Broad-spectrum activity of membranolytic cationic macrocyclic peptides against multi-drug resistant bacteria and fungi.

The emergence of multidrug-resistant (MDR) strains causes severe problems in the treatment of microbial infections owing to limited treatment options. Antimicrobial peptides (AMPs) are drawing considerable attention as promising antibiotic alternative candidates to combat MDR bacterial and fungal infections. Herein, we present a series of small amphiphilic membrane-active cyclic peptides composed, in part, of various nongenetically encoded hydrophilic and hydrophobic amino acids. Notably, lead cyclic peptides 3b and 4b showed broad-spectrum activity against drug-resistant Gram-positive (MIC = 1.5-6.2 µg/mL) and Gram-negative (MIC = 12.5-25 µg/mL) bacteria, and fungi (MIC = 3.1-12.5 µg/mL). Furthermore, lead peptides displayed substantial antibiofilm action comparable to standard antibiotics. Hemolysis (HC50 = 230 µg/mL) and cytotoxicity (>70 % cell viability against four different mammalian cells at 100 µg/mL) assay results demonstrated the selective lethal action of 3b against microbes over mammalian cells. A calcein dye leakage experiment substantiated the membranolytic effect of 3b and 4b, which was further confirmed by scanning electron microscopy. The behavior of 3b and 4b in aqueous solution and interaction with phospholipid bilayers were assessed by employing nuclear magnetic resonance (NMR) spectroscopy in conjunction with molecular dynamics (MD) simulations, providing a solid structural basis for understanding their membranolytic action. Moreover, 3b exhibited stability in human blood plasma (t1/2 = 13 h) and demonstrated no signs of resistance development against antibiotic-resistant S. aureus and E. coli. These findings underscore the potential of these newly designed amphiphilic cyclic peptides as promising anti-infective agents, especially against Gram-positive bacteria.

Modifying peptide/lipid-associated nucleic acids (PLANAs) for CRISPR/Cas9 ribonucleoprotein delivery.

With the first reports on the possibility of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas)9 surfacing in 2005, the enthusiasm for protein silencing via nucleic acid delivery experienced a resurgence following a period of diminished enthusiasm due to challenges in delivering small interfering RNAs (siRNA), especially in vivo. However, delivering the components necessary for this approach into the nucleus is challenging, maybe even more than the cytoplasmic delivery of siRNA. We previously reported the birth of peptide/lipid-associated nucleic acids (PLANAs) for siRNA delivery. This project was designed to investigate the efficiency of these nanoparticles for in vitro delivery of CRISPR/Cas9 ribonucleoproteins. Our initial experiments indicated higher toxicity for PLANAs with the more efficient reverse transfection method. Therefore, polyethylene glycol (PEG) was added to the composition for PEGylation of the nanoparticles by partially replacing two of the lipid components with the PEG-conjugated counterparts. The results indicated a more significant reduction in the toxicity of the nanoparticle, less compromise in encapsulation efficiency and more PEGylation of the surface of the nanoparticles using DOPE-PEG2000 at 50 % replacement of the naïve lipid. The cell internalization and transfection efficiency showed a comparable efficiency for the PEGylated and non-PEGylated PLANAs and the commercially available Lipofectamine™ CRISPRMAX™. Next Generation Sequencing of the cloned cells showed a variety of indels in the transfected cell population. Overall, our results indicate the efficiency and safety of PEGylated PLANAs for in vitro transfection with CRISPR/Cas9 ribonucleoproteins. PEGylation has been studied extensively for in vivo delivery, and PEGylated PLANAs will be candidates for future in vivo studies.

Development of a Cyclic, Cell Penetrating Peptide Compatible with In Vitro Selection Strategies.

A key limitation for the development of peptides as therapeutics is their lack of cell permeability. Recent work has shown that short, arginine-rich macrocyclic peptides containing hydrophobic amino acids are able to penetrate cells and reach the cytosol. Here, we have developed a new strategy for developing cyclic cell penetrating peptides (CPPs) that shifts some of the hydrophobic character to the peptide cyclization linker, allowing us to do a linker screen to find cyclic CPPs with improved cellular uptake. We demonstrate that both hydrophobicity and position of the alkylation points on the linker affect uptake of macrocyclic cell penetrating peptides (CPPs). Our best peptide, 4i, is on par with or better than prototypical CPPs Arg9 (R9) and CPP12 under assays measuring total cellular uptake and cytosolic delivery. 4i was also able to carry a peptide previously discovered from an in vitro selection, 8.6, and a cytotoxic peptide into the cytosol. A bicyclic variant of 4i showed even better cytosolic entry than 4i, highlighting the plasticity of this class of peptides toward modifications. Since our CPPs are cyclized via their side chains (as opposed to head-to-tail cyclization), they are compatible with powerful technologies for peptide ligand discovery including phage display and mRNA display. Access to diverse libraries with inherent cell permeability will afford the ability to find cell permeable hits to many challenging intracellular targets.

Modified Linear Peptides Effectively Silence STAT-3 in Breast Cancer and Ovarian Cancer Cell Lines.

RNA interference (RNAi) has drawn enormous attention as a powerful tool because of its capability to interfere with mRNA and protein production. However, designing a safe and efficient delivery system in RNAi therapeutics remains challenging. Herein, we have designed and synthesized several linear peptides containing tryptophan (W) and arginine (R) residues separated by the ß-alanine (ßA) spacer and attached to a lipophilic fatty acyl chain, cholesterol, or PEG. The peptide backbone sequences were: Ac-C-ßA-ßA-W4-ßA-ßA-R4-CO-NH2 and Ac-K-ßA-ßA-W4-ßA-ßA-R4-CO-NH2, with only a difference in N-terminal amino acid. The cysteine side chain in the first sequence was used for the conjugation with PEG2000 and PEG550. Alternatively, the side chain of lysine in the second sequence was used for conjugation with cholesterol or oleic acid. We hypothesized that amphiphilic peptides and optimum fatty acyl chain or PEG could function as an effective siRNA carrier by complementing each structural component's self-assembly and membrane internalization properties. None of the designed peptides showed cytotoxicity up to 10 µM. Serum stability studies suggested that the newly designed peptides efficiently protected siRNA against early degradation by nucleases. Flow cytometry analysis indicated 50-90% cellular uptake of siRNA using the newly developed modified linear peptides (MLPs). Western blot results revealed more than 90% protein downregulation after targeting STAT3 in MDA-MB-231 and SKOV-3 cell lines. In summary, a new peptide class was developed to safely and efficiently deliver siRNA.

Cyclic and Linear Peptides Containing Alternate WW and RR Residues as Molecular Cargo Delivery Tools.

Cell-impermeable and negatively charged compounds' cellular uptake across the cell membranes remains challenging. Herein, the synthesis of four linear [(WWRR)2, (WWRR)3, (WWRR)4, and (WWRR)5] and four cyclic ([WWRR]2, [WWRR]3, [WWRR]4, and [WWRR]5) peptides containing alternate two tryptophan (WW) and two arginine (RR) residues and their biological evaluation as molecular transporters are reported. The peptides did not show any significant cytotoxicity in different cell lines (MDA-MB-23, SK-OV-3, and HEK 293) at a concentration of 5 µM and after 3 h of incubation time. The uptake of fluorescence-labeled cargo molecules (F'-GpYEEI, F'-siRNA, and F'-3TC) in the presence of the peptides was monitored in different cell lines (SK-OV-3 and MDA-MB-231) with fluorescence-activated cell sorting. Among all the peptides, [WWRR]5 (C4) showed the highest cellular uptake of cargo molecules, indicating it can act as effective molecular transporter. Confocal microscopy in MDA-MB-231 cells showed the cellular uptake of F'-GpYEEI in the presence of C4 and the intracellular localization of fluorescence-labeled C4 (F'-C4) in the cytosol. The F'-C4 cellular uptake was found to be concentration- and time-dependent, as shown by flow cytometry in MDA-MB-231 cells. Confocal microscopy and flow cytometry of F'-C4 in MDA-MB-231 cells were examined alone and in the presence of different endocytosis inhibitors (chlorpromazine, methyl-ß-cyclodextrin, chloroquine, and nystatin). The data showed that the cellular uptake of F'-C4 in the presence of chlorpromazine, chloroquine, and methyl-ß-cyclodextrin was reduced but not completely eliminated, indicating that both energy-independent and energy-dependent pathways contributed to the cellular uptake of F'-C4. Similar results were obtained using the confocal microscopy of C4 and F'-GpYEEI in the presence of endocytosis inhibitors (chlorpromazine, methyl-ß-cyclodextrin, chloroquine, and nystatin). These data indicate that C4 has the potential to be used as a cell-penetrating peptide and cargo transporter.

Structure-Based Rational Design of Small α-Helical Peptides with Broad-Spectrum Activity against Multidrug-Resistant Pathogens.

A series of small (7-12 mer) amphipathic cationic peptides were designed and synthesized to create short helical peptides with broad-range bactericidal activity and selectivity toward the bacterial cells. The analysis identified a lead 12-mer peptide 8b with broad-spectrum activity against Gram-positive (MIC = 3.1-6.2 µg/mL) and Gram-negative (MIC = 6.2-12.5 µg/mL) bacteria and selectivity toward prokaryotic versus eukaryotic cells (HC50 = 280 µg/mL, >75% cell viability at 150 µg/mL). The rapid membranolytic action of 8b was demonstrated by a calcein dye leakage assay and confirmed using scanning electron microscopy. According to circular dichroism and NMR spectroscopy, the peptides have an irregular spatial structure in water. A lipid bilayer induced an amphipathic helix only in 12-mer peptides, including 8b. Molecular dynamics simulations provided detailed information about the interaction of 8b and its closest analogues with bacterial and mammalian membranes and revealed the roles of particular amino acids in the activity and selectivity of 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.

Oleyl Conjugated Histidine-Arginine Cell-Penetrating Peptides as Promising Agents for siRNA Delivery.

Recent approvals of siRNA-based products motivated the scientific community to explore siRNA as a treatment option for several intractable ailments, especially cancer. The success of approved siRNA therapy requires a suitable and safer drug delivery agent. Herein, we report a series of oleyl conjugated histidine-arginine peptides as a promising nonviral siRNA delivery tool. The conjugated peptides were found to bind with the siRNA at N/P ratio ≥ 2 and demonstrated complete protection for the siRNA from early enzymatic degradation at N/P ratio ≥ 20. Oleyl-conjugated peptide -siRNA complexes were found to be noncytotoxic in breast cancer cells (MCF-7 and MDA-MB-231) and normal breast epithelial cells (MCF 10A) at N/P ratio of ~40. The oleyl-R3-(HR)4 and oleyl-R4-(HR)4 showed ~80-fold increased cellular uptake in MDA-MB-231 cells at N/P 40. Moreover, the conjugated peptides-siRNA complexes form nanocomplexes (~115 nm in size) and have an appropriate surface charge to interact with the cell membrane and cause cellular internalization. Furthermore, this study provides a proof-of-concept that oleyl-R5-(HR)4 can efficiently silence STAT-3 gene (~80% inhibition) in MDA-MB-231 cells with similar effectiveness to Lipofectamine. Further exploration of this approach holds a great promise in discovering a successful in vivo siRNA delivery agent with a favorable pharmacokinetic profile.

Amphiphilic Cell-Penetrating Peptides Containing Natural and Unnatural Amino Acids as Drug Delivery Agents.

A series of cyclic peptides, [(DipR)(WR)4], [(DipR)2(WR)3], [(DipR)3(WR)2], [(DipR)4(WR)], and [DipR]5, and their linear counterparts containing arginine (R) as positively charged residues and tryptophan (W) or diphenylalanine (Dip) as hydrophobic residues, were synthesized and evaluated for their molecular transporter efficiency. The in vitro cytotoxicity of the synthesized peptides was determined in human epithelial ovary adenocarcinoma cells (SK-OV-3), human lymphoblast peripheral blood cells (CCRF-CEM), human embryonic epithelial kidney healthy cells (HEK-293), human epithelial mammary gland adenocarcinoma cells (MDA-MB-468), pig epithelial kidney normal cells (LLC-PK1), and human epithelial fibroblast uterine sarcoma cells (MES-SA). A concentration of 5-10 µM and 3 h incubation were selected in uptake studies. The cellular uptake of a fluorescent-labeled phosphopeptide, stavudine, lamivudine, emtricitabine, and siRNA was determined in the presence of peptides via flow cytometry. Among the peptides, [DipR]5 (10 µM) was found to be the most efficient transporter and significantly improved the uptake of F'-GpYEEI, i.e., by approximately 130-fold after 3 h incubation in CCRF-CEM cells. Confocal microscopy further confirmed the improved delivery of fluorescent-labeled [DipR]5 (F'-[K(DipR)5]) alone and F'-GpYEEI in the presence of [DipR]5 in MDA-MB-231 cells. The uptake of fluorescent-labeled siRNA (F'-siRNA) in the presence of [DipR]5 with N/P ratios of 10 and 20 was found to be 30- and 50-fold higher, respectively, compared with the cells exposed to F'-siRNA alone. The presence of endocytosis inhibitors, i.e., nystatin, chlorpromazine, chloroquine, and methyl ß-cyclodextrin, did not completely inhibit the cellular uptake of F'-[K(DipR)5] alone or F'-GpYEEI in the presence of [DipR]5, suggesting that a combination of mechanisms contributes to uptake. Circular dichroism was utilized to determine the secondary structure, while transmission electron microscopy was used to evaluate the particle sizes and morphology of the peptides. The data suggest the remarkable membrane transporter property of [DipR]5 for improving the delivery of various small molecules and cell-impermeable negatively charged molecules (e.g., siRNA and phosphopeptide).

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.

Combination of Amphiphilic Cyclic Peptide [R4W4] and Levofloxacin against Multidrug-Resistant Bacteria.

Bacterial resistance is a growing global concern necessitating the discovery and development of antibiotics effective against the drug-resistant bacterial strain. Previously, we reported a cyclic antimicrobial peptide [R4W4] containing arginine (R) and tryptophan (W) with a MIC of 2.67 µg/mL (1.95 µM) against methicillin-resistant Staphylococcus aureus (MRSA). Herein, we investigated the cyclic peptides [R4W4] or linear (R4W4) and their conjugates (covalent or noncovalent) with levofloxacin (Levo) with the intent to improve their potency to target drug-resistant bacteria. The physical mixture of the Levo with the cyclic [R4W4] proved to be significantly effective against all strains of bacteria used in the study as compared to covalent conjugation. Furthermore, the checkerboard assay revealed the significant synergistic effect of the peptides against all studied strains except for the wild type S. aureus, in which the partial synergy was observed. The hemolysis assay revealed less cytotoxicity of the physical mixture of the Levo with [R4W4] (22%) as compared to [R4W4] alone (80%). The linear peptide (R4W4) and the cyclic [R4W4] demonstrated ~90% and 85% cell viability at 300 µg/mL in the triple-negative breast cancer cells (MDA-MB-231) and the normal kidney cells (HEK-293), respectively. Similar trends were also observed in the cell viability of Levo-conjugates on these cell lines. Furthermore, the time-kill kinetic study of the combination of [R4W4] and Levo demonstrate rapid killing action at 4 h for MRSA (ATCC BAA-1556) and 12 h for E. coli (ATCC BAA-2452), P. aeruginosa (ATCC BAA-1744), and K. pneumoniae (ATCC BAA-1705). These results provide the effectiveness of a combination of Levo with cyclic [R4W4] peptide, which may provide an opportunity to solve the intriguing puzzle of treating bacterial resistance.

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.

Small Amphiphilic Peptides: Activity Against a Broad Range of Drug-Resistant Bacteria and Structural Insight into Membranolytic Properties.

We report the synthesis and antibacterial activities of a series of amphiphilic membrane-active peptides composed, in part, of various nongenetically coded hydrophobic amino acids. The lead cyclic peptides, 8C and 9C, showed broad-spectrum activity against drug-resistant Gram-positive (minimum inhibitory concentration (MIC) = 1.5-6.2 µg/mL) and Gram-negative (MIC = 12.5-25 µg/mL) bacteria. The cytotoxicity study showed the predominant lethal action of the peptides against bacteria as compared with mammalian cells. A plasma stability study revealed approximately 2-fold higher stability of lead cyclic peptides as compared to their linear counterparts after 24 h of incubation. A calcein dye leakage experiment revealed the membranolytic effect of the cyclic peptides. Nuclear magnetic resonance spectroscopy and molecular dynamics simulation studies of the interaction of the peptides with the phospholipid bilayer provided a solid structural basis to explain the membranolytic action of the peptides with atomistic details. These results highlight the potential of newly designed amphiphilic peptides as the next generation of peptide-based antibiotics.

Cyclic Peptide-Gadolinium Nanocomplexes as siRNA Delivery Tools.

We have recently reported that a cyclic peptide containing five tryptophan, five arginine, and one cysteine amino acids [(WR)5C], was able to produce peptide-capped gadolinium nanoparticles, [(WR)5C]-GdNPs, in the range of 240 to 260 nm upon mixing with an aqueous solution of GdCl3. Herein, we report [(WR)5C]-GdNPs as an efficient siRNA delivery system. The peptide-based gadolinium nanoparticles (50 µM) did not exhibit significant cytotoxicity (~93% cell viability at 50 µM) in human leukemia T lymphoblast cells (CCRF-CEM) and triple-negative breast cancer cells (MDA-MB-231) after 48 h. Fluorescence-activated cell sorting (FACS) analysis indicated that the cellular uptakes of Alexa-488-labeled siRNA were found to be enhanced by more than 10 folds in the presence of [(WR)5C]-GdNPs compared with siRNA alone in CCRF-CEM and MDA-MB-231 cells after 6 h of incubation at 37 °C. The gene silencing efficacy of the nanoparticles was determined via the western blot technique using an over-expressed gene, STAT-3 protein, in MDA-MB-231 cells. The results showed ~62% reduction of STAT-3 was observed in MDA-MB-231 with [(WR)5C]-GdNPs at N/P 40. The integrity of the cellular membrane of CCRF-CEM cells was found to be intact when incubated with [(WR)5C]-Gd nanoparticles (50 µM) for 2 h. Confocal microscopy reveals higher internalization of siRNA in MDA-MB-231 cells using [(WR)5C]-GdNPs at N/P 40. These results provided insight about the use of the [(WR)5C]-GdNPs complex as a potent intracellular siRNA transporter that could be a nontoxic choice to be used as a transfection agent for nucleic-acid-based therapeutics.

Synthesis and antiviral activity of fatty acyl conjugates of remdesivir against severe acute respiratory syndrome coronavirus 2 and Ebola virus.

We report here the synthesis, purification, and characterization of mono- and di-fatty acyl conjugates of remdesivir (RDV) and their in vitro antiviral activity against SAR-CoV-2, an Ebola virus transcription- and replication-competent virus-like particle (trVLP) system, and infectious Ebola virus. The most potent monofatty acyl conjugate was 4b, containing a 4-oxatetradecanolyl at the 3' position. Monofatty acyl conjugates, 3'-O-tetradecanoyl (4a) (IC50(VeroE6) = 2.3 µM; IC50(Calu3) = 0.24 µM), 3'-O-4-oxatetradodecanoyl (4b) (IC50(VeroE6) = 2.0 µM; IC50(Calu3) = 0.18 µM), and 3'-O-(12-ethylthiododecanoyl) (4e) (IC50(VeroE6) = 2.4 µM; IC50(Calu3) = 0.25 µM) derivatives exhibited less activity than RDV (IC50(VeroE6) = 0.85 µM; IC50(Calu3) = 0.06 µM) in both VeroE6 and Calu3 cells. Difatty acylation led to a significant reduction in the antiviral activity of RDV (as shown in conjugates 5a and 5b) against SARS-CoV-2 when compared with monofatty acylation (3a-e and 4a-e). About 77.9% of 4c remained intact after 4 h incubation with human plasma while only 47% of parent RDV was observed at the 2 h time point. The results clearly indicate the effectiveness of fatty acylation to improve the half-life of RDV. The antiviral activities of a number of monofatty acyl conjugates of RDV, such as 3b, 3e, and 4b, were comparable with RDV against the Ebola trVLP system. Meanwhile, the corresponding physical mixtures of RDV and fatty acids 6a and 6b showed 1.6 to 2.2 times less antiviral activity than the corresponding conjugates, 4a and 4c, respectively, against SARS-CoV-2 in VeroE6 cells. A significant reduction in viral RNA synthesis was observed for selected compounds 3a and 4b consistent with the IC50 results. These studies indicate the potential of these compounds as long-acting antiviral agents or prodrugs of RDV.

Targeted Delivery of Cabazitaxel Using Cyclic Cell-Penetrating Peptide and Biomarkers of Extracellular Matrix for Prostate and Breast Cancer Therapy.

Targeted drug delivery for cancer therapy is an emerging area of research. Cancer cells overexpress certain biomarkers that can be exploited for their targeted therapy. Cyclic cell-penetrating peptides (cCPP) are increasingly assessed for intracellular cargo delivery in cancer cells. In this study, we have conjugated cabazitaxel (CBT) to the cCPP via an ester bond to assist CBT release in the tumor's acidic environment. Integrin targeting (RGDC, TP1) and extra domain B of fibronectin (EDB-Fn) targeting (CTVRTSAD, TP2) peptides were linked to the peptide-drug conjugate (cCPP-CBT) via a disulfide bond to provide targeting ability to the conjugates until they reach the tumor site. Conjugate 11 (TP1-cCPP-CBT) and conjugate 16 (TP2-cCPP-CBT) showed approximately 3-4-fold less antiproliferative activity on integrin and EDB-FN overexpressing cancer cell lines as compared to the CBT analogue used for comparison (CBT-GA, 5). Conjugates (11 and 16) were less toxic (31-34-fold less antiproliferative activity) to the normal human embryonic kidney (HEK-293) cells as compared to CBT. The flow cytometry and quantitative confocal microscopy data further confirm the selective efficacy of conjugates (TP1-cCPP-FAM (10) and TP1-cCPP-FAM (15)) toward biomarker overexpressing cancer cells. Furthermore, the stability and release studies of conjugate 11 revealed its therapeutic potential under different conditions, such as human plasma, different pHs, and redox conditions. This conjugation strategy was proven to enhance chemotherapeutics agents' efficacy and targeting and can be applied to other chemotherapeutic agents.

siRNA Therapeutics for the Therapy of COVID-19 and Other Coronaviruses.

The ongoing pandemic of global concern has killed about three million humans and affected around 151 million people worldwide, as of April 30, 2021. Although recently approved vaccines for COVID-19 are engendering hope, finding new ways to cure the viral pandemic is still a quest for researchers worldwide. Major pandemics in history have been of viral origin, such as SARS, MERS, H1NI, Spanish flu, and so on. A larger emphasis has been on discovering potential vaccines, novel antiviral drugs, and agents that can mitigate the viral infection symptoms; however, a relatively new area, RNA interference (RNAi), has proven effective as an antiviral agent. The RNAi phenomenon has been largely exploited to cure cancer, neurodegenerative diseases, and some rare diseases. The U.S. Food and Drug Administration has recently approved three siRNA products for human use that garner significant hope in siRNA therapeutics for coronaviruses. There have been some commentaries and communications addressing this area. We have summarized and illustrated the significance and the potential of the siRNA therapeutics available as of April 30, 2021 to combat the ongoing viral pandemic and the emerging new variants such as B.1.1.7 and B.1.351. Numerous successful in vitro studies and several investigations to address the clinical application of siRNA therapeutics provide great hope in this field. This seminal Review describes the significance of siRNA-based therapy to treat diverse viral infections in addition to the current coronavirus challenge. In addition, we have thoroughly reviewed the patents approved for coronaviruses, the major challenges in siRNA therapy, and the potential approaches to address them, followed by innovation and prospects.

Cyclic Peptide-Gadolinium Nanoparticles for Enhanced Intracellular Delivery.

A cyclic peptide containing one cysteine and five alternating tryptophan and arginine amino acids [(WR)5C] was synthesized using Fmoc/tBu solid-phase methodology. The ability of the synthesized cyclic peptide to produce gadolinium nanoparticles through an in situ one-pot mixing of an aqueous solution of GdCl3 with [(WR)5C] peptide solution was evaluated. Transmission electron microscopy showed the formed peptide-Gd nanoparticles in star-shape morphology with a size of ~250 nm. Flow cytometry investigation showed that the cellular uptake of a cell-impermeable fluorescence-labeled phosphopeptide (F'-GpYEEI, where F' = fluorescein) was approximately six times higher in the presence of [(WR)5C]-Gd nanoparticles than those of F'-GpYEEI alone in human leukemia adenocarcinoma (CCRF-CEM) cells after 2 h incubation. The antiproliferative activities of cisplatin and carboplatin (5 µM) were increased in the presence of [(WR)5C]-GdNPs (50 µM) by 41% and 18%, respectively, after 72-h incubation in CCRF-CEM cells. The intracellular release of epirubicin, an anticancer drug, from the complex showed that 15% and 60% of the drug was released intracellularly within 12 and 48 h, respectively. This report provides insight about using a non-toxic MRI agent, gadolinium nanoparticles, for the delivery of various types of molecular cargos.

In Vitro and In Vivo Evaluation of Small Cationic Abiotic Lipopeptides as Novel Antifungal Agents.

We investigated the antifungal potential of short lipopeptides against clinical fungal isolates with an objective to evaluate their clinical feasibility. All tested lipopeptides exhibit good antifungal activity with negligible difference between the MICs against susceptible and drug-resistant clinical fungal isolates. The MTT assay results revealed the lower cytotoxicity of lipopeptides toward mammalian cells (NRK-52E). In particular, LP24 displayed highest potency against most of the tested fungal isolates with MICs in the range of 1.5-4.5 µg/mL. Calcein dye leakage experiments with model membrane suggested the membrane-active mode of action for LP24. Extending our work from model membranes to intact Aspergillus fumigatus in scanning electron micrographs, we could visualize surface perturbation caused by LP24. LP24 (5 mg/kg) significantly reduces the A. fumigatus burden among the various organs of infected animals, and 70% of the infected mice survived when observed for 28 days. This study underscores the potential of small cationic abiotic lipopeptides to develop into the next-generation antimicrobial therapy.

Development of novel membrane active lipidated peptidomimetics active against drug resistant clinical isolates.

A new series of small cationic lipidated peptidomimetics have been synthesized and found to be highly active against several susceptible as well as drug resistant clinical isolates of bacteria and fungi. All lipidated peptidomimetics do not cause significant lysis of human erythrocytes (HC50>200µg/mL). Calcein dye leakage experiment revealed membranolytic effect of LPEP08 which was further confirmed by scanning electron microscopy (SEM). The involvement of intracellular targets as an alternate mode of action was precluded by DNA retardation assay. Additionally, LPEP08 exhibit high proteolytic stability and dose not elicit resistance against drug resistant clinical isolate of Staphylococcusaureus, even after 16 rounds of passaging. These results demonstrate the potential of lipidated peptidomimetics as biocompatible anti-infective therapeutics.