Maojun Zhou

Conotoxins exhibit great potential as neuropharmacology tools and therapeutic candidates due to their high affinity and specificity for ion channels, neurotransmitter receptors or transporters. The traditional methods to discover new conotoxins are peptide purification from the crude venom or gene amplification from the venom duct.

Conotoxins are promising neuropharmacological tools and drug candidates due to their high efficiency and specificity in targeting ion channels or neurotransmitter receptors. In this study, a novel O₂‐superfamily conotoxin, Lt7b, was synthesized and its pharmacological functions were evaluated. Lt7b with three modified amino acids and three disulfide bonds was successfully synthesized. CD spectra showed that Lt7b had a typical α‐helix in the secondary structure. Patch clamp experiments on rat DRG neurons showed that Lt7b could significantly inhibit calcium currents with an IC₅₀ value of 856 ± 95 nM. Meanwhile, 10 μM Lt7b could significantly increase the sodium currents by 77 ± 8%, but it had no obvious effects on the potassium currents in DRG neurons. In addition, patch clamp experiments on ion channel subtypes showed that 10 μM Lt7b could inhibit 7.0 ± 1.2%, 8.0 ± 1.5%, 4.6 ± 3.4%, and 9.5 ± 0.1% of the hCa_v1.2, hCa_v2.1, hCa_v2.2, and hCa_v3.2 currents, respectively, while it did not increase the rNa_v1.7, rNa_v1.8, hNa_v1.5, hNa_v1.7, and hNa_v1.8 currents. Lt7b had no obvious toxicity to HaCaT and ND7/23 cells up to 1 mM and significantly increased the pain threshold at the testing time of 0.5–4 h in a dose‐dependent manner in the mouse hotplate assay. This novel conotoxin Lt7b may be a useful tool for ion channel studies and analgesic drug development.

Skin aging usually leads to the excessive deterioration of the dermal extracellular matrix, loss of antimicrobial function, loss of skin barrier function, and a series of inflammatory processes. Bioactive peptides have been widely used in cosmetics due to their protective effects on skin and efficient absorption. Combination of different peptides may lead to synergistic or antagonistic effects, so different formulas need to be designed and tested properly. In this study, 5 functional cosmeceutical peptides were tested on their individual and mixed activities to detect a suitable anti-aging and protective formula from our experiments. After the individual activity test, the optimal concentration is 200 μg/mL of carnosine for the superoxide dismutase (SOD) activity, 200 μg/mL of GHK peptide for the hydroxyproline (HYP) content activity, 100 μg/mL of acetyl tetrapeptide-5 for the angiotensin-converting enzyme 1 activity, 400 μg/mL of hexapeptide-11 for the HYP content activity, and 400 μg/mL of acetyl hexapeptide-3 for the catecholamine content activity. According to the optimal concentration of these 5 cosmeceutical peptides, 6 formulations of peptide mixtures were designed and tested for their anti-aging activities and protective effects against hydrogen peroxide-induced premature senescence in human skin fibroblasts. One of the cosmeceutical peptide mixtures (carnosine + acetyl tetrapeptide-5 + hexapeptide-11 + acetyl hexapeptide-3) significantly reduced the intracellular malondialdehyde and hydroxyl free radical contents and increased the HYP and human elastin contents as well as the enzymatic activities of SOD and glutathione peroxidase. Our study suggests that this formula of cosmeceutical peptide mixtures could be a promising agent for use in anti-aging and protective cosmetics.

Conotoxins, which target ion channels or neurotransmitter receptors with high specificity, are valuable in drug development for pain, epilepsy and other neurological diseases. However, the toxicology of conotoxins is rarely reported. In this study, we primarily researched parts of the pharmacological and toxicological properties of an analgesic conotoxin lt14a. Three doses of lt14a (1, 5 and 10 mg/kg) could prolong the pentobarbital‐induced sleep time of mice and showed no significant effect on the spontaneous locomotor activity of mice. Three doses of lt14a (50, 100 and 200 mg/kg) did not increase micronucleus rate in the micronucleus test. In addition, three doses of lt14a (200, 500 and 1000 mg/kg) showed no pathological change on the heart or brain of mice in the acute toxicity test. The high dose of lt14a (1000 times the effective analgesic dose) had a certain damaging effect on the liver and lung according to serological detection and histopathology. As part of the preclinical studies, our results provide acute toxicity and mutagenicity evaluation of the promising analgesic conotoxin lt14a.

Conotoxins have become a research hotspot in the neuropharmacology field for their high activity and specificity in targeting ion channels and neurotransmitter receptors. There have been reports of a conotoxin acting on two ion channels, but rare reports of a conotoxin acting on three ion channels. Vr3a, a proline-rich M-superfamily conotoxin from a worm-hunting Conus varius, was obtained by solid-phase synthesis and identified by mass spectrometry. The effects of synthesized Vr3a on sodium, potassium and calcium currents were tested on rat DRG cells by patch clamp experiments. The further effects of Vr3a on human Cav1.2 and Cav2.2 currents were tested on HEK293 cells. About 10 μM Vr3a has no effects on the peak sodium currents, but can induce a ~10 mV shift in a polarizing direction in the current-voltage relationship. In addition, 10 μM Vr3a can increase 19.61 ± 5.12% of the peak potassium currents and do not induce a shift in the current-voltage relationship. An amount of 10 μM Vr3a can inhibit 31.26% ± 4.53% of the peak calcium currents and do not induce a shift in the current-voltage relationship. The IC50 value of Vr3a on calcium channel currents in rat DRG neurons is 19.28 ± 4.32 μM. Moreover, 10 μM Vr3a can inhibit 15.32% ± 5.41% of the human Cav1.2 currents and 12.86% ± 4.93% of the human Cav2.2 currents. Vr3a can simultaneously affect sodium, potassium and calcium currents. This novel triple-target conotoxin Vr3a expands understanding of conotoxin functions.

Proliferation of Propionibacterium acnes (P. acnes) is one of the main pathogenetic mechanisms of acne. Antimicrobial peptides with low‐drug resistance and nonresidual are potential anti‐acne agents. In this study, two antimicrobial peptides named temporin‐1Dra and moronecidin were synthesized and tested their antimicrobial activity against P. acnes in vitro and in vivo. These two peptides inhibited the growth of Escherichia coli, Staphylococcus aureus, Candida albicans, and P. acnes. The minimal inhibitory concentrations (MICs) of temporin‐1Dra and moronecidin to P. acnes were 30 and 10 μM, respectively. Both peptides exhibited strong resistance to heat and pH, but no obvious cytotoxicity to HaCaT cells. They also displayed persistent antimicrobial activities in the microbial challenge test. In the P. acnes‐induced inflammation mouse model, moronecidin significantly decreased the ear swelling thickness in a concentration‐dependent manner. At the 14th day after injection, 20 μg/day moronecidin reduced the ear swelling thickness to 46.15 ± 5.23% compared with the normal cream group. Tissue staining showed that moronecidin effectively reduced abscess and thickness of the dermis layer. Our results indicate that the antimicrobial peptide moronecidin could be developed as a potential natural anti‐acne agent in the cosmetics or pharmaceutical industries.

Previous studies have indicated that each conotoxin precursor has a hyperconserved signal region, a rather conserved pro region and a hypervariable mature region, and nucleotide mutations are the main driven factor. However, in this study, we made an in-depth analysis on the M-superfamily conotoxin precursors and found that the diversity of the signal, pro and mature regions are more complicated than previous findings. Different conotoxin precursors can have same signal, pro and/or mature regions, especially different conotoxin precursors with same mature region but different signal and pro regions. In addition, insertions and deletions (indels) were detected in conotoxin precursors. Indels are infrequent in the signal region but frequent in the pro and mature regions. In contrast to deletions that dominate in the pro region, insertions dominate in the mature region. The number of amino acids is crucial for the physiological functions of mature conotoxins, therefore indels, especially insertions in the mature region, play an important role in the sequence and function diversity of conotoxins.