Methanethiosulfonate derivatives as ligands of STAT3-SH2 domain

It is well known that inflammatory conditions in selected organs increase the risk of cancer. Compounds of the inflammatory tumor microenvironment include leukocytes, cytokines, complement components, are orchestrated by transcription factors, such as STAT-3 (Signal Transducer and Activator of Transcription 3) and NF-kB. Therefore drugs able to inhibit one or both transcription factors could be useful tools to treat cancer disease. Two main approaches have been explored to inhibit STAT-3 signalling: \u2022 indirect, inhibiting the upstream tyrosine kinases that are responsible for STAT-3 activation or blocking factors such as JAK, Src, Bcr-Abl, FLT3 and EGFR that are involved in the activation of STAT-3 signalling. This kind of inhibition induces tumour-cell apoptosis but is poor selective. \u2022 direct, by interaction of small molecules with the protein. In this selective approach the starting point is the crystallographic structure of STAT-3 SH2 domain. S-methyl methanethiosulfonate, isolated from cauliflower has been shown to inhibit colon tumor incidence when administered to rats during the post-initiation phase of carcinogenesis [1]. Recently, a new methanethiosulfonate derivative of valproic acid (ACS33) was reported by some of us to show good in vitro antiproliferative activity and to inhibit in vivo the growth of PC3 in subcutaneous xenograft mice models [2]. Fig.1: Structures of the studied thiosulfonate hybrids. Since the influence of methanethiosulfonates on STAT-3 activity has not been yet studied, we decided to synthesize a set of thiosulfonate-drug hybrids (Fig.1) and to submit them and their parent compounds to the AlphaScreen-based assay, to investigate their ability to bind STAT-3 SH2 domain. Moreover, in order to check the selectivity of our molecules on STAT-3, other SH2-containing proteins, such as STAT-1, exhibiting a high degree of sequence homology to STAT-3, have also been tested. Results showed that most of the synthesized thiosulfonate-hybrids are able to strongly and selectively bind STAT-3 SH2 domain, whereas the parent drugs were completely devoid of this ability. Studies are ongoing to better define the profile of our new methanethiosulfonate derivatives as potential dual STAT-3/NFkB inhibitors. References 1. Reddy, B. S.; Kawamori, T.; Lubet, R.; Steele, V.; Kelloff, G.; Rao, C. V. Chemopreventive effect of S-methylmethane thiosulfonate and sulindac administered together during the promotion/progression stages of colon carcinogenesis Carcinogenesis 1999, 20, 1645-8. 2. Wedel S. A.; Sparatore A.; Del Soldato P.; Al-Batran S. E.; Atmaca A.; Juengel E.; Hudak L.; Jonas D.; Blaheta R. A. New histone deacetylase inhibitors as potential therapeutics tools for advanced prostate carcinoma. J. Cell. Mol Med 2008, 12, 2457-66

Synthesis of new dithiolethione and methanethiosulfonate systems endowed with pharmaceutical interest

Here we report synthetic methodology affording in the most efficient way the rapid preparation of new dithiolethiones (DTTs) and methanethiosulfonates (MTSs). These were evaluated as STAT3 inhibitors since these electrophilic systems could react with thiol groups of STAT3-SH2 domain. The results showed that MTSs strongly interacted with the SH2 domain, whereas the corresponding DTTs possessed lower affinity, independently from the nature of the linked heterocyclic scaffold

Anti-inflammatory, but not osteoprotective, effect of the TRAF6/CD40 inhibitor 6877002 in rodent models of local and systemic osteolysis

NFκB plays a key role in inflammation and skeletal disorders. Previously, we reported that pharmacological inhibition of NFκB at the level of TRAF6 suppressed RANKL, CD40L and IL1β-induced osteoclastogenesis and attenuated cancer-induced bone disease. TNFα is also known to regulate TRAF6/NFκB signalling, however the anti-inflammatory and osteoprotective effects associated with inhibition of the TNFα/TRAF6/NFκB axis have not been investigated. Here, we show that in vitro and ex vivo exposure to the verified small-molecule inhibitor of TRAF6, 6877002 prevented TNFα-induced NFκB activation, osteoclastogenesis and calvarial osteolysis, but it had no effects on TNFα-induced apoptosis or growth inhibition in osteoblasts. Additionally, 6877002 disrupted T-cells support for osteoclast formation and synoviocyte motility, without affecting the viability of osteoblasts in the presence of T-cells derived factors. Using the collagen-induced arthritis model, we show that oral and intraperitoneal administration of 6877002 in mice reduced joint inflammation and arthritis score. Unexpectedly, no difference in trabecular and cortical bone parameters were detected between vehicle and 6877002 treated mice, indicating lack of osteoprotection by 6877002 in the arthritis model described. Using two independent rodent models of osteolysis, we confirmed that 6877002 had no effect on trabecular and cortical bone loss in both osteoporotic rats or RANKL- treated mice. In contrast, the classic anti-osteolytic alendronate offered complete osteoprotection in RANKL- treated mice. In conclusion, TRAF6 inhibitors may be of value in the management of the inflammatory component of bone disorders, but may not offer protection against local or systemic bone loss, unless combined with anti-resorptive therapy such as bisphosphonates

Drug-mediated inhibition of Fli-1 for the treatment of leukemia

The Ets transcription factor, Fli-1 is activated in murine erythroleukemia and overexpressed in various human malignancies including Ewing's sarcoma, induced by the oncogenic fusion protein EWS/Fli-1. Recent studies by our group and others have demonstrated that Fli-1 plays a key role in tumorigenesis, and disrupting its oncogenic function may serve as a potential treatment option for malignancies associated with its overexpression. Herein, we describe the discovery of 30 anti-Fli-1 compounds, characterized into six functional groups. Treatment of murine and human leukemic cell lines with select compounds inhibits Fli-1 protein or mRNA expression, resulting in proliferation arrest and apoptosis. This anti-cancer effect was mediated, at least in part through direct inhibition of Fli-1 function, as anti-Fli-1 drug treatment inhibited Fli-1 DNA binding to target genes, such as SHIP-1 and gata-1, governing hematopoietic differentiation and proliferation. Furthermore, treatment with select Fli-1 inhibitors revealed a positive relationship between the loss of DNA-binding activity and Fli-1 phosphorylation. Accordingly, anti-Fli-1 drug treatment significantly inhibited leukemogenesis in a murine erythroleukemia model overexpressing Fli-1. This study demonstrates the ability of this drug-screening strategy to isolate effective anti-Fli-1 inhibitors and highlights their potential use for the treatment of malignancies overexpressing this oncogene

Second Generation Steroidal 4-Aminoquinolines Are Potent, Dual-Target Inhibitors of the Botulinum Neurotoxin Serotype A Metalloprotease and P. falciparum Malaria

Significantly more potent second generation 4-amino-7-chloroquinoline (4,7-ACQ) based inhibitors of the botulinum neurotoxin serotype A (BoNT/A) light chain were synthesized. Introducing an amino group at the C(3) position of the cholate component markedly increased potency (IC50 values for such derivatives ranged from 0.81 to 2.27 mu M). Two additional subclasses were prepared: bis(steroidal)-4,7-ACQ derivatives and bis(4,7-ACQ)cholate derivatives; both classes provided inhibitors with nanomolar-range potencies (e.g., the K-i of compound 67 is 0.10 mu M). During BoNT/A challenge using primary neurons, select derivatives protected SNAP-25 by up to 89%. Docking simulations were performed to rationalize the compounds' in vitro potencies. In addition to specific residue contacts, coordination of the enzyme's catalytic zinc and expulsion of the enzyme's catalytic water were a consistent theme. With respect to antimalarial activity, the compounds provided better IC90 activities against chloroquine resistant (CQR) malaria than CQ, and seven compounds were more active than mefloquine against CQR strain W2

2-{4-[\u3c9-[4-(2-methoxyphenyl)-1-piperazinyl]alkoxy]phenyl}-2H-benzotriazole s and their N-oxides as ligands for some 5-hydroxytryptamine, dopamine and adrenergic receptor subtypes

We have prepared and studied some new 2-methoxyphenylpiperazine derivatives as combined ligands for 5-hydroxytryptamine 5-HT(1A) and dopamine D3 receptor subtypes. The compounds displayed affinity for 5-HT(1A) and D3 receptors, which improved with the lengthening of the intermediate aliphatic chain. Conversely, binding to 5-HT(2A), D2 and \u3b11-receptor subtypes was affected in an irregular, and mainly negative, manner by the chain length. Benzotriazole derivatives with 4-5 methylenes exhibited good or excellent selectivity for 5-HT(1A) and D3 vs 5-HT(2A), D2 and \u3b11-receptors

2-{4-[\u3c9-[4-(2-methoxyphenyl)-1-piperazinyl] alkoxy] phenyl}phthalimides as ligands for some 5-HT, dopamine and adrenergic receptor subtypes

We have prepared and studied some new 2-methoxyphenylpiperazine derivatives as combined ligands for 5-HT1A and D3 receptor subtypes. The introduction of a phenyloxy moiety between the phthalimido group and the polymethylene chain of nanserone does not impair the typical high affinity for 5-HT1A receptors, but reduces in different measures the affinity for 5-HT2, D2 and \u3b11-receptors. Good affinity for the D3-receptor subtype has also been observed. The polymethylene chain length remains a critical factor in determining optimal affinity and selectivity

Synthesis and preliminary pharmacological investigation of some 2-[4-(dialkylaminoalkoxy)phenyl]benzotriazoles and their N-oxides

A set of 2-[4-(dialkylaminoalkoxy)phenyl]benzotriazoles and corresponding N-oxides was prepared. In a preliminary pharmacological investigation concerning some of these compounds, several in vitro and in vivo activities were shown. At concentrations in the range of 3-10 microM all tested compounds strongly inhibited (50-100%) the guinea pig ileum contractions induced either electrically or by means of several agonists; of particular interest was the antagonism to leukotriene D4. Compound 5b inhibited platelet aggregation induced by thromboxane A2, PAF and ADF (but not by arachidonic acid) and increased the bleeding time in mice. Compounds 5b and 6b protected mice from potassium cyanide hypoxia and exerted anti-hypercholesterolemic action; the first compound produced a ratio between HPL and total serum cholesterol concentrations below 0.92, thus indicating a potential anti-atherogenic activity