Tripeptide linked dispiro cyclotriphosphazene conjugates: Synthesis, molecular docking analysis of compounds binding within cancer cell line receptors and in vitro cytotoxic and genotoxic activities.

The novel dioxybiphenyl bridged-cyclotriphosphazenes (DPP) bearing tripeptide were synthesized and investigated for their molecular docking analysis, visualizing their binding profiles within various cancer cell line receptors and in vitro cytotoxic and genotoxic properties. The dipeptide compound (Tyr-Phe) was treated with various amino acids to obtain the tripeptide compounds (Tyr-Phe-Gly, Tyr-Phe-Ala, Tyr-Phe-Val, Tyr-Phe-Phe, and Tyr-Phe-Leu). These synthesized tripeptides were subsequently treated with DPP to obtain novel phosphazene compounds bearing tripeptide structures. As a result, the synthesis of target molecules with phosphazene compound in the center and biphenyl and tripeptide groups in the side arms was obtained for the first time in this study. Examining the cytotoxic studies in vitro of our newly synthesized compounds demonstrated the anticancer properties against four selected human cancer cell lines, including breast (MCF-7), ovarian (A2780), prostate (PC-3), and colon (Caco-2) cancer cells. The Comet Assay analysis determined that the cell death mechanism of most of the compounds with cytotoxic activity stemmed from the DNA damage mechanism. Among the compounds, the DPP-Tyr-Phe-Phe compound seems to have the best anticancer activity against the subjected cell lines (Except for A2780) with IC50 values equal to 20.18, 72.14, 12.21, and 5.17 µM against breast, ovarian, prostate, and colon cancer cell lines, respectively. For this reason, the molecular docking analysis was conducted for the DTPP compound to visualize its binding geometry and profile within the target enzyme's binding site associated with the specific cancer cell line. The analysis revealed that the DTPP derivative exhibited an optimal binding conformation and characteristics within the target enzyme's binding site, aligning well with the experimental data. Based on the data, these compounds are believed to be strong candidate molecules for both pharmaceutical and clinical applications.

Ile-Pro-Pro attenuates sympathetic activity and hypertension.

Isoleucine-proline-proline (Ile-Pro-Pro, IPP) is a natural food source tripeptide that inhibits angiotensin-converting enzyme (ACE) activity. The aim of this study was to determine the central and peripheral roles of IPP in attenuating sympathetic activity, oxidative stress and hypertension. Male Sprague-Dawley rats were subjected to sham-operated surgery (Sham) or two-kidney one-clip (2K1C) surgery to induce renovascular hypertension. Renal sympathetic nerve activity and blood pressure were recorded. Bilateral microinjections of IPP to hypothalamic paraventricular nucleus (PVN) attenuated sympathetic activity (-16.1 ± 2.5%, P < 0.001) and hypertension (-8.7 ± 1.5 mmHg, P < 0.01) in 2K1C rats by inhibiting ACE activity and subsequent angiotensin II and superoxide production in the PVN. Intravenous injections of IPP also attenuated sympathetic activity (-15.1 ± 2.1%, P < 0.001) and hypertension (-16.8 ± 2.3 mmHg, P < 0.001) via inhibiting ACE activity and oxidative stress in both PVN and arteries of 2K1C rats. The duration of the effects of the intravenous IPP was longer than those of the PVN microinjection, but the sympatho-inhibitory effect of intravenous injections occurred later than that of the PVN microinjection. Intraperitoneal injection of IPP (400 pmol/day for 20 days) attenuated hypertension and vascular remodeling via inhibiting ACE activity and oxidative stress in both PVN and arteries of 2K1C rats. These results indicate that IPP attenuates hypertension and sympathetic activity by inhibiting ACE activity and oxidative stress. The sympathoinhibitory effect of peripheral IPP is mainly caused by the ACE inhibition in PVN, and the antihypertensive effect is related to the sympathoinhibition and the arterial ACE inhibition. Long-term intraperitoneal IPP therapy attenuates hypertension, oxidative stress and vascular remodeling.

Therapeutic Effect of Anti-Inflammatory Tripeptide Cream in Hand-Foot Syndrome/Skin Reaction Related to Anticancer Drugs: a Randomized, Double-Blind, Placebo-Controlled Pilot Trial.

Purpose: Hand-foot syndrome (HFS) and hand-foot skin reaction (HFSR) are relatively common toxicities that interfere with the quality of life (QoL) of patients with cancer. Anti-inflammatory tripeptide cream (ATPC) is a complex formulation of anti-inflammatory tripeptides, the CD99-agonist BinterinTM and the Wnt-antagonist WinhibinTM. The present study aimed to assess the therapeutic effects of ATPC in HFS/HFSR associated with anticancer drugs. Materials and Methods: This was a single-center, randomized, double-blind, placebo-controlled trial. Patients who developed grade 1 HFS/HFSR after systemic anticancer treatments were enrolled, and randomly assigned to receive either ATPC or placebo cream (PC) and followed up at 3-week intervals for up to nine weeks. Primary endpoint was the development of grade ≥ 2 HFS/HFSR. Results: Between April 2019 and July 2022, 60 patients (31 in the ATPC and 29 in the PC group) completed the study. The incidence of grade ≥ 2 HFS/HFSR was significantly lower in the ATPC than in the PC group (25.8% vs. 51.7%, p=0.039). The ATPC showed trends towards a better QoL score, assessed by a HFSR and QoL questionnaire at 9 weeks (26.0 vs. 29.9, p=0.574), and a lower frequency of discontinuation, interruption, or dose reduction of anticancer drugs (51.6% vs. 58.6%, p=0.586) than the PC group over 9 weeks, though without statistical significance. Conclusion: Our results showed that ATPC significantly decreased the development of grade ≥ 2 HFS/HFSR in patients already with HFS/HFSR. Therefore, ATPC may be an effective treatment for HFS/HFSR associated with anticancer drugs.

Relationships between conformational and vibrational features of tryptophan characteristic Raman markers.

Tryptophan (Trp) residue provides characteristic vibrational markers to the middle wavenumber spectral region of the Raman spectra recorded from peptides and proteins. In this report, we were particularly interested in eight Trp Raman markers, referred to as Wi (i = 1,…,8). All responsible for pronounced Raman lines, these markers originate from indole moiety, a bicyclic conjugated segment involved in the Trp structure. Numerous investigations have previously attempted to relate the variations observed in the spectral features of these markers to the environmental changes of Trp residues. To emphasize the most important points we can mention (i) the variations in the Raman profile of W4 (∼1360 cm-1) and W5 (∼1340 cm-1), frequently observed as a doublet with variable intensity ratio. These two markers were thought to result from a Fermi-resonance effect between certain planar and nonplanar modes; (ii) the changes observed in the wavenumbers and relative intensities of W4, W7 (∼880 cm-1) and W8 (∼760 cm-1) were supposed to be related to the accessibility of Trp to surrounding water molecules; and (iii) the wavenumber fluctuations of W3 (∼1550 cm-1), taken as a Trp side chain orientational marker. However, some ambiguities still exist regarding the interpretation of these markers, needing further clarification. Herein, upon a joint experimental and theoretical analysis based on a multiconformational approach, attention was paid to the relationships between structural and vibrational features of three indole-containing compounds with increasing structural complexity, i.e., skatole (3-methylindole), tryptophan, and tripeptide Gly-Trp-Gly. This study clearly shows that the existing assignments given to certain Trp Raman markers should be reconsidered, especially those based on the Fermi-resonance origin of W4-W5 (∼1360-1340 cm-1) doublet, as well as the purely environmental dependence of W7 and W8 markers.

Microemulsions and Nanoemulsions for Topical Delivery of Tripeptide-3: From Design of Experiment to Anti-Sebum Efficacy on Facial Skin.

The targeted delivery of a hydrophilic Tripeptide-3 to the skin using microemulsions or nanoemulsions for facial oil reduction was the focus of this study. The impact factors affecting oil/water transparent dispersion formation, such as the surfactant system, HLB value, and co-solvent, were identified through the water titration method and pseudoternary phase diagram plots. The interfacial tension between caprylic/capric triglyceride (CCT oil) and water was significantly reduced by the surfactant/co-surfactant combination (Smix) of Cremophore® RH40 and a double-tails co-surfactant, polyglycerol-3-diisostearate, at an HLB of 13 together with a water-to-co-solvent (PG) ratio of 1:1. A two-level full factorial design of experiment (FFD-DoE) emphasized the independent variables of the HLB value, co-solvent, and CCT oil contents affecting the optimal compositions for micro- or nanoemulsion formation. The low-energy spontaneous emulsification of the optimized combination at a low Smix content (10%) yielded the translucent oil-in-water Tripeptide-3 nanoemulsions with an internal droplet size of 25.7 ± 1.20 nm, a narrow polydispersity index of 0.237 ± 0.129, and 70.6 ± 0.58% transmittance. The in vitro skin permeation study revealed a significantly higher skin penetration and retention of the Tripeptide-3 nanoemulsions compared to the high surfactant microemulsions and coarse emulsions. Skin irritation and oil control efficacy were evaluated in healthy volunteers before and after product application for 28 days. The obtained nanoemulsions not only decreased sebum production but also enhanced skin moisture levels. In conclusion, the meticulously designed nanoemulsions, incorporating suitable excipients, show a promising delivery system for hydrophilic peptides to control sebum overproduction in oily facial skin.

Advances in Bone-Targeting Drug Delivery: Emerging Strategies Using Adeno-Associated Virus.

The development of bone-targeting drug delivery systems holds immense promise for improving the treatment of skeletal diseases. By precisely delivering therapeutic agents to the affected areas of bone, these strategies can enhance drug efficacy, minimize off-target effects, and promote patient adherence, ultimately leading to improved treatment outcomes and an enhanced quality of life for patients. This review aims to provide an overview of the current state of affinity-based bone-targeting agents and recent breakthroughs in innovative bone-targeting adeno-associated virus (AAV) strategies to treat skeletal diseases in mice. In particular, this review will delve into advanced AAV engineering, including AAV serotype selection for bone targeting and capsid modifications for bone-specific tropism. Additionally, we will highlight recent advancements in AAV-mediated gene therapy for skeletal diseases and discuss challenges and future directions of this promising therapeutic approach.

Understanding the Transepithelial Transport and Transbilayer Diffusion of the Antihypertensive Peptide Asn-Cys-Trp: Insights from Caco-2 Cell Monolayers and the DPPC Model Membrane.

Understanding the transport mechanism of the peptide Asn-Cys-Trp (NCW) is crucial to improving its intestinal absorption and bioavailability. This study investigated the absorption of NCW through Caco-2 cell monolayers and its interaction with the DPPC bilayers. Results revealed that after a 3 h incubation, the Papp (AP-BL) and Papp (BL-AP) values of NCW at a concentration of 5 mmol/L were (22.24 ± 4.52) × 10-7 and (6.63 ± 2.31) × 10-7 cm/s, respectively, with the transport rates of 1.59 ± 0.32 and 0.62 ± 0.20%, indicating its moderate absorption. NCW was found to be transported via PepT1 and paracellular transport pathways, as evidenced by the significant impact of Gly-Pro and cytochalasin D on the Papp values. Moreover, NCW upregulated ZO-1 mRNA expression. Further investigation of the ZO-1-mediated interaction between NCW and tight junction proteins will contribute to a better understanding of the paracellular transport mechanism of NCW. The interaction between NCW and the DPPC bilayers was predominantly driven by entropy. NCW permeated the bilayers through electrostatic, hydrogen bonding, and hydrophobic interactions, resulting in increased fluidity, flexibility, and disorder as well as phase transition and phase separation of the bilayers.

Prediction of protein N-terminal acetylation modification sites based on CNN-BiLSTM-attention model.

N-terminal acetylation is one of the most common and important post-translational modifications (PTM) of eukaryotic proteins. PTM plays a crucial role in various cellular processes and disease pathogenesis. Thus, the accurate identification of N-terminal acetylation modifications is important to gain insight into cellular processes and other possible functional mechanisms. Although some algorithmic models have been proposed, most have been developed based on traditional machine learning algorithms and small training datasets. Their practical applications are limited. Nevertheless, deep learning algorithmic models are better at handling high-throughput and complex data. In this study, DeepCBA, a model based on the hybrid framework of convolutional neural network (CNN), bidirectional long short-term memory network (BiLSTM), and attention mechanism deep learning, was constructed to detect the N-terminal acetylation sites. The DeepCBA was built as follows: First, a benchmark dataset was generated by selecting low-redundant protein sequences from the Uniport database and further reducing the redundancy of the protein sequences using the CD-HIT tool. Subsequently, based on the skip-gram model in the word2vec algorithm, tripeptide word vector features were generated on the benchmark dataset. Finally, the CNN, BiLSTM, and attention mechanism were combined, and the tripeptide word vector features were fed into the stacked model for multiple rounds of training. The model performed excellently on independent dataset test, with accuracy and area under the curve of 80.51% and 87.36%, respectively. Altogether, DeepCBA achieved superior performance compared with the baseline model, and significantly outperformed most existing predictors. Additionally, our model can be used to identify disease loci and drug targets.

Synthesis and mechanistic study of ultrashort peptides that inhibits Alzheimer's Aß-aggregation-induced neurotoxicity.

Misfolding/aggregation of ß-amyloid peptide lead to the formation of toxic oligomers or accumulation of amyloid plaques, which is a seminal step in the progression of Alzheimer's disease (AD). Despite continuous efforts in the development of therapeutic agents, the cure for AD remains a major challenge. Owing to specific binding affinity of structure-based peptides, we report the synthesis of new peptide-based inhibitors derived from the C-terminal sequences, Aß38-40 and Aß40-42. Preliminary screening using MTT cell viability assay and corroborative results from ThT fluorescence assay revealed a tripeptide showing significantly effective inhibition towards Aß1-42 aggregation and induced toxicity. Peptide 3 exhibited excellent cell viability of 94.3 % at 2 µM and of 100 % at 4 µM and 10 µM. CD study showed that peptide 3 restrict the conformation transition of Aß1-42 peptide towards cross-ß-sheet structure and electron microscopy validated the absence of Aß aggregates as indicated by the altered morphology of Aß1-42 in the presence of peptide 3. The HRMS-ESI, DLS and ANS studies were performed to gain mechanistic insights into the effect of inhibitor against Aß aggregation. This Aß-derived ultrashort motif provides impetus for the development of peptide-based anti-AD agents.

Development of a Novel Covalently Bonded Conjugate of Caprylic Acid Tripeptide (Isoleucine-Leucine-Aspartic Acid) for Wound-Compatible and Injectable Hydrogel to Accelerate Healing.

Third-degree burn injuries pose a significant health threat. Safer, easier-to-use, and more effective techniques are urgently needed for their treatment. We hypothesized that covalently bonded conjugates of fatty acids and tripeptides can form wound-compatible hydrogels that can accelerate healing. We first designed conjugated structures as fatty acid-aminoacid1-amonoacid2-aspartate amphiphiles (Cn acid-AA1-AA2-D), which were potentially capable of self-assembling into hydrogels according to the structure and properties of each moiety. We then generated 14 novel conjugates based on this design by using two Fmoc/tBu solid-phase peptide synthesis techniques; we verified their structures and purities through liquid chromatography with tandem mass spectrometry and nuclear magnetic resonance spectroscopy. Of them, 13 conjugates formed hydrogels at low concentrations (≥0.25% w/v), but C8 acid-ILD-NH2 showed the best hydrogelation and was investigated further. Scanning electron microscopy revealed that C8 acid-ILD-NH2 formed fibrous network structures and rapidly formed hydrogels that were stable in phosphate-buffered saline (pH 2-8, 37 °C), a typical pathophysiological condition. Injection and rheological studies revealed that the hydrogels manifested important wound treatment properties, including injectability, shear thinning, rapid re-gelation, and wound-compatible mechanics (e.g., moduli G″ and G', ~0.5-15 kPa). The C8 acid-ILD-NH2(2) hydrogel markedly accelerated the healing of third-degree burn wounds on C57BL/6J mice. Taken together, our findings demonstrated the potential of the Cn fatty acid-AA1-AA2-D molecular template to form hydrogels capable of promoting the wound healing of third-degree burns.

Water-Based Synthesis of ß-Sheet-Like Supramolecular Metallohydrogel Organized by Using a Native Ultrashort Peptide Sequence.

Designing supramolecular hydrogels using short peptides is challenging. To control self-assembly, a certain amount of organic solvent is typically added to the system, or the short peptide is modified with a functional group that is hydrophobic, hydrophilic, or highly coordinative. We discovered that l-His-l-Ile-l-Thr (HIT), a very short unmodified "native" tripeptide, selectively responds to Cu2+ ions in pure water to form a transparent supramolecular metallohydrogel. Circular dichroism analysis revealed that Cu2+ ions, but no other metal species, caused HIT to change from a random-coil-like to a ß-sheet-like structure. Other spectroscopic methods were used to characterize the properties of the supramolecular metallohydrogel. These results are expected to facilitate the development of native short peptides as advanced functional biomaterials.

The chromatin remodeling factors EP300 and TRRAP are novel SMYD3 interactors involved in the emerging 'nonmutational epigenetic reprogramming' cancer hallmark.

SMDY3 is a histone-lysine N-methyltransferase involved in several oncogenic processes and is believed to play a major role in various cancer hallmarks. Recently, we identified ATM, BRCA2, CHK2, MTOR, BLM, MET, AMPK, and p130 as direct SMYD3 interactors by taking advantage of a library of rare tripeptides, which we first tested for their in vitro binding affinity to SMYD3 and then used as in silico probes to systematically search the human proteome. Here, we used this innovative approach to identify further SMYD3-interacting proteins involved in crucial cancer pathways and found that the chromatin remodeling factors EP300 and TRRAP interact directly with SMYD3, thus linking SMYD3 to the emerging 'nonmutational epigenetic reprogramming' cancer hallmark. Of note, we validated these interactions in gastrointestinal cancer cell lines, including HCT-116 cells, which harbor a C-terminal truncating mutation in EP300, suggesting that EP300 binds to SMYD3 via its N-terminal region. While additional studies are required to ascertain the functional mechanisms underlying these interactions and their significance, the identification of two novel SMYD3 interactors involved in epigenetic cancer hallmark pathways adds important pieces to the puzzle of how SMYD3 exerts its oncogenic role.

Hydrolyzed Collagen Tripeptide Promotes Longitudinal Bone Growth in Childhood Rats via Increases in Insulin-Like Growth Factor-1 and Bone Morphogenetic Proteins.

Previous studies have reported that collagen tripeptide (CTP) derived from collagen hydrolysate has various beneficial effects on health by protecting against skin aging and improving bone formation and cartilage regeneration. Collagen-Tripep20TM (CTP20), which is a low-molecular-weight CTP derived from fish skin, contains a bioactive CTP, Gly-Pro-Hyp >3.2% with a tripeptide content >20%. Herein, we investigated the osteogenic effects and mechanisms of CTP20 (<500 Da) on MG-63 osteoblast-like cells and SW1353 chondrocytes. And we measured promoting ratio of the longitudinal bone growth in childhood rats. First, CTP20 at 100 µg/mL elevated the proliferation (15.0% and 28.2%), alkaline phosphatase activity (29.3% and 32.0%), collagen synthesis (1.25- and 1.14-fold), and calcium deposition (1.18- and 1.15-fold) in MG-63 cells and SW1353, respectively. In addition, we found that CTP20 could promote the longitudinal growth and height of the growth plate of the tibia in childhood rats. CTP20 enhanced the protein expression of insulin-like growth factor-1 (IGF-1) in MG-63 and SW1353 cells, and in the growth plate of childhood rats, along with Janus Kinase 2, and signal transducer and activator of transcription 5 activation in MG-63 and SW1353 cells. CTP20 also elevated the expression levels of bone morphogenetic proteins (BMPs) in MG-63 and SW1353 cells and in the growth plates of childhood rats. These results indicate that CTP20 may promote the endochondral ossification and longitudinal bone growth, through enhancing of IGF-1 and BMPs. (Clinical Trial Registration number: smecae 19-09-01).

Effect of Peptides on the Synthesis, Properties and Wound Healing Capacity of Silver Nanoparticles.

The aim of this study is the synthesis of novel peptide-silver nanoparticle conjugates with enhanced wound healing capacity. Peptide-silver nanoparticle conjugates were synthesized using myristoyl tetrapeptide 6 (MT6) or copper tripeptide 1 (CuTP1). Peptide-free silver nanoparticles (AgNP) were synthesized using NaBH4 and sodium citrate and were used as control. The addition of the peptides during or after the synthesis of nanoparticles and its impact on the properties of the synthesized peptide-silver nanoparticle conjugates were assessed. The monitoring of the synthesis of nanoparticles was achieved using ultraviolet-visible spectrophotometry (UV-/Vis). The characteristics and colloidal stability of the nanoparticles (size and ζ-potential distribution, morphology, composition and structure) were monitored using dynamic laser scattering (DLS), transmission electron microscopy (TEM), atomic absorption spectroscopy (AAS) and X-ray diffraction (XRD). The wound healing capacity of the peptide-silver nanoparticle conjugates was assessed using scratch test assay on fibroblasts (NIH/3T3). The results indicated that the addition of the peptides during the synthesis of nanoparticles lead to better yield of the reaction and more effective capping while the size distribution and ζ-potential of the conjugates indicated long-term colloidal stability. The MT6-AgNP conjugate exhibited 71.97 ± 4.35% wound closure, which was about 5.48-fold higher (p < 0.05) than the corresponding free MT6. The CuTP1-AgNP conjugate exhibited 62.37 ± 18.33% wound closure that was better by 2.82 fold (p < 0.05) compared to the corresponding free CuTP1. Both peptides led to the synthesis of silver nanoparticle conjugates with enhanced wound healing capacity compared to the respective free peptide or to the peptide-free AgNP (29.53 ± 4.71% wound closure, p < 0.05). Our findings demonstrated that the synthetized peptide-silver nanoparticle conjugates are promising ingredients for wound care formulation.

Liposomes as Carriers of GHK-Cu Tripeptide for Cosmetic Application.

Liposomes are self-assembled spherical systems composed of amphiphilic phospholipids. They can be used as carriers of both hydrophobic and hydrophilic substances, such as the anti-aging and wound-healing copper-binding peptide, GHK-Cu (glycyl-L-histidyl-L-lysine). Anionic (AL) and cationic (CL) hydrogenated lecithin-based liposomes were obtained as GHK-Cu skin delivery systems using the thin-film hydration method combined with freeze-thaw cycles and the extrusion process. The influence of total lipid content, lipid composition and GHK-Cu concentration on the physicochemical properties of liposomes was studied. The lipid bilayer fluidity and the peptide encapsulation efficiency (EE) were also determined. Moreover, in vitro assays of tyrosinase and elastase inhibition were performed. Stable GHK-Cu-loaded liposome systems of small sizes (approx. 100 nm) were obtained. The bilayer fluidity was higher in the case of cationic liposomes. As the best carriers, 25 mg/cm3 CL and AL hydrated with 0.5 mg/cm3 GHK-Cu were selected with EE of 31.7 ± 0.9% and 20.0 ± 2.8%, respectively. The obtained results confirmed that the liposomes can be used as carriers for biomimetic peptides such as copper-binding peptide and that the GHK-Cu did not significantly affect the tyrosinase activity but led to 48.90 ± 2.50% elastase inhibition, thus reducing the rate of elastin degeneration and supporting the structural integrity of the skin.

Clinical and histologic findings after use of a novel combined retinol, tripeptide, and glaucine containing cream in the treatment of photo-aged skin.

BACKGROUND: Neck skin is thinner and has a more delicate dermal layer than facial skin. The studied product was specifically formulated for the neck combining a hydrating delivery system with a trifunctional corrective technology composed of 0.2% pure retinol, 2.5% tripeptide concentrate, and 5.0% glaucine complex to help improvement in signs of aging. OBJECTIVES: To evaluate cosmetic and histologic changes 3 months after treatment using immunostains for Type I collagen, Type III collagen, and glycosaminoglycan (GAGS). In addition, overall clinical improvement in photoaged skin was measured by both Griffith's photonumeric photoaging scale, photographic improvement, and questionnaires. METHODS: This study was an open-label, blinded clinical trial evaluating a combined retinol, tripeptide, and glaucine containing cream in the treatment of photo-aged skin. The study enrolled a total of 20 healthy male or female subjects, who applied the product for 3 months to their face and neck. RESULTS: Clinical as well histologic changes were consistent with improvement in all 20 subjects. CONCLUSION: Use of a combined retinol, tripeptide, and glaucine containing cream led to both clinical and histologic improvement of phototoaging.

A 90-day toxicity study of tripeptide, arginine-alanine-lysine.

There is a growing global interest in using peptides in the health industry for pharmaceuticals, cosmetics, and natural food products. Peptides contain two or more linked amino acids, whereas more than 50 amino acids are classified as polypeptides. Although there is a growing level of interest in the use of peptides in the health and wellness industry, there is a lack of literature pertaining to a specific tripeptide derived from arginine, alanine, and lysine (RAK) that is of interest for human dietary use. Therefore, a 90-day repeated-dose toxicity study was performed in rats to evaluate the subchronic oral toxicity of RAK. Eighty Han:WIST rats were administered RAK by gavage at doses of 0, 250, 500, or 1000 mg/kg bw/day. There were no mortalities or other treatment related effects, and no target organs were identified. A no-observed-adverse-effect-level (NOAEL) of 1000 mg/kg bw/day, the highest dose tested, was determined. This study will contribute to the body of research in regard to the safety of the use of RAK.

A short oxazolidine-2-one containing peptide forms supramolecular hydrogels under controlled conditions.

Low-molecular-weight hydrogels are made of a small percentage of small organic molecules dispersed in an aqueous medium, which may aggregate in several manners using different methods. However, often the organic gelator in water has poor solubility, so the addition of a solubilising agent is required. In the case of acidic gelators, this mainly consists of the addition of a strong base, that is sodium hydroxide, that deprotonates the acidic moiety, so the gelator molecules become more soluble and tend to assemble into micelles, forming a dispersion. Some gelators, however, are sensitive to the harsh pH and get hydrolysed. This is the case of some molecules presenting carbamates in their features, like Fmoc-protected or oxazolidinone-containing peptides. In this paper, we present a valid alternative to sodium hydroxide, by dissolving a tripeptide containing an oxazolidinone moiety in a phosphate buffer (PB) medium at pH 7.4. The results obtained with the NaOH dissolution are compared with the ones with PB, as both methods present advantages and drawbacks. The use of NaOH produces transparent but weak hydrogels, as it exposes the gelator to harsh conditions that end up in its partial hydrolysis, which is more pronounced at high concentrations (≥10 mM). Using PB to dissolve the gelator, this problem is completely avoided as no hydrolysis product has been detected in the hydrogels, which are very stiff although more opaque. By tuning the preparation conditions, we can obtain a wide variety of hydrogels, with the properties required by the final application.

Enhanced conformational exploration of protein loops using a global parameterization of the backbone geometry.

Flexible loops are paramount to protein functions, with action modes ranging from localized dynamics contributing to the free energy of the system, to large amplitude conformational changes accounting for the repositioning whole secondary structure elements or protein domains. However, generating diverse and low energy loops remains a difficult problem. This work introduces a novel paradigm to sample loop conformations, in the spirit of the hit-and-run (HAR) Markov chain Monte Carlo technique. The algorithm uses a decomposition of the loop into tripeptides, and a novel characterization of necessary conditions for Tripeptide Loop Closure to admit solutions. Denoting m the number of tripeptides, the algorithm works in an angular space of dimension 12 m. In this space, the hyper-surfaces associated with the aforementioned necessary conditions are used to run a HAR-like sampling technique. On classical loop cases up to 15 amino acids, our parameter free method compares favorably to previous work, generating more diverse conformational ensembles. We also report experiments on a 30 amino acids long loop, a size not processed in any previous work.

Functionalized Fluorescent Nanostructures Generated from Self-Assembly of a Cationic Tripeptide Direct Cell-Selective Chemotherapeutic Drug Delivery.

Nanodrug delivery systems (NDDs) capable of conveying chemotherapeutics directly into malignant cells without harming healthy ones are of significant interest in the field of cancer therapy. However, the development of nanostructures with the requisite biocompatibility, inherent optical properties, cellular penetration ability, encapsulation capability, and target selectivity has remained elusive. In an effort to develop cell-selective NDDs, we have synthesized a cationic tripeptide Boc-Arg-Trp-Phe-OMe (PA1), which self-assembles into well-ordered spheres in 100% aqueous medium. The inherent fluorescence properties of the peptide PA1 were shifted from the ultraviolet to the visible region by the self-assembly. These fluorescent nanostructures are proteolytically stable, photostable, and biocompatible, with characteristic blue fluorescence signals that permit us to monitor their intracellular entry in real time. We also demonstrate that these tripeptide spherical structures (TPSS) have the capacity to entrap the chemotherapeutic drug doxorubicin (Dox), shuttle the encapsulated drug within cancerous cells, and initiate the DNA damage signaling cascade, which culminates in apoptosis. Next, we functionalized the TPSS with an epithelial-cell-specific epithelial cell adhesion molecule aptamer. Aptamer-conjugated PA1 (PA1-Apt) facilitated efficient Dox delivery into the breast cancer epithelial cell line MCF7, resulting in cell death. However, cells of the human cardiomyocyte cell line AC16 were resistant to the cell killing actions of PA1-Apt. Together, these data demonstrate that not only can the self-assembly of cationic tripeptides like PA1 be exploited for efficient drug encapsulation and delivery but their unique chemistry also allows for functional modifications, which can improve the selectivity of these versatile NDDs.