Structural Analysis and Activity Correlation of Amphiphilic Cyclic Antimicrobial Peptides Derived from the [W4R4] Scaffold.

In our ongoing quest to design effective antimicrobial peptides (AMPs), this study aimed to elucidate the mechanisms governing cyclic amphiphilic AMPs and their interactions with membranes. The objective was to discern the nature of these interactions and understand how peptide sequence and structure influence antimicrobial activity. We introduced modifications into the established cyclic AMP peptide, [W4R4], incorporating an extra aromatic hydrophobic residue (W), a positively charged residue (R), or the unique 2,5-diketopiperazine (DKP). This study systematically explored the structure-activity relationships (SARs) of a series of cyclic peptides derived from the [W4R4] scaffold, including the first synthesis and evaluation of [W4R4(DKP)]. Structural, dynamic, hydrophobic, and membrane-binding properties of four cyclic peptides ([W4R4], [W5R4], [W4R5], [W4R4(DKP)]) were explored using molecular dynamics simulations within a DOPC/DOPG lipid bilayer that mimics the bacterial membrane. The results revealed distinct SARs linking antimicrobial activity to parameters such as conformational plasticity, immersion depth in the bilayer, and population of the membrane binding mode. Notably, [W4R5] exhibited an optimal "activity/binding to the bacterial membrane" pattern. This multidisciplinary approach efficiently decoded finely regulated SAR profiles, laying a foundation for the rational design of novel antimicrobial peptides.

Membrane-Active Cyclic Amphiphilic Peptides: Broad-Spectrum Antibacterial Activity Alone and in Combination with Antibiotics.

We designed a library of 24 cyclic peptides containing arginine (R) and tryptophan (W) residues in a sequential manner [RnWn] (n = 2-7) to study the impact of the hydrophilic/hydrophobic ratio, charge, and ring size on the antibacterial activity against Gram-positive and Gram-negative strains. Among peptides, 5a and 6a demonstrated the highest antimicrobial activity. In combination with 11 commercially available antibiotics, 5a and 6a showed remarkable synergism against a large panel of resistant pathogens. Hemolysis (HC50 = 340 µg/mL) and cell viability against mammalian cells demonstrated the selective lethal action of 5a against bacteria over mammalian cells. Calcein dye leakage and scanning electron microscopy studies revealed the membranolytic effect of 5a. Moreover, the stability in human plasma (t1/2 = 3 h) and the negligible ability of pathogens to develop resistance further reflect the potential of 5a for further development as a peptide-based antibiotic.

Combined Nasal, Oropharyngeal Povidone Iodine Plus Glycyrrhizic Acid Sprays, Accelerate Clinical and Laboratory Recovery and Reduces Household Transmission of SARS-CoV-2: A Randomized Placebo-Controlled Clinical Trial.

The COVID-19 pandemic is still posing challenging health and economic problems. Effective broad-spectrum antiviral therapy is urgently needed for the control of early SARS-CoV-2 infection to limit its spread and mutations. In this randomized placebo-controlled clinical study, we tested the effects of intranasal and oropharyngeal delivery of a compound of povidone-iodine 0.5% and glycyrrhizic acid 2.5 mg/ml on the laboratory (PCR) and clinical recovery from SARS-CoV-2 patients and their household contacts. 353 patients suspected of having COVID-19 infection were screened by chest CT and nasopharyngeal swab tests (PCR). 200 patients were randomly allocated to two equal groups: treatment and placebo groups. Treatment accelerated the recovery of PCR on days 4, 7, and 10, as evidenced by PCR-positive patients (70, vs. 99%, 20 vs. 65%, 1 vs. 10%) in both the treated and placebo groups, respectively. Treatment enhanced the early recovery of symptoms [day 7.6 ± 2 (CI 7:8.3) vs. 8.9 ± 2 (CI 8.3:9.6)]. Treatment promoted early recovery of anosmia and ageusia [5.6 ± 1 (CI, 4.8:6.4) vs. 11 ± 3 days, (CI, 10.8:12)] in both the treated and control groups (P < 0.0001). There was a notable reduction in transmission of the virus among the household close contacts in the treatment group (4%) vs. 76% in the placebo group. Combined PVI-GA nasal and oropharyngeal spray accelerates both laboratory and clinical recovery of SARS-CoV-2 infected patients in the early phases of the disease and reduces the household spread of the virus; thus, it may play an important role in controlling coronavirus outbreaks. Clinical Trial Registration: https://pactr.samrc.ac.za, PACTR202101875903773.

Amphiphilic cyclic peptide [W4KR5]-Antibiotics combinations as broad-spectrum antimicrobial agents.

Linear and cyclic amphiphilic peptides, (W4KR5) and [W4KR5], were evaluated as antibacterial agents against Gram-positive and Gram-negative bacteria, including four multi-drug resistant strains and the corresponding four non-resistant strains. Cyclic peptide [W4KR5] showed higher antibacterial activity than the linear (W4KR5) counterpart. Cyclic [W4KR5] was subjected to combination (physical mixture or covalent conjugation) with meropenem as a model antibiotic to study the impact of the combination on antimicrobial activity. A physical mixture of meropenem and [W4KR5] showed synergistic antibacterial activity against Gram-negative P. aeruginosa (ATCC BAA-1744) and P. aeruginosa (ATCC 27883) strains. [W4KR5] was further subjected to extensive antibacterial studies against additional 10 bacteria strains, showing significant antibacterial efficacy against Gram-positive bacteria strains. Combinations studies of [W4KR5] with an additional 9 commercially available antibiotics showed significant enhancement in antibacterial activity for all tested combinations, especially with tetracycline, tobramycin, levofloxacin, clindamycin, daptomycin, polymyxin, kanamycin, and vancomycin. Time-kill kinetics assay and flow cytometry results exhibited that [W4KR5] had a time-dependent synergistic effect and membrane disruption property. These data indicate that [W4KR5] improves the antibacterial activity, presumably by facilitating the internalization of antibiotics and their interaction with the intracellular targets. This study introduces a potential strategy for treating multidrug-resistant pathogens by combining [W4KR5] and a variety of classical antibiotics to improve the antibacterial effectiveness.

Redox-Responsive Disulfide Cyclic Peptides: A New Strategy for siRNA Delivery.

RNA interference (RNAi) is a powerful tool capable of targeting virtually any protein without time-consuming and expensive drug development studies. However, due to obstacles facing efficient and safe delivery, RNAi-based therapeutic approach remains a challenge. Herein, we have designed and synthesized a number of disulfide-constraining cyclic and hybrid peptides using tryptophan and arginine residues. Our hypothesis was that peptide structures would undergo reduction by intracellular glutathione (more abundant in cancer cells) and unpack the small interfering RNA (siRNA) from the peptide/siRNA complexes. A subset of newly developed peptides (specifically, C4 and H4) exhibited effective cellular internalization of siRNA (∼70% of the cell population; monitored by flow cytometry and confocal microscopy), the capability of protecting siRNA against early degradation by nucleases (monitored by gel electrophoresis), minimal cytotoxicity in selected cell lines (studied by cell viability and LC50 calculations), and efficient protein silencing by 70-75% reduction in the expression of targeting signal transducer and activator of transcription 3 (STAT3) in human triple-negative breast cancer (TNBC) MDA-MB-231 cells, analyzed using the Western blot technique. Our results indicate the birth of a promising new family of siRNA delivery systems that are capable of safe and efficient delivery, even in the presence of nucleases.

Cyclic Dipeptides: The Biological and Structural Landscape with Special Focus on the Anti-Cancer Proline-Based Scaffold.

Cyclic dipeptides, also know as diketopiperazines (DKP), the simplest cyclic forms of peptides widespread in nature, are unsurpassed in their structural and bio-functional diversity. DKPs, especially those containing proline, due to their unique features such as, inter alia, extra-rigid conformation, high resistance to enzyme degradation, increased cell permeability, and expandable ability to bind a diverse of targets with better affinity, have emerged in the last years as biologically pre-validated platforms for the drug discovery. Recent advances have revealed their enormous potential in the development of next-generation theranostics, smart delivery systems, and biomaterials. Here, we present an updated review on the biological and structural profile of these appealing biomolecules, with a particular emphasis on those with anticancer properties, since cancers are the main cause of death all over the world. Additionally, we provide a consideration on supramolecular structuring and synthons, based on the proline-based DKP privileged scaffold, for inspiration in the design of compound libraries in search of ideal ligands, innovative self-assembled nanomaterials, and bio-functional architectures.

Hydrophobic interactions between the HA helix and S4-S5 linker modulate apparent Ca2+ sensitivity of SK2 channels.

AIM: Small-conductance Ca2+ -activated potassium (SK) channels are activated exclusively by increases in intracellular Ca2+ that binds to calmodulin constitutively associated with the channel. Wild-type SK2 channels are activated by Ca2+ with an EC50 value of ~0.3 µmol/L. Here, we investigate hydrophobic interactions between the HA helix and the S4-S5 linker as a major determinant of channel apparent Ca2+ sensitivity. METHODS: Site-directed mutagenesis, electrophysiological recordings and molecular dynamic (MD) simulations were utilized. RESULTS: Mutations that decrease hydrophobicity at the HA-S4-S5 interface lead to Ca2+ hyposensitivity of SK2 channels. Mutations that increase hydrophobicity result in hypersensitivity to Ca2+ . The Ca2+ hypersensitivity of the V407F mutant relies on the interaction of the cognate phenylalanine with the S4-S5 linker in the SK2 channel. Replacing the S4-S5 linker of the SK2 channel with the S4-S5 linker of the SK4 channel results in loss of the hypersensitivity caused by V407F. This difference between the S4-S5 linkers of SK2 and SK4 channels can be partially attributed to I295 equivalent to a valine in the SK4 channel. A N293A mutation in the S4-S5 linker also increases hydrophobicity at the HA-S4-S5 interface and elevates the channel apparent Ca2+ sensitivity. The double N293A/V407F mutations generate a highly Ca2+ sensitive channel, with an EC50 of 0.02 µmol/L. The MD simulations of this double-mutant channel revealed a larger channel cytoplasmic gate. CONCLUSION: The electrophysiological data and MD simulations collectively suggest a crucial role of the interactions between the HA helix and S4-S5 linker in the apparent Ca2+ sensitivity of SK2 channels.

Comparative Molecular Transporter Properties of Cyclic Peptides Containing Tryptophan and Arginine Residues Formed through Disulfide Cyclization.

We have previously reported cyclic cell-penetrating peptides [WR]5 and [WR]4 as molecular transporters. To optimize further the utility of our developed peptides for targeted therapy in cancer cells using the redox condition, we designed a new generation of peptides and evaluated their cytotoxicity as well as uptake behavior against different cancer cell lines. Thus, cyclic [C(WR)xC] and linear counterparts (C(WR)xC), where x = 4-5, were synthesized using Fmoc/tBu solid-phase peptide synthesis, purified, and characterized. The compounds did not show any significant cytotoxicity (at 25 µM) against ovarian (SK-OV-3), leukemia (CCRF-CEM), gastric adenocarcinoma (CRL-1739), breast carcinoma (MDA-MB-231), and normal kidney (LLCPK) cells after 24 and 72 h incubation. Both cyclic [C(WR)5C] and linear (C(WR)5C) demonstrated comparable molecular transporter properties versus [WR]5 in the delivery of a phosphopeptide (F'-GpYEEI) in CCRF-CEM cells. The uptake of F'-GpYEEI in the presence of 1,4-dithiothreitol (DTT) as the reducing agent was significantly improved in case of l(C(WR)5C), while it was not changed by [C(WR)5C]. Fluorescence microscopy also demonstrated a significant uptake of F'-GpYEEI in the presence of l(C(WR)5C). Cyclic [C(WR)5C] improved the uptake of the fluorescent-labeled anti-HIV drugs F'-d4T, F'-3TC, and F'-FTC by 3.0-4.9-fold. These data indicate that both [C(WR)5C] and linear (C(WR)5C) peptides can act as molecular transporters.

Anticancer properties of N-alkyl-2, 4-diphenylimidazo [1, 2-a] quinoxalin-1-amine derivatives; kinase inhibitors.

Structure activity correlation revealed that the quinoxaline ring is a satisfactory backbone for anticancer activity and a specific functional group at position 1 and 2 can improve the activity. In this basis, besides quinoxaline, imidazoles as potential anticancer agents were used as a supplementary agents for cancer treatment. In this paper, a new series of N-alkyl-2, 4-diphenylimidazo [1, 2-a] quinoxalin-1-amine derivatives were synthesized in a simple and efficient step. The products are fully characterized by 1H NMR, 13C NMR, FT-IR, HRMS, and CHN elemental analysis. Several starting materials with different functionalities have been used for the synthesis of the final products with high isolated yields. The biological activities of the synthesized compounds were evaluated in kinase inhibition and cytotoxic activity in several cancerous cell lines. All compounds (6) were evaluated for inhibition of the cell proliferation using 4 cancerous cell lines. Five of the more active compounds were studied for determination of IC50%. Compounds 6(32-34) showed good activity on some of cancerous cell lines. The results showed that compound 6-32 has the highest biological activity (IC50% 9.77 for K562 cell line). An IC50% value of 15.84 µM was observed for 6-34. Furthermore 6-34 exhibited inhibition of ABL1 and c-Src kinases with an IC50% value of 5.25 µM and 3.94 µM respectively. Docking simulation was performed to position active synthesized compounds 6-32, 6-33, and 6-34 over the ABL1 active site in two different wild-type (DFG-in and DFG-out motif conformer) and T315I mutant to determine the probable binding orientation, conformation and mode of interaction. According to docking study, the docked location in wild type forms is similar and can be found near the P-loop region while in the case of T315I mutant form, the compounds have a distinct docked location which is close to the αC helix and activation loop. Also, it concluded the role of R1 substituent on phenyl ring produced higher interaction energy. Additionally, the detailed inter-molecular energy and types of non-bonding interaction of these compounds over the wild-type and mutant form of ABL1.