated high-affinity receptors, probably protein kinase C. Two other types of potent tumor-promoting substances, aplysiatoxin and teleocidin, appear to act also by binding to and activating protein kinase C, even though their chemical structures are quite different. Therefore, we have undertaken computer modeling of the special relationship of various functional groups in these three chemical classes of tumor promoters in an attempt to explain how these diverse structures bind to the same receptor molecule. We propose a stereochemical model in which the oxygens in TPA at C-3, C-4, C-9, and C-20 (O-3, O-4, O-9, and O-20) correspond to the O-11, N-13, N-1, and O-24 positions in teleocidin and the O-27, O-3, O-11, and O-30 oxygens in aplysiatoxin, respectively. In this model all distances with respect to overlap of the corresponding atoms are less than 1 A. In addition, all three types of molecules have their hydrophobic moieties oriented in a similar position. This model is further discussed with respect to other compounds showing various degrees of activity as tumor promoters, including mezerein, ingenol, and 4 alpha-TPA. The model explains how chemically diverse structures can have similar biological activity as tumor promoters and provides a basis for designing both agonists and antagonists of tumor promoters.

Specificity and function of the individual amino acids of an important determinant of human immunodeficiency virus type 1 that induces neutralizing activity.

An important antigenic determinant of human immunodeficiency virus type 1 that induces neutralizing activity in infected humans and chimpanzees was previously mapped with nonapeptides between amino acids 307 and 320 on the external envelope glycoprotein (gp 120) of strain HTLV-IIIB (molecular clone BH10) and amino acids 320 to 330 of strain HTLV-IIIRF. Using different sera we found different reactive nonapeptides that overlapped and shared a tetrapeptide, GPGR. This tetrapeptide, which is the same in HTLV-IIIB and HTLV-IIIRF, is flanked by amino acids that vary between virus strains. Because GPGR is predicted to form a beta-turn and is flanked by two cysteine residues that may form a disulphide bridge, a hairpin-like structure is suggested for this part of gp120. The tetrapeptide GPGR and the reactive peptides are located on top of this structure, well exposed to antibodies. We determined the role of the individual amino acids in antibody binding using three sets of peptide analogues derived from three reactive nonapeptides (two of strain HTLV-IIIB which overlapped and one of strain HTLV-IIIRF). Each set contained peptide analogues in which each amino acid was replaced, one at a time, by all genetically encoded amino acids. At least five consecutive amino acids in each nonapeptide were essential for antibody binding. They include amino acids of GPGR and potentially provide the virus with ample opportunity to escape immune surveillance.

Solid-phase synthesis of oligourea peptidomimetics employing the Fmoc protection strategy.

A solid-phase-Fmoc-based-synthesis strategy is described for oligourea peptidomimetics as well as a convenient general synthesis approach for the preparation of the required building blocks 5a-j and 5k. These are suitable for use in peptide or robot synthesizers, which is illustrated by the synthesis of oligourea peptidomimetics of part of Leu-enkephalin (10) and a neurotensin derivative (17).

Synthesis of cyclic peptides by ring-closing metathesis.

The synthesis of a series of "amide to amide" cyclized peptides by ring-closing metathesis (RCM) as well as a convenient synthesis for the linear precursors is described. In addition, the influence of the length of the alkene substituents and the influence of the peptide sequence is investigated, leading to a set of general rules to obtain "amide to amide" cyclized peptides by RCM.

Combinatorial chemistry of hydantoins.

Access to combinatorial chemistry of hydantoins is provided by convenient and versatile methods for the solid phase synthesis of libraries of 3,5-, 1,3- and 1,3,5-substituted hydantoins. The preparation of trisubstituted hydantoins features a Mitsunobu reaction for introduction of the substituent on N-1.

Inhibition of protein kinase C and calmodulin by the geometric isomers cis- and trans-tamoxifen.

The triphenylethylene antiestrogen trans-tamoxifen is an effective antitumor agent used in the treatment of human breast cancer. While the antiestrogenic activity of trans-tamoxifen clearly plays an important role in its tumoricidal action, some of the biological effects of trans-tamoxifen are independent of estrogen. Therapeutic concentrations of trans-tamoxifen inhibit protein kinase C (PKC) and calmodulin-dependent enzymes. PKC and calmodulin play critical roles in growth regulation, and there is evidence that inhibition of PKC and calmodulin by trans-tamoxifen may contribute to the antitumor activity of the drug in vivo. The geometric isomers cis- and trans-tamoxifen have a number of opposing biological activities that have been attributed to their interactions with the estrogen receptor. Cis-tamoxifen is generally estrogenic, whereas trans-tamoxifen is generally antiestrogenic. In this report, we compared the effects of cis- and trans-tamoxifen on PKC activity and on calmodulin-dependent cAMP phosphodiesterase activity. Cis- and trans-tamoxifen inhibited the Ca2(+)- and phosphatidylserine- (PS-) dependent activity of purified rat brain PKC with indistinguishable potencies, but cis-tamoxifen was somewhat more potent than the trans isomer in the inhibition of the Ca2(+)- and PS-independent activity of PKC. In addition, cis-tamoxifen was the more potent isomer in the inhibition of T lymphocyte activation, an event that entails a PKC-requiring signal transduction pathway. A modest preference for the cis isomer was also observed in the inhibition of a calmodulin-dependent cAMP phosphodiesterase. These results suggest a congruence between triphenylethylene binding sites on PKC and on the activated calmodulin-cAMP phosphodiesterase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of CD3 expression in a protein kinase C isozyme-deficient T-cell line.

Using the T-cell lymphoma line HuT 78, and a clone derived from HuT 78, designated K-4, the role of protein kinase C (PKC) isozymes in the expression of a variety of T-cell surface antigens was investigated. HuT 78 expresses PKC isozymes alpha and beta while K-4 expresses only PKC alpha. Flow cytometric analysis revealed that incubation of HuT 78 cells with phorbol 12-myristate 13-acetate (PMA) results in significant down-regulation of surface expression of CD3. While K-4 cells expressed reduced amounts of CD3, a similar reduction in CD3 expression was not observed when these cells were stimulated with PMA. The regulation of expression of CD11a (LFA-1), CD44, CD45RA and CD45RO and of the class II molecules DR and DP in response to PMA, was similar in both cell lines.

Computer-aided molecular modeling and design of DNA-inserting molecules.

Intercalators are molecules capable of sliding between base pairs without disturbing the overall stacking pattern. In addition, there may exist molecules capable of inserting into a base pair thereby disrupting the hydrogen bonds and replacing them with new hydrogen bonds. A molecule probably capable of inserting, i.e., an insertor, is the diketopiperazine cyclo-[Gly-Gly] (1). A barbiturate (2), alloxan (3), a pyrimidine derivative (4) and a hydantoin (5) were also studied as possible insertors. Furthermore, molecules such as ethyleneurea (6), succinimide (7), as well as a malonamide derivative (8) and oxamide derivatives (9-11) were studied in order to investigate the arrangement and the number of hydrogen bonds necessary for insertion. Molecules 12-14 were designed and studied for their capacity to act as bisinsertors and/or bisintercalators. These molecules feature two diketopiperazine moieties which are connected via a diphenyl(thio)ether, i.e., 12 and 13, or a bisphenol A spacer, i.e., 14. The latter molecule (14) seems a promising candidate as a bisinsertor.

Molecular diversity of peptidomimetics: approaches to the solid-phase synthesis of peptidosulfonamides.

In order to use the potential molecular diversity of the peptidosulfonamide peptidomimetics ultimately in libraries, approaches towards the solid-phase synthesis of peptidosulfonamides are a prerequisite. It is shown that peptidosulfonamides can be synthesized by solid-phase synthesis methods using either a Merrifield or a Tentagel resin. Better and more reproducible results are obtained using the latter resin. The possibility to prepare cyclic peptidosulfonamides was illustrated by the synthesis of cyclo-phenylalanyl psi[CH2S(O)2N]-glycine. However, translation of synthesis of peptidosulfonamides in solution to a solid-phase method was rather laborious and still requires careful optimization.