AGSE: A Novel Grape Seed Extract Enriched for PP2A Activating Flavonoids That Combats Oxidative Stress and Promotes Skin Health.

Environmental stimuli attack the skin daily resulting in the generation of reactive oxygen species (ROS) and inflammation. One pathway that regulates oxidative stress in skin involves Protein Phosphatase 2A (PP2A), a phosphatase which has been previously linked to Alzheimer's Disease and aging. Oxidative stress decreases PP2A methylation in normal human dermal fibroblasts (NHDFs). Thus, we hypothesize agents that increase PP2A methylation and activity will promote skin health and combat aging. To discover novel inhibitors of PP2A demethylation activity, we screened a library of 32 natural botanical extracts. We discovered Grape Seed Extract (GSE), which has previously been reported to have several benefits for skin, to be the most potent PP2A demethylating extract. Via several fractionation and extraction steps we developed a novel grape seed extract called Activated Grape Seed Extract (AGSE), which is enriched for PP2A activating flavonoids that increase potency in preventing PP2A demethylation when compared to commercial GSE. We then determined that 1% AGSE and 1% commercial GSE exhibit distinct gene expression profiles when topically applied to a 3D human skin model. To begin to characterize AGSE's activity, we investigated its antioxidant potential and demonstrate it reduces ROS levels in NHDFs and cell-free assays equal to or better than Vitamin C and E. Moreover, AGSE shows anti-inflammatory properties, dose-dependently inhibiting UVA, UVB and chemical-induced inflammation. These results demonstrate AGSE is a novel, multi-functional extract that modulates methylation levels of PP2A and supports the hypothesis of PP2A as a master regulator for oxidative stress signaling and aging in skin.

HYVIA™: A novel, topical chia seed extract that improves skin hydration.

BACKGROUND: Chia seeds have gained importance as it is the highest known plant source of omega-3 (ω3) polyunsaturated fatty acids. Specifically, chia seeds possess ω3 α-linolenic acid (ALA) and ω6 linoleic acid (LA), together known as Vitamin F, which play an important role in maintaining skin function. Protein phosphatase 2A (PP2A) is a master regulatory protein that plays a critical role in skin barrier function and its activity is modulated by natural lipids. AIMS: Obtain a chia seed extract (HYVIA™) with significant higher levels of Vitamin F, determine in vitro PP2A activity and skin hydration markers compared to other commercial chia seed extracts (CCSEs), and evaluate the potential skin hydration benefits clinically in human subjects. METHODS: A PP2A demethylation assay was utilized to assess PP2A activity. In vitro studies utilizing normal human epidermal keratinocytes (NHEKs) were treated with HYVIA™ and gene expression of hydration markers (AQP3, HAS2) were measured by quantitative PCR (qPCR). A 16-subject clinical trial was performed with 0.1% HYVIA™ formulated in a cream and applied topically to assess its skin moisturizing potential. RESULTS: We demonstrate here that HYVIA™, ALA, and LA inhibit PP2A demethylation, boosting PP2A activity, while most other CCSEs do not. Unlike other CCSEs, HYVIA™ increases keratinocyte hydration factors aquaporin-3 and hyaluronic acid synthase-2 in vitro. Clinical assessment of 0.1% HYVIA™ cream shows that HYVIA™ improves skin hydration. CONCLUSIONS: HYVIA™ is a novel chia seed extract, enriched for Vitamin F, that modulates PP2A activity and clinically improves skin hydration and barrier function.

SIG-1273 protects skin against urban air pollution and when formulated in AgeIQ™ Night Cream anti-aging benefits clinically demonstrated.

BACKGROUND: SIG-1273 is a novel cosmetic active that provides a broad spectrum of benefits to the skin. Considering the chronic skin exposure to pollution in urban areas, we sought to determine if SIG-1273 could provide additional protection against skin aging by inhibiting pollutant-induced cytotoxicity and inflammation. OBJECTIVE: Determine if SIG-1273 possesses antipollution properties in vitro and evaluate the potential anti-aging benefits of Age IQ™ Night Cream clinically in human subjects. METHODS: In vitro studies utilizing normal human epidermal keratinocytes (NHEKs), were co-treated with urban dust (SRM 1649b) and SIG-1273 (toxicity protection measured by MTS assay). A water-soluble fraction of urban dust (UD-WS) induces pro-inflammatory cytokine release (IL-8) from NHEKs (measured via ELISA). An 8-week, 37-subject clinical trial was performed with 0.05% SIG-1273 formulated in Age IQ™ Night Cream and applied topically to assess its potential to reduce the appearance of aging. RESULTS: In vitro studies using NHEKs demonstrate SIG-1273 protects against urban dust-induced cell toxicity, reducing cell death by 66% and concentration dependently inhibits UD-WS-induced IL-8 production (IC50  = 20 nmol/L), outperforming niacinamide, ascorbic acid, and α-tocopherol, commonly used actives in antipollution skin-care products. Clinical assessment of Age IQ™ Night Cream shows it is effective in improving the appearance of facial skin aging including fine lines and wrinkles, skin texture, skin clarity/brightness, and firmness/elasticity. CONCLUSIONS: SIG-1273, is demonstrated here for the first time to possess antipollution properties. Included as a key active ingredient in Age IQ™ Night Cream, this novel topical formulation provides benefits to individuals with aging skin.

SIG1459: A novel phytyl-cysteine derived TLR2 modulator with in vitro and clinical anti-acne activity.

Cutibacterium (formerly Propionibacterium acnes) is a major contributor to the pathogenesis of acne. C. acnes initiates an innate immune response in keratinocytes via recognition and activation of toll-like receptor-2 (TLR2), a key step in comedogenesis. Tetramethyl-hexadecenyl-cysteine-formylprolinate (SIG1459), a novel anti-acne isoprenylcysteine (IPC) small molecule, is shown in this study to have direct antibacterial activity and inhibit TLR2 inflammatory signalling. In vitro antibacterial activity of SIG1459 against C. acnes was established demonstrating minimal inhibitory concentration (MIC = 8.5 µmol\L), minimal bactericidal concentration (MBC = 16.1 µmol\L) and minimal biofilm eradication concentration (MBEC = 12.5 µmol\L). To assess SIG1459's anti-inflammatory activity, human keratinocytes were exposed to C. acnes and different TLR2 ligands (peptidoglycan, FSL-1, Pam3CSK4) that induce pro-inflammatory cytokine IL-8 and IL-1α production. Results demonstrate SIG1459 inhibits TLR2-induced IL-8 release from TLR2/TLR2 (IC50  = 0.086 µmol\L), TLR2/6 (IC50  = 0.209 µmol\L) and IL-1α from TLR2/TLR2 (IC50  = 0.050 µmol\L). To assess the safety and in vivo anti-acne activity of SIG1459, a vehicle controlled clinical study was conducted applying 1% SIG1459 topically (n = 35 subjects) in a head-to-head comparison against 3% BPO (n = 15 subjects). Utilizing the Investigator Global Assessment scale for acne as primary endpoint, results demonstrate 1% SIG1459 significantly outperformed 3% BPO over 8 weeks, resulting in 79% improvement as compared to 56% for BPO. Additionally, 1% SIG1459 was well tolerated. Thus, SIG1459 and phytyl IPC compounds represent a novel anti-acne technology that provides a safe dual modulating benefit by killing C. acnes and reducing the inflammation it triggers via TLR2 signalling.

Caspase-9 CARD : core domain interactions require a properly formed active site.

Caspase-9 is a critical factor in the initiation of apoptosis and as a result is tightly regulated by many mechanisms. Caspase-9 contains a Caspase Activation and Recruitment Domain (CARD), which enables caspase-9 to form a tight interaction with the apoptosome, a heptameric activating platform. The caspase-9 CARD has been thought to be principally involved in recruitment to the apoptosome, but its roles outside this interaction have yet to be uncovered. In this work, we show that the CARD is involved in physical interactions with the catalytic core of caspase-9 in the absence of the apoptosome; this interaction requires a properly formed caspase-9 active site. The active sites of caspases are composed of four extremely mobile loops. When the active-site loops are not properly ordered, the CARD and core domains of caspase-9 do not interact and behave independently, like loosely tethered beads. When the active-site loop bundle is properly ordered, the CARD domain interacts with the catalytic core, forming a single folding unit. Taken together, these findings provide mechanistic insights into a new level of caspase-9 regulation, prompting speculation that the CARD may also play a role in the recruitment or recognition of substrate.

Eicosanoyl-5-hydroxytryptamide (EHT) prevents Alzheimer's disease-related cognitive and electrophysiological impairments in mice exposed to elevated concentrations of oligomeric beta-amyloid.

Soluble forms of oligomeric beta-amyloid (Aß) are thought to play a central role in Alzheimer's disease (AD). Transgenic manipulation of methylation of the serine/threonine protein phosphatase, PP2A, was recently shown to alter the sensitivity of mice to AD-related impairments resulting from acute exposure to elevated levels of Aß. In addition, eicosanoyl-5-hydroxytryptamide (EHT), a naturally occurring component from coffee beans that modulates PP2A methylation, was shown to confer therapeutic benefits in rodent models of AD and Parkinson's disease. Here, we tested the hypothesis that EHT protects animals from the pathological effects of exposure to elevated levels of soluble oligomeric Aß. We treated mice with EHT-containing food at two different doses and assessed the sensitivity of these animals to Aß-induced behavioral and electrophysiological impairments. We found that EHT administration protected animals from Aß-induced cognitive impairments in both a radial-arm water maze and contextual fear conditioning task. We also found that both chronic and acute EHT administration prevented Aß-induced impairments in long-term potentiation. These data add to the accumulating evidence suggesting that interventions with pharmacological agents, such as EHT, that target PP2A activity may be therapeutically beneficial for AD and other neurological conditions.

N-Acetylglutaminoyl-S-farnesyl-L-cysteine (SIG-1191): an anti-inflammatory molecule that increases the expression of the aquaglyceroporin, aquaporin-3, in human keratinocytes.

Isoprenylcysteine (IPC) small molecules were discovered as signal transduction modulating compounds ~25 years ago. More recently, IPC molecules have demonstrated antioxidant and anti-inflammatory properties in a variety of dermal cells as well as antimicrobial activity, representing a novel class of compounds to ameliorate skin conditions and disease. Here, we demonstrate a new IPC compound, N-acetylglutaminoyl-S-farnesyl-L-cysteine (SIG-1191), which inhibits UVB-induced inflammation blocking pro-inflammatory cytokine interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) production. To investigate further the previously reported hydrating potential of IPC compounds, SIG-1191 was tested for its ability to modulate aquaporin expression. Specifically, aquaporin 3 (AQP3) the most abundant aquaporin found in skin has been reported to play a key role in skin hydration, elasticity and barrier repair. Results show here for the first time that SIG-1191 increases AQP3 expression in both cultured normal human epidermal keratinocytes as well as when applied topically in a three-dimensional (3D) reconstructed human skin equivalent. Additionally, SIG-1191 dose dependently increased AQP3 protein levels, as determined by specific antibody staining, in the epidermis of the 3D skin equivalents. To begin to elucidate which signaling pathways SIG-1191 may be modulating to increase AQP3 levels, we used several pharmacological pathway inhibitors and determined that AQP3 expression is mediated by the Mitogen-activated protein kinase/Extracellular signal-regulated kinase kinase (MEK) pathway. Altogether, these data suggest SIG-1191 represents a new IPC derivative with anti-inflammatory activity that may also promote increased skin hydration based on its ability to increase AQP3 levels.

In vitro and clinical evaluation of SIG1273: a cosmetic functional ingredient with a broad spectrum of anti-aging and antioxidant activities.

BACKGROUND: Isoprenylcysteine (IPC) small molecules were identified as a new class of anti-inflammatory compounds over 20 years ago. Since then, they have been developed as novel cosmetic functional ingredients (CFI) and topical drug candidates. SIG1273 is a second generation CFI that has previously been shown to provide a broad spectrum of benefits for the skin through its anti-inflammatory and antimicrobial properties. OBJECTIVE: To determine whether SIG1273 possesses anti-aging properties in vitro and evaluate the tolerability and activity of SIG1273 when applied topically to human subjects. METHODS: To model photoaging in vitro, human dermal fibroblasts (HDFs) were exposed in culture to UVA to induce collagenase (MMP-1) production. An in vitro wound-healing model was based on the activation of HDF migration into cell-free tissue culture surface. Hydrogen peroxide-induced oxidative stress was performed using HDFs to measure intracellular ROS activity. Radical scavenging capacity was determined using a colorimetric antioxidant assay kit (ABTS method). Lastly, a 4-week, 29-subject study was performed in which SIG1273 was applied topically as a cream to assess its tolerance and activity in reducing the appearance of aging. RESULTS: In vitro studies demonstrate SIG1273 inhibits UVA-induced MMP-1 production, hydrogen peroxide-induced oxidative stress and promotes wound healing. Moreover, SIG1273 was shown to be a radical scavenging antioxidant. Clinical assessment of SIG1273 cream (0.25%) showed it was well tolerated with significant improvement in the appearance of fine lines, coarse wrinkles, radiance/luminosity, pore size, texture/smoothness, hydration and increased firmness. CONCLUSIONS: SIG1273 represents a novel CFI with antioxidant, anti-aging, and anti-inflammatory properties that when applied topically is well tolerated and provides benefits to individuals with aging skin.

Inhibition of caspase-9 by stabilized peptides targeting the dimerization interface.

Caspases comprise a family of dimeric cysteine proteases that control apoptotic programmed cell death and are therefore critical in both organismal development and disease. Specific inhibition of individual caspases has been repeatedly attempted, but has not yet been attained. Caspase-9 is an upstream or initiator caspase that is regulated differently from all other caspases, as interaction with natural inhibitor X-linked inhibitor of apoptosis protein (XIAP)-baculovirus inhibitory repeat 3 (BIR3) occurs at the dimer interface maintaining caspase-9 in an inactive monomeric state. One route to caspase-9-specific inhibition is to mimic this interaction, which has been localized to the α5 helix of XIAP-BIR3. We have developed three types of stabilized peptides derived from the α5 helix, using incorporation of aminoisobutyric acid, the avian pancreatic polypeptide (aPP)-scaffold or aliphatic staples. The stabilized peptides are helical in solution and achieve up to 32 µM inhibition, indicating that this allosteric site at the caspase-9 dimerization interface is regulatable with low-molecular weight synthetic ligands and is thus a druggable site. The most potent peptides against caspase-9 activity are the aPP-scaffolded peptides. Other caspases, which are not regulated by dimerization, should not be inactivated by these peptides. Given that all of the peptides attain helical structures but cannot recapitulate the high-affinity inhibition of the intact BIR3 domain, it has become clear that interactions of caspase-9 with the BIR3 exosite are essential for high-affinity binding. These results explain why the full XIAP-BIR3 domain is required for maximal inhibition and suggest a path forward for achieving allosteric inhibition at the dimerization interface using peptides or small molecules.

Mechanism of zinc-mediated inhibition of caspase-9.

Zinc-mediated inhibition is implicated in global caspase regulation, with relief of zinc-mediated inhibition central to both small-molecule and natively induced caspase activation. As an initiator, caspase-9 regulates the upstream stages of the apoptotic caspase cascade, making it a critical control point. Here we identify two distinct zinc-binding sites on caspase-9. The first site, composed of H237, C239, and C287, includes the active site dyad and is primarily responsible for zinc-mediated inhibition. The second binding site at C272 is distal from the active site. Given the amino-acid conservation in both regions, these sites appear to be present across the caspase family underscoring the importance of zinc-mediated regulation of this class of enzymes.

Robust production of a peptide library using methodological synchronization.

Peptide libraries have proven to be useful in applications such as substrate profiling, drug candidate screening and identifying protein-protein interaction partners. However, issues of fidelity, peptide length, and purity have been encountered when peptide libraries are chemically synthesized. Biochemically produced libraries, on the other hand, circumvent many of these issues due to the fidelity of the protein synthesis machinery. Using thioredoxin as an expression partner, a stably folded peptide scaffold (avian pancreatic polypeptide) and a compatible cleavage site for human rhinovirus 3C protease, we report a method that allows robust expression of a genetically encoded peptide library, which yields peptides of high purity. In addition, we report the use of methodological synchronization, an experimental design created for the production of a library, from initial cloning to peptide characterization, within a 5-week period of time. Total peptide yields ranged from 0.8% to 16%, which corresponds to 2-70 mg of pure peptide. Additionally, no correlation was observed between the ability to be expressed or overall yield of peptide-fusions and the intrinsic chemical characteristics of the peptides, indicating that this system can be used for a wide variety of peptide sequences with a range of chemical characteristics.