This study investigated the role of TRPA1 in the development and maintenance of mechanical and cold hyperalgesia in persistent inflammation induced by Complete Freund's Adjuvant (CFA) in mice. The intraplantar (i.pl.) injection of CFA induced a long lasting (28 days) hyperalgesia for both mechanical and thermal (cold) stimuli. The intraperitoneal (i.p., 30-300 mg/kg), intraplantar (i.pl., 100 microg/site) or intrathecal (i.t., 10 microg/site) injection of the TRPA1 selective antagonist HC-030031 significantly reduced the mechanical hyperalgesia evaluated by the von Frey hair test. The effect of HC-030031 was evidenced on the day after CFA injection and was kept throughout the test. However, the intracerebroventricular (i.c.v., 10 microg/site) injection of HC-030031 did not interfere with CFA-induced hyperalgesia. Treatment with HC-030031 (300 mg/kg, i.p.) completely inhibited the noxious cold hyperalgesia induced by tetrafluoroethane in mice that received CFA. The pre-treatment with the TRPA1 oligonucleotide antisense (AS-ODN, i.t.) consistently prevented both mechanical and cold hyperalgesia. Interestingly, both TRPA1 protein expression and mRNA were over-expressed in spinal cord and dorsal root ganglia (DRG) of mice treated with CFA, an effect that was fully prevented by the pre-treatment with the TRPA1 antagonist HC-030031. Collectively, the present results showed that TRPA1 present at either peripheral or spinal sites play a relevant role in the development and maintenance of both mechanical and cold hyperalgesia during CFA-induced inflammation. Thus, TRPA1 selective antagonists represent promising candidates to treat hyperalgesia in persistent inflammatory states.

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Although neutrophils are strongly implicated in eliminating pathogens, excessive recruitment may cause tissue damage. Therefore, reducing cell influx during an inflammatory process may be a potential target for treating inflammatory bowel diseases (IBD). As CXCR2 is involved in neutrophil migration, this study aimed to evaluate whether the systemic therapeutic treatment with selective CXCR2 antagonist SB225002 ameliorates experimental colitis, which was induced in mice by 2,4,6-trinitrobenzene sulfonic acid (TNBS). After colitis establishment (24 h), mice were treated with SB225002. At later time-points, up to 72 h, mice were monitored for body weight loss and overall mortality. At the time of sacrifice, colonic tissues were scored for macro- and microscopic damage, and cytokine levels, myeloperoxidase (MPO) activity, and protein expression were analyzed. TNBS administration induced macro- and microscopic damage in colon tissue, leading in most cases to animal death. Curative treatment with SB225002 significantly reduced all of the parameters analyzed, leading to an improvement of inflammatory signs. SB225002 reduced neutrophil influx, MPO activity, IL-1beta, MIP-2, and keratinocyte-derived chemokine (KC) levels and the expression of vascular endothelial growth factor, inducible NO synthase, and cyclooxygenase-2 proteins into the colon tissue. Levels of IL-4 and IL-10 were increased significantly in the colons of animals treated with SB225002. Additionally, curative treatment with mouse anti-KC significantly reduced MPO activity and colonic damage. These results taken together demonstrate that a selective blockade of CXCR2 consistently reduced TNBS-induced colitis, suggesting that the use of SB225002 is a potential therapeutic approach for the treatment of IBD and other related inflammatory disorders.

The synthetic n-alkyl esters of gallic acid (GA), also known as gallates, especially propyl, octyl and dodecyl gallates, are widely employed as antioxidants by food and pharmaceutical industries. The inhibitory effects of GA and 15 gallates on Herpes Simplex Virus type 1 (HSV-1) and Human Immunodeficiency Virus (HIV-1) replication were investigated here. After a preliminary screening of these compounds, GA and pentyl gallate (PG) seemed to be the most active compounds against HSV-1 replication and their mode of action was characterized through a set of assays, which attempted to localize the step of the viral multiplication cycle where impairment occurred. The detected anti-HSV-1 activity was mediated by the inhibition of virus attachment to and penetration into cells, and by virucidal properties. Furthermore, an anti-HIV-1 activity was also found, to different degrees. In summary, our results suggest that both compounds could be regarded as promising candidates for the development of topical anti-HSV-1 agents, and further studies concerning the anti-HIV-1 activity of this group of molecules are merited.Key words: antiviral -HSV-1 -HIV-1 -gallic acid -pentyl gallate Herpes Simplex Virus type 1 (HSV-1) is an enveloped DNA virus that causes one of the most common viral infections in humans, leading to a variety of diseases ranging from mild to severe and sometimes life-threatening (White & Fenner 1994, Whitley & Rozman 2001. Although several nucleoside analogues have been approved for clinical use, such as acyclovir, immunocompromised patients are at increased risk of severity and recurrent infections, since resistant strains have recently been observed (Brady & Bernstein 2004). Therefore, it is desirable to develop new antiviral agents in order to substitute or complement currently available drugs.The synthetic n-alkyl esters of gallic acid (GA), also known as gallates, especially propyl, octyl and dodecyl gallates, are widely employed as antioxidants by the food and pharmaceutical industries (van der Heijden et al. 1986, Kubo et al. 2002a. Besides the antioxidant activity, other biological activities have been described for this group of molecules, mainly anticancer mechanisms (Fiuza et al. 2004, Kitagawa et al. 2005, Frey et al. 2006, Veluri et al. 2006 as well as antibacterial and antifungal properties (Fujita & Kubo 2002, Kubo et al. 2002b, c, 2003, Stapleton et al. 2004). However, there are few reports about the antiviral activity of these compounds. In 1988, a study described the inhibition of HSV-1 and HSV-2 replication by methyl gallate (Kane et al. 1988). In 2002, as part of the screening of phenolic compounds against HIV-1 integrase, GA was found to be active (Ahn et al. 2002). More recently, several biological activities of a group of gallates were described by our research group, and various structure-activity relationships regarding their anti-HSV-1, antioxidant and genotoxic effects were proposed (Savi et al. 2005). Furthermore, the pronounced anti-HSV-1 activity of octyl gallate, and its inhibitory...

Herpes simplex virus (HSV) is a DNA-containing enveloped virus that causes common viral infections in humans worldwide leading to a variety of diseases. HSV-1 and HSV-2 can be distinguished on the basis of clinical manifestations (the former is more frequently associated with oral cold sores, while the later causes genital ulcers) and biochemical and serological examinations. In most cases, HSV infection is usually benign or asymptomatic in immunocompetent individuals; however, in patients with an immature or compromised immune system, the infection can be serious and sometimes life-threatening. 1,2) Several nucleoside analogues have been approved for clinical use. Among those, acyclovir is widely used for the systemic treatment of HSV infections. It is a highly selective antiviral agent because it is specifically phosphorylated by viral thymidine kinase in infected cells. However, acyclovir-resistant HSV infection in immunocompromised patients such as transplanted patients and patients with AIDS has recently been observed. Therefore, it is desirable to develop new anti-HSV agents in order to substitute or complement the antiviral drugs available. 3,4)The synthetic n-alkyl esters of gallic acid (GA), also known as gallates, especially propyl, octyl and dodecyl gallates, are widely employed as antioxidants by food and pharmaceutical industries. 5,6) Besides the antioxidant activity, other biological activities have been described for this group of molecules, mainly anticancer, 7-10) antibacterial and antifungal properties. [11][12][13][14][15][16] There are few reports about the antiviral activity of these compounds. In 1988, the potent inhibition of HSV-1 and HSV-2 by methyl gallate was described. 17) In 2000, as part of the screening of phenolic compounds against HIV-1 integrase, gallic acid was found to be active. 18)More recently, the anti-HSV activity of several gallates was described by our research group, which proposed various structure-activity relationships regarding the antiviral, antioxidant and genotoxic effects.19) Furthermore, the pronounced anti-HSV-1 activity of octyl gallate, and its inhibitory effect against RNA viruses were also recently described. 20,21) In the present study, the anti-HSV-2 activity of gallic acid and pentyl gallate was evaluated followed by the determination of the site of antiviral activity of these compounds. MATERIALS AND METHODSCompounds GA and pentyl gallate (PG) (Fig. 1) were synthesized as previously described.19) The compounds (50 mM) were dissolved in dimethyl sulfoxide, stored at Ϫ20°C protected from light, and further diluted in culture medium prior to use.Cells and Virus African green monkey kidney cells (GMK AH1) were grown in Eagle's minimum essential medium (EMEM, Gibco BRL, Grand Island, NY, U.S.A.) supplemented with 2% fetal calf serum (FSC), 0.05% primaton substance (Kraft Inc., Norwich, CT, U.S.A.), 100 U/ml Göteborg, Sweden. Received November 23, 2007; accepted February 5, 2008; published online February 20, 2008 The synthetic n-alkyl esters of galli...

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Falcipain-2 (FP-2) is a key cysteine protease from the malaria parasite Plasmodium falciparum. Many previous studies have identified FP-2 inhibitors; however, none has yet met the criteria for an antimalarial drug candidate. In this work, we assayed an in-house library of non-peptidic organic compounds, including (E)-chalcones, (E)-N'-benzylidene-benzohydrazides and alkylesters of gallic acid, and assessed the activity toward FP-2 and their mechanisms of inhibition. The (E)-chalcones 48, 54 and 66 showed the lowest IC 50 values (8.5 ± 0.8 mM, 9.5 ± 0.2 mM and 4.9 ± 1.3 mM, respectively). The best inhibitor (compound 66) demonstrated non-competitive inhibition, and using mass spectrometry and fluorescence spectroscopy assays, we suggest a potential allosteric site for the interaction of this compound, located between the catalytic site and the hemoglobin binding arm in FP-2. We combined structural biology tools and mass spectrometry to characterize the inhibition mechanisms of novel compounds targeting FP-2.