Synthesis and anti-HIV activity of trivalent CD4-mimetic miniproteins

A series of trivalent CD4-mimetic miniproteins was synthesized, in which three CD4M9 miniprotein moieties were tethered on a threefold-symmetric scaffold. The trivalent miniproteins were designed to target the CD4-binding sites displayed in the trimeric gp120 complex of HIV-1. The synthesis takes advantage of the highly efficient ligation between a cysteine-tagged CD4M9 miniprotein and a suitable trivalent maleimide that varied in the nature and length of spacer. Antiviral assay revealed that most of the synthetic trivalent miniproteins demonstrated significantly enhanced anti-HIV activities over the monomeric CD4M9 against both R5- and X4-tropic viruses, indicating the beneficial multivalent effects. One compound that possesses a hydrophobic linker was shown to be 140-fold more active than CD4M9 against HIV-1(Bal) infection, implicating a positive contribution of the lipid portion to the antiviral activity. It was also found that most of the trivalent miniproteins showed comparable anti-HIV activities in comparison with a typical bivalent miniprotein, regardless of the length of the linker. The results implicate a novel mechanism of the interactions between the multivalent inhibitors and the trimeric gp120 complex.     

Use of sulfated linked cyclitols as heparan sulfate mimetics to probe the heparin/heparan sulfate binding specificity of proteins

Heparin and heparan sulfate (HS) are structurally diverse glycosaminoglycans (GAG) that are known to interact, via unique structural motifs, with a wide range of functionally distinct proteins and modulate their biological activity. To define the GAG motifs that interact with proteins, we assessed the ability of 15 totally synthetic HS mimetics to interact with 10 functionally diverse proteins that bind heparin/HS. The HS mimetics consisted of cyclitol-based pseudo-sugars coupled by linkers of variable chain length, flexibility, orientation, and hydrophobicity, with variations in sulfation also being introduced into some molecules. Three of the proteins tested, namely hepatocyte growth factor, eotaxin, and elastase, failed to interact with any of the sulfated linked cyclitols. In contrast, each of the remaining seven proteins tested exhibited a unique reactivity pattern with the panel of HS mimetics, with tetrameric cyclitols linked by different length alkyl chains being particularly informative. Thus, compounds with short alkyl spacers (2-3 carbon atoms) effectively blocked the interaction of fibroblast growth factor-1 (FGF-1) and lipoprotein lipase with heparin/HS, whereas longer chain spacers (7-10 carbon atoms) were required for optimal inhibition of FGF-2 and vascular endothelial growth factor binding. This effect was most pronounced with the chemokine, interleukin-8, where alkyl-linked tetrameric cyclitols were essentially inactive unless a spacer of >7 carbon atoms was used. The heparin-inhibitable enzymes heparanase and cathepsin G also displayed characteristic inhibition patterns, cathepsin G interacting promiscuously with most of the sulfated cyclitols but heparanase activity being inhibited most effectively by HS mimetics that structurally resemble a sulfated pentasaccharide. These data indicate that a simple panel of HS mimetics can be used to probe the HS binding specificity of proteins, with the position of anionic groups in the HS mimetics being critical.     

Novel catecholate-type siderophore analogs based on a myo-inositol scaffold

A novel 1,3,5-triamino-myo-inositol derivative ispresented as a readily available scaffold for the design oftripodal siderophore mimetics.Based on this scaffold, various hexadentate catecholate-type siderophoreanalogs weresynthesized by attaching the catechols to the inositol scaffold via spacer units of differentstruc-tureand length. The potential to tune the polarity of the inositol containing siderophoreanalogs has also beendemonstrated by varying the protection group strategy. The siderophoreactivity of the prepared siderophoreanalogs was examined by cross-feeding tests withvarious Gram-negative bacteria and mycobacteria.     

Antitumor activity of Mn(III) complexes in combination with phototherapy and antioxidant therapy

Manganese (Mn) is an essential trace element and trivalent Mn complexes have been used as oxidation catalysts and enzyme mimetics. We studied the cytotoxicity of Mn(III) derivatives of citrate, pyrophosphate and salicylene diamine (respectively, MnCit, MnPPi and EUK8) toward HeLa cells stressed by ultraviolet irradiation and the effect of the co-administration of ascorbate and para-amino salicylate (PAS) on cell viability. Metal complexes enhanced the lethality of irradiated cells, and this effect was even more pronounced when ascorbate was co-administered with Mn(III) species. The active role of Mn(III) compounds in the antitumor activity was demonstrated by the treatment of the cells with the chelator PAS, which restored the viability of both non-irradiated and UV-irradiated cells. The association of the Mn(III) metallodrugs with radiation and an antioxidant proved to be a very effective approach to chemotherapy.     

Presence of closely spaced protein thiols on the surface of mammalian cells

It has been proposed that certain cell-surface proteins undergo redox reactions, that is, transfer of hydrogens and electrons between closely spaced cysteine thiols that can lead to reduction, formation, or interchange of disulfide bonds. This concept was tested using a membrane-impermeable trivalent arsenical to identify closely spaced thiols in cell-surface proteins. We attached the trivalent arsenical, phenylarsenoxide, to the thiol of reduced glutathione to produce 4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide (GSAO). GSAO bound tightly to synthetic, peptide, and protein dithiols like thioredoxin, but not to monothiols. To identify cell-surface proteins that contain closely spaced thiols, we attached a biotin moiety through a spacer arm to the primary amino group of the gamma-glutamyl residue of GSAO (GSAO-B). Incorporation of GSAO-B into proteins was assessed by measuring the biotin using streptavidin-peroxidase. Up to 12 distinct proteins were labeled with GSAO-B on the surface of endothelial and fibrosarcoma cells. The pattern of labeled proteins differed between the different cell types. Protein disulfide isomerase was one of the proteins on the endothelial and fibrosarcoma cell surface that incorporated GSAO-B. These findings demonstrate that the cell-surface environment can support the existence of closely spaced protein thiols and suggest that at least some of these thiols are redox active.     

Modular synthesis of non-peptidic bivalent NPY Y1 receptor antagonists

According to a 'bivalent ligand approach' to increase the affinity of the potent argininamide-type NPY Y(1) receptor antagonist BIBP-3226, dimeric ligands were synthesized in which two molecules of the parent compound were linked by different spacers via N(G)-acylation at the guanidino groups. A synthetic route for the preparation of the title compounds was developed, which includes a copper(I)-catalyzed azide alkyne cycloaddition as the key step. Three bivalent analogues of BIBP-3226 were prepared showing nanomolar antagonistic activity and binding affinity to the NPY Y(1) receptor (calcium assay on HEL cells, radioligand binding assay on SK-N-MC cells), but these ligands were not superior to the parent compound and there was no correlation with the length or the chemical nature of the spacer. A trivalent BIBP-3226 derivate showed, surprisingly, no affinity to the NPY Y(1) receptor at all.     

Novel use of fluorescent glucose analogues to identify a new class of triazine-based insulin mimetics possessing useful secondary effects

There is an urgent need to discover new compounds that effectively treat diabetes by mimicking the action of insulin (insulin mimetics). Traditional approaches to studying anti-diabetic agents in cells are inconvenient for screening chemical libraries to identify insulin mimetics. 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) and 6-NBDG are fluorescent analogues of glucose that could be employed in screening. However, there are no published data about the use of these analogues to identify new insulin mimetics. We have developed a screening system based on 6-NBDG using 3T3-L1 adipocytes in a 96-well culture plate format. 6-NBDG was found to produce a larger signal than 2-NBDG in this screening system. 6-NBDG uptake in 3T3-L1 adipocytes was sensitive to insulin, known insulin mimetics, inhibitors of glucose transport and insulin-sensitizing compounds. To validate our screening system, a chemical library of 576 tagged, triazine-based small molecules was screened. The screening results were identical to that obtained from a commercial enzyme-based glucose assay. Two inducers of glucose uptake were shown to be non-cytotoxic and confirmed as insulin mimetic compounds by their inhibition of epinephrine-stimulated free fatty acid release from adipocytes. These novel insulin mimetics functioned at a markedly lower concentration than two widely studied insulin mimetics, zinc(ii) complexes and vanadium compounds, and also showed novel, beneficial effects on endothelial cell function (a key determinant of secondary complications in diabetes). The discovery of new insulin mimetics using 6-NBDG validates the use of this probe in the development of large-scale, cell-based screening systems based on the uptake of fluorescent-tagged glucose analogues. This research should aid the development of novel strategies to discover new drugs and drug targets for combating the increasing prevalence of diabetes.     

Antibody mimetics: promising complementary agents to animal-sourced antibodies

Despite their wide use as therapeutic, diagnostic and detection agents, the limitations of polyclonal and monoclonal antibodies have inspired scientists to design the next generation biomedical agents, so-called antibody mimetics that offer many advantages over conventional antibodies. Antibody mimetics can be constructed by protein-directed evolution or fusion of complementarity-determining regions through intervening framework regions. Substantial progress in exploiting human, butterfly (Pieris brassicae) and bacterial systems to design and select mimetics using display technologies has been made in the past 10 years, and one of these mimetics [Kalbitor® (Dyax)] has made its way to market. Many challenges lie ahead to develop mimetics for various biomedical applications, especially those for which conventional antibodies are ineffective, and this review describes the current characteristics, construction and applications of antibody mimetics compared to animal-sourced antibodies. The possible limitations of mimetics and future perspectives are also discussed.     

Synthesis,biological evaluation,X-ray molecular structure and molecular docking studies of RGD mimetics containing 6-amino-2,3-dihydroisoindolin-1-one fragment as ligands of integrin αIIbβ3

A series of novel RGD mimetics containing phthalimidine fragment was designed and synthesized. Their antiaggregative activity determined by Born’s method was shown to be due to inhibition of fibrinogen binding to α_IIbβ₃. Molecular docking of RGD mimetics to α_IIbβ₃ receptor showed the key interactions in this complex, and also some correlations have been observed between values of biological activity and docking scores. The single crystal X-ray data were obtained for five mimetics.     

Examination of novel non-phosphorus-containing phosphotyrosyl mimetics against protein-tyrosine phosphatase-1B and demonstration of differential affinities toward Grb2 SH2 domains

Inhibitory potencies were compared of several mono- and dicarboxy-based pTyr mimetics in Grb2 SH2 domain versus PTP1B assays. Although in both systems pTyr residues provide critical binding elements, significant differences in the manner of recognition exist between the two. This is reflected in the current study, where marked variation in relative potencies was observed between the two systems. Of particular note was the poor potency of all monocarboxy-based pTyr mimetics against PTP1B when incorporated into a hexapeptide platform. The recently reported high PTP1B inhibitory potency of similar phenylphosphate mimicking moieties displayed in small molecule, non-peptide structures, raises questions on the limitations of using peptides as platforms for pTyr mimetics in the discovery of small molecule inhibitors.     

Effects of superoxide dismutase/catalase mimetics on life span and oxidative stress resistance in the housefly,Musca domestica

The purpose of this study was to determine whether superoxide dismutase/catalase mimetics lengthen the life span of the housefly, Musca domestica, as previously demonstrated for the nematode Caenorhabditis elegans. Various concentrations of Eukarion-8 or Eukarion-134 were administered via the drinking water and the effects on the life span of the flies and amounts of protein carbonyls were determined under normoxic and hyperoxic conditions. These SOD/catalase mimetics neither extended the life span of the flies nor attenuated the protein carbonyl content under normoxic conditions and shortened life span under hyperoxic conditions. Thus, the effect of these SOD/catalase mimetics on the life span of animals seem to be species-specific.     

Smac mimetics activate the E3 ligase activity of cIAP1 protein by promoting RING domain dimerization

The inhibitor of apoptosis (IAP) proteins are important ubiquitin E3 ligases that regulate cell survival and oncogenesis. The cIAP1 and cIAP2 paralogs bear three N-terminal baculoviral IAP repeat (BIR) domains and a C-terminal E3 ligase RING domain. IAP antagonist compounds, also known as Smac mimetics, bind the BIR domains of IAPs and trigger rapid RING-dependent autoubiquitylation, but the mechanism is unknown. We show that RING dimerization is essential for the E3 ligase activity of cIAP1 and cIAP2 because monomeric RING mutants could not interact with the ubiquitin-charged E2 enzyme and were resistant to Smac mimetic-induced autoubiquitylation. Unexpectedly, the BIR domains inhibited cIAP1 RING dimerization, and cIAP1 existed predominantly as an inactive monomer. However, addition of either mono- or bivalent Smac mimetics relieved this inhibition, thereby allowing dimer formation and promoting E3 ligase activation. In contrast, the cIAP2 dimer was more stable, had higher intrinsic E3 ligase activity, and was not highly activated by Smac mimetics. These results explain how Smac mimetics promote rapid destruction of cIAP1 and suggest mechanisms for activating cIAP1 in other pathways.     

Farnesyltransferase inhibitors: CAAX mimetics based on different biaryl scaffolds

Mimetics of the C-terminal CAAX tetrapeptide of Ras protein were designed as farnesyltransferase (FTase) inhibitors (FTIs) by replacing AA with o-aryl or o-heteroaryl substituted p-hydroxy- or p-aminobenzoic acid, while maintaining the replacement of C with 1,4-benzodioxan-2-ylmethyl or 2-amino-4-thiazolylacetyl residue as in previous CAAX mimetics. Both FTase inhibition and antiproliferative effect were showed by two thiazole derivatives, namely those with 1-naphthyl (10 and 10a) or 3-furanyl (15 and 15a) in the central spacer, and by the benzodioxane derivative with 2-thienyl (6 and 6a) in the same position. Accumulation of unprenylated RAS was demonstrated in cells incubated with 15a. Consistently with FTIs literature, such results delineate the biaryl scaffold not only as a spacer but also as a sensible area of these mimetic molecules, where modifications at the branching aromatic ring are not indifferent and should be matter of further investigation.     

Modelling, synthesis and biological evaluation of novel glucuronide-based probes of Vibrio cholerae sialidase

The development of sialidase inhibitors is an area of continuing interest due to their potential use as therapeutic agents to combat viral and bacterial infections. Herein, we report our studies involving the sialidase from the pathogen Vibrio cholerae, through the modelling, synthesis and biological evaluation of mimetics of 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enonic acid (Neu5Ac2en, 1), a naturally occurring sialidase inhibitor. These mimetics are O- and S-glycosides of N-acetyl-D-glucosaminuronic acid in which the aglycone portion effectively replaces the C-6 glycerol side chain of Neu5Ac2en (1). The choice of aglycones was aided by use of the X-ray crystal structure of V. cholerae sialidase complexed with Neu5Ac2en (1). All Neu5Ac2en mimetics tested were found to inhibit V. cholerae sialidase as determined using a standard fluorometric assay.     

Small-molecule inhibitors of Bcl-2 family proteins are able to induce tumor regression in a mouse model of pre-B-cell acute lymphocytic lymphoma

The overexpression of prosurvival members of the Bcl-2 family is commonly associated with the enhanced malignancy of hematological tumors. There has been great interest in a novel set of agents that are able to mimic the function of the BH3 domain by binding to the groove of Bcl-2-like proteins and initiating the cell death sequence. We sought to examine the efficacy of BH3 mimetics in a spontaneous mouse model of B-cell neoplasia. We evaluated the ability of the BH3 mimetics to preferentially target tumor cells while sparing normal cells. In addition, we examined the contributions of Bim and Puma to the sensitivity of tumor cells to the BH3 mimetics. We report here that two BH3 mimetics (HA-14-1 and BH3-I-2') were able to induce apoptosis of murine B-cell lymphoma cells in vitro and in vivo. Tumors that arose from transplantation of primary lymphoma cells regressed following 7 days of treatment with BH3-mimetic drugs. The long-term benefits of the transient treatment of tumor-bearing mice with the BH3 mimetics, however, could not be properly evaluated, due to the high levels of toxicity we observed in vivo with these drugs. Decreased expression of either Bim or Puma from B-cell tumor cells was able to protect these cells from the apoptosis induced by these BH3 mimetics, suggesting that they function through other means. We conclude that while the BH3-mimetic drugs are effective at inducing cell death of lymphoma cells in vitro and in vivo, their unclear molecular specificity and their ability to kill normal cells may limit their therapeutic uses in humans.     

Identification of an N-cadherin motif that can interact with the fibroblast growth factor receptor and is required for axonal growth

In this study, we show that the neurite outgrowth response stimulated by N-cadherin is inhibited by a recently developed and highly specific fibroblast growth factor receptor (FGFR) antagonist. To test whether the N-cadherin response also requires FGF function, we developed peptide mimetics of the receptor binding sites on FGFs. Most mimetics inhibit the neurite outgrowth response stimulated by FGF in the absence of any effect on the N-cadherin response. The exceptions to this result were two mimetics of a short FGF1 sequence, which has been shown to interact with the region of the FGFR containing the histidine-alanine-valine motif. These peptides inhibited FGF and N-cadherin responses with similar efficacy. The histidine-alanine-valine region of the FGFR has previously been implicated in the N-cadherin response, and a candidate interaction site has been identified in extracellular domain 4 of N-cadherin. We now show that antibodies directed to this site on N-cadherin inhibit the neurite outgrowth response stimulated by N-cadherin, and peptide mimetics of the site inhibit N-cadherin and FGF responses. Thus, we can conclude that N-cadherin contains a novel motility motif in extracellular domain 4, and that peptide mimetics of this motif can interact with the FGFR.     

Engineering of substrate mimetics as novel-type substrates for glutamic acid-specific endopeptidases: design, synthesis, and application

This account reports on the development and function of novel substrate mimetics as artificial substrates for Glu-specific endopeptidases. Firstly, in an empirical way, various aliphatic and aromatic analogs of the already established carboxymethyl thioester-substrate mimetics were designed from simple structure-function relationship studies. The specificity of the newly developed substrates for Staphylococcus aureus V8 protease-catalyzed reactions have been examined by steady-state hydrolysis kinetic studies. Additionally, these studies were expanded to the use of the equally Glu-specific endopeptidase from Bacillus licheniformis (BL-GSE) which can easily be purified from alcalase in high yields. Finally, the novel substrate mimetics were used as acyl donor components in BL-GSE- and V8 protease-catalyzed model acyl transfer reactions. The results clarify the newly developed substrate mimetics as efficient acyl donors as well as BL-GSE as an attractive alternative to V8 protease for enzymatic peptide synthesis.     

Development of Noxa-like BH3 mimetics for apoptosis-based therapeutic strategy in chronic lymphocytic leukemia

Despite real advances made in chemoimmunotherapy, chronic lymphocytic leukemia (CLL) is still an incurable disease. New therapeutic strategies based on the restoration of the cell death program seemed relevant. Some members of the Bcl-2 family are critical players in the defective apoptotic program in CLL cells and/or targets of apoptosis inducers in vitro. The concept of BH3 mimetics has led to the characterization of small molecules mimicking proapoptotic BH3-only members of the Bcl-2 family by their ability to bind and antagonize the prosurvival members. Some putative or actual BH3 mimetics are already being tested in clinical trials with somewhat promising results. However, none of them has a high enough interaction affinity with Mcl-1, a crucial antiapoptotic factor in CLL. It has been suggested that resistance to BH3 mimetics can be overcome by using inhibitors of Mcl-1 expression. An alternative and more direct strategy is to design mimetics of the Noxa BH3 domain, which is a specific antagonistic Mcl-1 ligand. The development of such Noxa-like BH3 mimetics, capable of directly interacting with Mcl-1 and efficiently neutralizing its antiapoptotic activity, is extremely important to evaluate their impact on the clinical outcome of patients with CLL.     

Design, synthesis and pharmacological evaluation of cyclic mimetics of the insulin-like peptide 3 (INSL3) B-chain.

Insulin-like peptide 3 (INSL3) is a peptide hormone belonging to the relaxin-insulin superfamily of peptides that plays important roles in testes descent, oocyte maturation and the control of male germ cell apoptosis. These actions are mediated via a specific G-protein coupled receptor, LGR8. Previous structure-activity studies have shown that the key binding site of INSL3 is situated within its B-chain. Recent studies in our laboratory have led to the identification of a cyclic peptide mimetic 2 of the INSL3 B-chain, which we have shown to compete with the binding of [33P]-relaxin to LGR8 expressed in HEK293T cells, and to inhibit cAMP-mediated signaling in these cells, i.e. it is an antagonist of INSL3. In order to further define the structure-activity relationships of cyclic analogues of the INSL3 B-chain, we used a structure-based approach to design a series of cyclic, disulfide-constrained INSL3 B-chain mimetics. To do this, we first created a model of the 3D structure of INSL3 using the crystal structure of human relaxin as a template. This model of INSL3 was then used as a template to design a series of disulfide-constrained mimetics of the INSL3 B-chain. The peptides were synthesized by solid-phase peptide synthesis using pseudoproline dipeptides to improve the synthesis outcome. Of the seven prepared INSL3 B-chain mimetics, three compounds were found to have partial displacement activity, while four were able to completely displace [33P]-relaxin from LGR8, including compounds that were markedly shorter than compound 2. The best of these, mimetic 6, showed significantly greater affinity for LGR8 than compound 2, but still displayed around 1000-fold less affinity for LGR8 than native INSL3. Analysis of selected mimetics for their alpha-helical content using circular dichroism (CD) spectroscopy revealed that, generally, the mimetics showed less than expected helicity. The inability of the compounds to display true native INSL3 structure is likely contributing to their reduced receptor binding affinity. We are currently examining alternative INSL3 B-chain mimetics that might better present key receptor binding residues in the native INSL3-like conformation.