Preformulation and stability in biological fluids of the retrocyclin RC-101, a potential anti-HIV topical microbicide

Sassi, Alexandra B.; Bunge, Katherine; Hood, Brian L.; Conrads, Thomas P.; Cole, Alexander M.; Gupta, Phalguni; Rohan, Lisa C.

doi:10.1186/1742-6405-8-27

Cited by 18 publications

(14 citation statements)

References 28 publications

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“…Therefore, initially introduced as promising anti-viral humanized synthetic peptides, analogous to those that are still active in old-world primates [6] and widely proposed as a topical microbicide [48–50], RCs should be seriously considered as first-line antidotes against a broad range of bacterial toxins. We predict that the newly discovered mechanism of RC anti-toxin activity will further promote their development as therapeutic agents.…”

Section: Discussionmentioning

confidence: 99%

Retrocyclins neutralize bacterial toxins by potentiating their unfolding

Kudryashova

Seveau

et al. 2015

Biochemical Journal

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Defensins are a class of immune peptides with a broad range of activities against bacterial, fungal and viral pathogens. Besides exerting direct anti-microbial activity via dis-organization of bacterial membranes, defensins are also able to neutralize various unrelated bacterial toxins. Recently, we have demonstrated that in the case of human α- and β-defensins, this later ability is achieved through exploiting toxins’ marginal thermodynamic stability, i.e. defensins act as molecular anti-chaperones unfolding toxin molecules and exposing their hydrophobic regions and thus promoting toxin precipitation and inactivation [Kudryashova et al. (2014) Immunity 41, 709–721]. Retrocyclins (RCs) are humanized synthetic θ-defensin peptides that possess unique cyclic structure, differentiating them from α- and β-defensins. Importantly, RCs are more potent against some bacterial and viral pathogens and more stable than their linear counterparts. However, the mechanism of bacterial toxin inactivation by RCs is not known. In the present study, we demonstrate that RCs facilitate unfolding of bacterial toxins. Using differential scanning fluorimetry (DSF), limited proteolysis and collisional quenching of internal tryptophan fluorescence, we show that hydrophobic regions of toxins normally buried in the molecule interior become more exposed to solvents and accessible to proteolytic cleavage in the presence of RCs. The RC-induced unfolding of toxins led to their precipitation and abrogated activity. Toxin inactivation by RCs was strongly diminished under reducing conditions, but preserved at physiological salt and serum concentrations. Therefore, despite significant structural diversity, α-, β- and θ-defensins employ similar mechanisms of toxin inactivation, which may be shared by anti-microbial peptides from other families.

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Section: Discussionmentioning

confidence: 99%

Retrocyclins neutralize bacterial toxins by potentiating their unfolding

Kudryashova

Seveau

et al. 2015

Biochemical Journal

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“…In prior studies, exposure of RC-101 to human vaginal fluid samples taken from women with BV caused a marked reduction in activity through an unknown mechanism. 8 This finding represents a potential challenge to development of RC-101 for use in the setting of BV and requires …”

Section: Discussionmentioning

confidence: 99%

Retrocyclin inhibits Gardnerella vaginalis biofilm formation and toxin activity

Hooven

Randis

Hymes

et al. 2012

Journal of Antimicrobial Chemotherapy

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Background: Retrocyclins are cyclic antimicrobial peptides that have been shown to be both broadly active and safe in animal models. RC-101, a synthetic retrocyclin, targets important human pathogens and is a candidate vaginal microbicide. Its activity against microbes associated with bacterial vaginosis is unknown. Methods:We investigated the effect of RC-101 on toxin activity, bacterial growth and biofilm formation of Gardnerella vaginalis in vitro.Results: RC-101 potently inhibits the cytolytic activity of vaginolysin, the Gardnerella vaginalis toxin, on both erythrocytes and nucleated cells. RC-101 lacks inhibitory activity against planktonic G. vaginalis but markedly decreases biofilm formation.Conclusions: These dual properties, toxin inhibition and biofilm retardation, justify further exploration of RC-101 as a candidate agent for bacterial vaginosis prevention.Keywords: defensin, vaginolysin, bacterial vaginosis, biofilm IntroductionBacterial vaginosis (BV) is a highly prevalent vaginal dysbiosis that has been linked to adverse pregnancy outcomes and enhanced transmission of sexually transmitted infections (STIs). The precise pathogenesis of BV remains unclear. However, loss of vaginal Lactobacillus and overgrowth of Gardnerella vaginalis are thought to be key elements of the disease process. G. vaginalis forms biofilms at the vaginal mucosal surface in vivo, and these appear to contribute to persistence and the frequent failure of antimicrobial therapy for BV.1,2 G. vaginalis biofilms may also be found in male partners of women with BV and may represent a transmissible state for this organism.3 In addition, G. vaginalis produces vaginolysin (VLY), a human-specific cholesterol-dependent cytolysin that lyses epithelial cells and erythrocytes and is thought to play an important role in BV pathogenesis. 4 VLY is inactive at normal vaginal pH (,4.5) but fully active at the higher pH levels noted in the BV milieu. 5Defensins are antimicrobial peptides that have important roles in immune defence and in shaping microbial ecology at mucosal surfaces.6 Q-Defensins, also known as retrocyclins, are 18-residue circular peptides related to the a-and b-defensins. Retrocyclins have broad antibacterial and antiviral activity, but the human retrocyclin gene does not produce a functional product owing to a premature stop codon.7 Based on several studies showing that retrocyclins prevent HIV and herpes simplex virus (HSV) infection, efforts are now under way to develop a topical intravaginal preparation of synthetic retrocyclin RC-101 for STI prophylaxis.8 RC-101 has been shown to lack significant effects on lactobacilli that normally colonize the vaginal tract and to be non-toxic to human cells. We hypothesized that RC-101 might have antibacterial and antitoxin effects against G. vaginalis and VLY that could make it useful for BV prophylaxis. Materials and methods Bacterial strains and growth conditionsGardnerella vaginalis strain 49145 was purchased from ATCC and was grown on solid human blood-bilayer Tween agar (BD Bios...

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“…For example, retrocyclin (RC101) and protegrin-1 (PG1) have high antimicrobial activity or stability when cyclized [20] or form hairpin structure [21] with formation of disulfide bonds. RC101 is highly stable at pH 3, 4, 7 and temperature 25°C to 37°C as well as in human vaginal fluid for 48 hours [22], while the antimicrobial activity was maintained for up to six months [23]. Likewise, PG1 is highly stable in salt or human fluids [24,25] but potency is lost when linearized.…”

Section: Introductionmentioning

confidence: 99%

Topical delivery of low-cost protein drug candidates made in chloroplasts for biofilm disruption and uptake by oral epithelial cells

et al. 2016

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Protein drugs (PD) are minimally utilized in dental medicine due to high cost and invasive surgical delivery. There is limited clinical advancement in disrupting virulent oral biofilms, despite their high prevalence in causing dental caries. Poor efficacy of antimicrobials following topical treatments or to penetrate and disrupt formed biofilms is a major challenge. We report an exciting low-cost approach using plant-made antimicrobial peptides (PMAMPs) retrocyclin or protegrin with complex secondary structures (cyclic/hairpin) for topical use to control biofilms. The PMAMPs rapidly killed the pathogen Streptococcus mutans and impaired biofilm formation following a single topical application of tooth-mimetic surface. Furthermore, we developed a synergistic approach using PMAMPs combined with matrix-degrading enzymes to facilitate their access into biofilms and kill the embedded bacteria. In addition, we identified a novel role for PMAMPs in delivering drugs to periodontal and gingival cells, 13–48 folds more efficiently than any other tested cell penetrating peptides. Therefore, PDs fused with protegrin expressed in plant cells could potentially play a dual role in delivering therapeutic proteins to gum tissues while killing pathogenic bacteria when delivered as topical oral formulations or in chewing gums. Recent FDA approval of plant-produced PDs augurs well for clinical advancement of this novel concept.

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Preformulation and stability in biological fluids of the retrocyclin RC-101, a potential anti-HIV topical microbicide

Cited by 18 publications

References 28 publications

Retrocyclins neutralize bacterial toxins by potentiating their unfolding

Retrocyclins neutralize bacterial toxins by potentiating their unfolding

Retrocyclin inhibits Gardnerella vaginalis biofilm formation and toxin activity

Topical delivery of low-cost protein drug candidates made in chloroplasts for biofilm disruption and uptake by oral epithelial cells

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