Novel PARP-1 inhibitors based on a 2-propanoyl-3H-quinazolin-4-one scaffold.

Poly(ADP-ribose)polymerase-I (PARP-1) enzyme is involved in maintaining DNA integrity and programmed cell death. A virtual screening of commercial libraries led to the identification of five novel scaffolds with inhibitory profile in the low nanomolar range. A hit-to-lead optimization led to the identification of a group of new potent PARP-1 inhibitors, acyl-piperazinylamides of 3-(4-oxo-3,4-dihydro-quinazolin-2-yl)-propionic acid. Molecular modeling studies highlighted the preponderant role of the propanoyl side chain.

Mutational analysis identifies residues crucial for homodimerization of myeloid differentiation factor 88 (MyD88) and for its function in immune cells.

Myeloid differentiation factor 88 (MyD88) is an adaptor protein that transduces intracellular signaling pathways evoked by the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 is composed of an N-terminal death domain (DD) and a C-terminal Toll/IL-1 receptor (TIR) domain, separated by a short region. Upon ligand binding, TLR/IL-1Rs hetero- or homodimerize and recruit MyD88 through their respective TIR domains. Then, MyD88 oligomerizes via its DD and TIR domain and interacts with the interleukin-1 receptor-associated kinases (IRAKs) to form the Myddosome complex. We performed site-directed mutagenesis of conserved residues that are located in exposed regions of the MyD88-TIR domain and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of Glu(183), Ser(244), and Arg(288) impaired homodimerization of the MyD88-TIR domain, recruitment of IRAKs, and activation of NF-κB. Moreover, overexpression of two green fluorescent protein (GFP)-tagged MyD88 mini-proteins (GFP-MyD88151-189 and GFP-MyD88168-189), comprising the Glu(183) residue, recapitulated these effects. Importantly, expression of these dominant negative MyD88 mini-proteins competed with the function of endogenous MyD88 and interfered with TLR2/4-mediated responses in a human monocytic cell line (THP-1) and in human primary monocyte-derived dendritic cells. Thus, our studies identify novel residues of the TIR domain that are crucially involved in MyD88 homodimerization and TLR signaling in immune cells.

Discovery of potent inhibitors of human and mouse fatty acid amide hydrolases.

Fatty acid amide hydrolase (FAAH, EC 3.5.1.99) is the main enzyme catabolizing endocannabinoid fatty acid amides. FAAH inactivation promotes beneficial effects upon pain and anxiety without the side effects accompanying agonists of type-1 cannabinoid receptors. Aiming at discovering new selective FAAH inhibitors, we developed a series of compounds (5a-u) characterized by a functionalized heteroaromatic scaffold. Particularly, 5c and 5d were identified as extremely potent, noncompetitive, and reversible FAAH inhibitors endowed with a remarkable selectivity profile and lacking interaction with the hERG channels. In vivo antinociceptive activity was demonstrated for 5c, 5d, and 5n at a dose much lower than that able to induce either striatal and limbic stereotypies or anxiolytic activity, thus outlining their potential to turn into optimum preclinical candidates. Aiming at improving pharmacokinetic properties and metabolic stability of 5d, we developed a subset of nanomolar dialyzable FAAH inhibitors (5v-z), functionalized by specific polyethereal lateral chains and fluorinated aromatic rings.

Isoxazolo(aza)naphthoquinones: a new class of cytotoxic Hsp90 inhibitors.

A series of 3-aryl-naphtho[2,3-d]isoxazole-4,9-diones and some of their 6-aza analogues were synthesized and found to inhibit the heat shock protein 90 (Hsp90). The compounds were tested for their binding to Hsp90 and for their effects on Hsp90 client proteins expression in a series of human tumour cell lines. Representative compounds (7f, 10c) downregulated the Hsp90 client proteins EGFR, Akt, Cdk4, Raf-1, and survivin, and upregulated Hsp70. Most of the compounds, in particular the alkylated 3-pyridyl derivatives, exhibited potent antiproliferative activity, down to two-digit nanomolar range. Preliminary results indicated in vivo activity of 7f against human epithelial carcinoma A431 model growing as tumour xenograft in nude mice, thus supporting the therapeutic potential of this novel series of Hsp90 inhibitors.

Novel 3,4-isoxazolediamides as potent inhibitors of chaperone heat shock protein 90.

A structural investigation on the isoxazole scaffold led to the discovery of 3,4-isoxazolediamide compounds endowed with potent Hsp90 inhibitory properties. We have found that compounds possessing a nitrogen atom directly attached to the C-4 heterocycle ring possess in vitro Hsp90 inhibitory properties at least comparable to those of the structurally related 4,5-diarylisoxazole derivatives. A group of compounds from this series of diamides combine potent binding affinity and cell growth inhibitory activity in both series of alkyl- and aryl- or heteroarylamides, with IC50 in the low nanomolar range. The 3,4-isoxazolediamides were also very effective in causing dramatic depletion of the examined client proteins and, as expected for the Hsp90 inhibitors, always induced a very strong increase in the expression levels of the chaperone Hsp70. In vivo studies against human epidermoid carcinoma A431 showed an antitumor effect of morpholine derivative 73 comparable to that induced by the reference compound 10.

Non-natural macrocyclic inhibitors of histone deacetylases: design, synthesis, and activity.

Nonpeptidic chiral macrocycles were designed on the basis of an analogue of suberoylanilide hydroxamic acid (2) (SAHA, vorinostat) and evaluated against 11 histone deacetylase (HDAC) isoforms. The identification of critical amino acid residues highly conserved in the cap region of HDACs guided the design of the suberoyl-based macrocycles, which were expected to bear a maximum common substructure required to target the whole HDAC panel. A nanomolar HDAC inhibitory profile was observed for several compounds, which was comparable, if not superior, to that of 2. A promising cytotoxic activity was found for selected macrocycles against lung and colon cancer cell lines. Further elaboration of selected candidates led to compounds with an improved selectivity against HDAC6 over the other isozymes. Pair-fitting analysis was used to compare one of the best candidates with the natural tetrapeptide apicidin, in an effort to define a general pharmacophore that might be useful in the design of surrogates of peptidic macrocycles as potent and isoform-selective inhibitors.

Pharmacological inhibition of TLR9 activation blocks autoantibody production in human B cells from SLE patients.

OBJECTIVES: Toll-like receptor 9 (TLR9), which recognizes hypomethylated DNA [cytosine-phosphate-guanine (CpG)], plays a role in the maintenance of serological memory and has been recently implicated in the pathogenesis of SLE. We previously reported that in vitro TLR9 triggers memory B-cell differentiation into antibody-producing cells, and that the MyD88-inhibitor ST2825 blocks TLR9-induced plasma cell (PC) generation. Here, we investigated whether memory B cells produce autoantibodies in SLE patients with active disease or in clinical remission, and whether ST2825 could inhibit PC generation in SLE patients. METHODS: Peripheral blood mononuclear cells from 10 SLE patients in clinical remission and 2 with active SLE were cultured in the presence of CpG with or without ST2825. Phenotypical analysis of CpG-stimulated cells was performed by flow cytometry. Supernatants were collected to measure antibody production by ELISA and to detect autoantibodies by IF. RESULTS: CpG-induced TLR9 stimulation caused autoantibody secretion in patients with active disease and in the majority of patients in clinical remission. Inhibition of MyD88 completely blocked the de novo generation of PCs and the secretion of autoantibodies. CONCLUSIONS: Autoreactive B cells persist in SLE patients during disease remission in the circulating B-cell memory pool. TLR9-dependent activation of memory B cells by pathogens could be one of the mechanisms triggering relapses in SLE. Compounds targeting the TLR/MyD88 pathway may be used as novel therapeutic tools to treat acute disease and to prevent relapses in SLE patients.

Vorinostat-like molecules as structural, stereochemical, and pharmacological tools.

The inhibitory activity of an ω-alkoxy analogue of the HDAC inhibitor, Vorinostat (SAHA), against the 11 isoforms of HDAC is described and evaluated with regard to structural biology information retrieved through computational methods. Preliminary absorption and metabolism studies were performed, which positioned this compound as a potential candidate for further preclinical studies and delineated measures for improving its pharmacokinetic profile.

Fibroblast growth factor 2-antagonist activity of a long-pentraxin 3-derived anti-angiogenic pentapeptide.

Fibroblast growth factor-2 (FGF2) plays a major role in angiogenesis. The pattern recognition receptor long-pentraxin 3 (PTX3) inhibits the angiogenic activity of FGF2. To identify novel FGF2-antagonistic peptide(s), four acetylated (Ac) synthetic peptides overlapping the FGF2-binding region PTX3-(97-110) were assessed for their FGF2-binding capacity. Among them, the shortest pentapeptide Ac-ARPCA-NH(2) (PTX3-[100-104]) inhibits the interaction of FGF2 with PTX3 immobilized to a BIAcore sensorchip and suppresses FGF2-dependent proliferation in endothelial cells, without affecting the activity of unrelated mitogens. Also, Ac-ARPCA-NH(2) inhibits angiogenesis triggered by FGF2 or by tumorigenic FGF2-overexpressing murine endothelial cells in chick and zebrafish embryos, respectively. Accordingly, the peptide hampers the binding of FGF2 to Chinese Hamster ovary cells overexpressing the tyrosine-kinase FGF receptor-1 (FGFR1) and to recombinant FGFR1 immobilized to a BIAcore sensorchip without affecting heparin interaction. In all the assays the mutated Ac-ARPSA-NH(2) peptide was ineffective. In keeping with the observation that hydrophobic interactions dominate the interface between FGF2 and the FGF-binding domain of the Ig-like loop D2 of FGFR1, amino acid substitutions in Ac-ARPCA-NH(2) and saturation transfer difference-nuclear magnetic resonance analysis of its mode of interaction with FGF2 implicate the hydrophobic methyl groups of the pentapeptide in FGF2 binding. These results will provide the basis for the design of novel PTX3-derived anti-angiogenic FGF2 antagonists.

Azetidinones as zinc-binding groups to design selective HDAC8 inhibitors.

2-Azetidinones, commonly known as beta-lactams, are well-known heterocyclic compounds. Herein we described the synthesis and biological evaluation of a series of novel beta-lactams. In vitro inhibition assays against HDAC isoforms showed an interesting isoform-selectivity of these compounds towards HDAC6 and HDAC8. The isoform selectivity changed in response to modification of the azetidinone-ring nitrogen atom substituent. The presence of an N-thiomethyl group is a prerequisite for the activity of these compounds in the micromolar range towards HDAC8.

Identification of critical residues of the MyD88 death domain involved in the recruitment of downstream kinases.

MyD88 couples the activation of the Toll-like receptors and interleukin-1 receptor superfamily with intracellular signaling pathways. Upon ligand binding, activated receptors recruit MyD88 via its Toll-interleukin-1 receptor domain. MyD88 then allows the recruitment of the interleukin-1 receptor-associated kinases (IRAKs). We performed a site-directed mutagenesis of MyD88 residues, conserved in death domains of the homologous FADD and Pelle proteins, and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of residues 52 (MyD88(E52A)) and 58 (MyD88(Y58A)) impaired recruitment of both IRAK1 and IRAK4, whereas mutation of residue 95 (MyD88(K95A)) only affected IRAK4 recruitment. Since all MyD88 mutants were defective in signaling, recruitment of both IRAKs appeared necessary for activation of the pathway. Moreover, overexpression of a green fluorescent protein (GFP)-tagged mini-MyD88 protein (GFP-MyD88-(27-72)), comprising the Glu(52) and Tyr(58) residues, interfered with recruitment of both IRAK1 and IRAK4 by MyD88 and suppressed NF-kappaB activation by the interleukin-1 receptor but not by the MyD88-independent TLR3. GFP-MyD88-(27-72) exerted its effect by titrating IRAK1 and suppressing IRAK1-dependent NF-kappaB activation. These experiments identify novel residues of MyD88 that are crucially involved in the recruitment of IRAK1 and IRAK4 and in downstream propagation of MyD88 signaling.

Synthesis and biological activity of fluorinated combretastatin analogues.

With the aim of understanding the influence of fluorine on the double bond of the cis-stilbene moiety of combretastatin derivatives and encouraged by a preliminary molecular modeling study showing a different biological environment on the interaction site with tubulin, we prepared, through various synthetic approaches, a small library of compounds in which one or both of the olefinic hydrogens were replaced with fluorine. X-ray analysis on the difluoro-CA-4 analogue demonstrated that the spatial arrangement of the molecule was not modified, compared to its nonfluorinated counterpart. SAR analysis confirmed the importance of the cis-stereochemistry of the stilbene scaffold. Nevertheless, some unpredicted results were observed on a few trans-fluorinated derivatives. The position of a fluorine atom on the double bond may affect the inhibition of tubulin polymerization and cytotoxic activity of these compounds.

Design, synthesis, and in vitro activity of peptidomimetic inhibitors of myeloid differentiation factor 88.

We describe the design and synthesis of a peptidomimetic library derived from the heptapeptide Ac-RDVLPGT-NH 2, belonging to the Toll/IL-1 receptor (TIR) domain of the adaptor protein MyD88 and effective in inhibiting its homodimerization. The ability of the peptidomimetics to inhibit protein-protein interaction was assessed by yeast 2-hybrid assay and further validated in a mammalian cell system by evaluating the inhibition of NF-kappaB activation, a transcription factor downstream of MyD88 signaling pathway that allows production of essential effector molecules for immune and inflammatory responses.

Structural characterization of PTX3 disulfide bond network and its multimeric status in cumulus matrix organization.

PTX3 is an acute phase glycoprotein that plays key roles in resistance to certain pathogens and in female fertility. PTX3 exerts its functions by interacting with a number of structurally unrelated molecules, a capacity that is likely to rely on its complex multimeric structure stabilized by interchain disulfide bonds. In this study, PAGE analyses performed under both native and denaturing conditions indicated that human recombinant PTX3 is mainly composed of covalently linked octamers. The network of disulfide bonds supporting this octameric assembly was resolved by mass spectrometry and Cys to Ser site-directed mutagenesis. Here we report that cysteine residues at positions 47, 49, and 103 in the N-terminal domain form three symmetric interchain disulfide bonds stabilizing four protein subunits in a tetrameric arrangement. Additional interchain disulfide bonds formed by the C-terminal domain cysteines Cys(317) and Cys(318) are responsible for linking the PTX3 tetramers into octamers. We also identified three intrachain disulfide bonds within the C-terminal domain that we used as structural constraints to build a new three-dimensional model for this domain. Previously it has been shown that PTX3 is a key component of the cumulus oophorus extracellular matrix, which forms around the oocyte prior to ovulation, because cumuli from PTX3(-/-) mice show defective matrix organization. Recombinant PTX3 is able to restore the normal phenotype ex vivo in cumuli from PTX3(-/-) mice. Here we demonstrate that PTX3 Cys to Ser mutants, mainly assembled into tetramers, exhibited wild type rescue activity, whereas a mutant, predominantly composed of dimers, had impaired functionality. These findings indicate that protein oligomerization is essential for PTX3 activity within the cumulus matrix and implicate PTX3 tetramers as the functional molecular units required for cumulus matrix organization and stabilization.

Synthesis of benzo[1,2-d;3,4-d']diimidazole and 1H-pyrazolo[4,3-b]pyridine as putative A2A receptor antagonists.

The synthesis and the binding affinity for the putative adenosine receptor antagonist 6-methyl-7-[1,2,3]triazol-2-yl-1,6-dihydrobenzo[1,2-d;3,4-d']diimidazole (10) and 5-oxazol-2-yl-1H-pyrazolo[4,3-b]pyridin-3-ylamine (16) are reported. The title compounds were prepared from commercially available 1-chloro-2,4-dinitrobenzene (1) and 2-chloro-6-methoxy-3nitropyridine (11), respectively, but proved devoid of affinity for the adenosine A1 and A2A receptors.

Pivotal Advance: Inhibition of MyD88 dimerization and recruitment of IRAK1 and IRAK4 by a novel peptidomimetic compound.

MyD88 is an adaptor protein, which plays an essential role in the intracellular signaling elicited by IL-1R and several TLRs. Central to its function is the ability of its Toll/IL-1R translation initiation region (TIR) domain to heterodimerize with the receptor and to homodimerize with another MyD88 molecule to favor the recruitment of downstream signaling molecules such as the serine/threonine kinases IL-1R-associated kinase 1 (IRAK1) and IRAK4. Herein, we have synthesized and tested the activity of a synthetic peptido-mimetic compound (ST2825) modeled after the structure of a heptapeptide in the BB-loop of the MyD88-TIR domain, which interferes with MyD88 signaling. ST2825 inhibited MyD88 dimerization in coimmunoprecipitation experiments. This effect was specific for homodimerization of the TIR domains and did not affect homodimerization of the death domains. Moreover, ST2825 interfered with recruitment of IRAK1 and IRAK4 by MyD88, causing inhibition of IL-1beta-mediated activation of NF-kappaB transcriptional activity. After oral administration, ST2825 dose-dependently inhibited IL-1beta-induced production of IL-6 in treated mice. Finally, we observed that ST2825 suppressed B cell proliferation and differentiation into plasma cells in response to CpG-induced activation of TLR9, a receptor that requires MyD88 for intracellular signaling. Our results indicate that ST2825 blocks IL-1R/TLR signaling by interfering with MyD88 homodimerization and suggest that it may have therapeutic potential in treatment of chronic inflammatory diseases.

Structure and function of the long pentraxin PTX3 glycosidic moiety: fine-tuning of the interaction with C1q and complement activation.

The prototypic long pentraxin PTX3 is a unique fluid-phase pattern recognition receptor that plays a nonredundant role in innate immunity and female fertility. The PTX3 C-terminal domain is required for C1q recognition and complement activation and contains a single N-glycosylation site on Asn 220. In the present study, we characterized the structure of the human PTX3 glycosidic moiety and investigated its relevance in C1q interaction and activation of the complement classical pathway. By specific endo and exoglycosidases digestion and direct mass spectrometric analysis, we found that both recombinant and naturally occurring PTX3 were N-linked to fucosylated and sialylated complex-type sugars. Interestingly, glycans showed heterogeneity mainly in the relative amount of bi, tri, and tetrantennary structures depending on the cell type and inflammatory stimulus. Enzymatic removal of sialic acid or the entire glycosidic moiety equally enhanced PTX3 binding to C1q compared to that in the native protein, thus indicating that glycosylation substantially contributes to modulate PTX3/C1q interaction and that sialic acid is the main determinant of this contribution. BIAcore kinetic measurements returned decreasing K(off) values as sugars were removed, pointing to a stabilization of the PTX3/C1q complex. No major rearrangement of PTX3 quaternary structure was observed after desialylation or deglycosylation as established by size exclusion chromatography. Consistent with C1q binding, PTX3 desialylation enhanced the activation of the classical complement pathway, as assessed by C4 and C3 deposition. In conclusion, our results provided evidence of an involvement of the PTX3 sugar moiety in C1q recognition and complement activation.

2-n-Butyl-9-methyl-8-[1,2,3]triazol-2-yl-9H-purin-6-ylamine and analogues as A2A adenosine receptor antagonists. Design, synthesis, and pharmacological characterization.

Two types of adenosine receptor ligands were designed, i.e., 9H-purine and 1H-imidazo[4,5-c]pyridines, to obtain selective A(2A) antagonists, and we report here their synthesis and binding affinities for the four adenosine receptor subtypes A(1), A(2A), A(2B) and A(3). The design was carried out on the basis of the molecular modeling of a number of potent adenosine receptor antagonists described in the literature. Three compounds (25b-d) showed an interesting affinity and selectivity for the A(2A) subtype. One of them, i.e., ST1535 (2-n-butyl-9-methyl-8-[1,2,3]triazol-2-yl-9H-purin-6-ylamine, 25b) (K(i) A(2A) = 6.6 nM, K(i) A(1)/A(2A) = 12; K(i) A(2B)/A(2A) = 58; K(i) A(3)/A(2A) > 160), was selected for in vivo study and shown to induce a dose-related increase in locomotor activity, suggestive of an A(2A) antagonist type of activity.