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Cytosolic delivery of functional siRNA remains the major challenge to develop siRNA-based therapeutics. Designing clinically safe and effective siRNA transporter to deliver functional siRNA across the plasma and endosomal membrane remains a key hurdle. With the aim of improving endosomal release, we have designed cyclic and linear peptide-based transporters having an Arg-DHis-Arg template. Computational studies show that the Arg-DHis-Arg template is also stabilized by the Arg-His side-chain hydrogen bonding interaction at physiological pH, which dissociates at lower pH. The overall atomistic interactions were examined by molecular dynamics simulations, which indicate that the extent of peptide_siRNA assembly formation depends greatly on physicochemical properties of the peptides. Our designed peptides having the Arg-DHis-Arg template and two lipidic moieties facilitate high yield of intracellular delivery of siRNA. Additionally, unsaturated lipid, linoleic acid moieties were introduced to promote fusogenicity and facilitate endosomal release and cytosolic delivery. Interestingly, such protease-resistant peptides provide serum stability to siRNA and exhibit high efficacy of erk1 and erk2 gene silencing in the triple negative breast cancer (TNBC) cell line. The peptide having two linoleyl moieties demonstrated comparable efficacy with commercial transfection reagent HiPerFect, as evidenced by the erk1 and erk2 gene knockdown experiment. Additionally, our study shows that ERK1/2 silencing siRNA and doxorubicin-loaded gramicidin-mediated combination therapy is more effective than siRNA-mediated gene silencing-based monotherapy for TNBC treatment.

Peptides have emerged as powerful platform technologies to engineer effective therapies against cancer. Peptides play significant role as clinically important molecules in developing next-generation cancer therapeutics by targeting signalling pathways, cell cycle, tumour suppressor protein, transcription factor, ion channels, several G-protein-coupled receptors and matrix metalloproteinases MMP 2 and MMP 9. Peptide-based cancer vaccines also hold huge promise in cancer treatment. Peptide-based biomarkers have immense potential to facilitate the early detection of cancer and thus increase the lifespan of cancer patients. Additionally, peptides also act as safe clinically suitable molecular medicine transporters for cancer treatment. Cell-penetrating cationic peptides, lipopeptides, amphipathic peptides and mini-proteins facilitate nucleic acid-based drug delivery and can be exploited for developing siRNA-based nanotherapeutics and combination therapy against cancer. Peptide-based medicines and molecular transporters play significant role in managing adaptive chemotherapeutic drug resistance and metastasis against cancer. Here we have discussed the current status and emerging opportunities of peptides as molecular medicines and molecular transporters.

Designing an effective peptide based molecular transporter for the intracellular delivery of hydrophilic therapeutic biomacromolecules remains a considerable challenge. Highly basic oligoarginine and lipidated arginine rich cell penetrating peptides have been reported in the literature as molecular transporters, which were extensively used for cellular internalization of significantly large biopharmaceuticals. However, oligoarginine based molecular transporters with l-arginine residues pose significant challenges due to proteolytic instability and limited stability of noncovalent peptide-cargo nanocomplexes. Exploiting the rational peptide designing strategy, we have engineered protease-resistant facial lipopeptide based molecular transporter having arginine-sarcosine-arginine moiety to minimize adjacent arginine-arginine pair repulsion. N-Methylated amino acid sarcosine was incorporated as a spacer between two adjacent arginine residues, which provides proteolytic stability to the designed peptide and minimizes intermolecular aggregation of peptides. Two stearyl moieties were incorporated to facilitate cellular internalization. Interestingly, our designed lipopeptide exhibits significantly enhanced cellular internalization with only six l-arginine residues compared to stearylated oligo-nona-arginine. Additionally, enhanced proteolytic stability of such class of molecular transporter enables increased cargo internalization, and we anticipate that our engineered multifunctional, proteolytically stable, nanostructured facial lipopeptide based molecular transporter can have major impact in advancing drug delivery technologies.

Designing biologically inspired nanoscale molecular assembly with desired functionality is a challenging endeavour. Here we report the designing of fibrin-inspired nanostructured peptide based sealants which facilitate remarkably fast entrapping of blood corpuscles (~28 seconds) in contrast to fibrin (~56 seconds). Our engineered sealants are stabilized by lysine-aspartate ionic interactions and also by Nε(γ-glutamyl) lysine isopeptide bond mediated covalent interaction. Each sealant is formed by two peptides having complementary charges to promote lysine-aspartate ionic interactions and designed isopeptide bond mediated interactions. Computational analysis reveals the isopeptide bond mediated energetically favourable peptide assemblies in sealants 1–3. Our designed sealants 2 and 3 mimic fibrin-mediated clot formation mechanism in presence of transglutaminase enzyme and blood corpuscles. These fibrin-inspired peptides assemble to form sealants having superior hemostatic activities than fibrin. Designed sealants feature mechanical properties, biocompatibility, biodegradability and high adhesive strength. Such nature-inspired robust sealants might be potentially translated into clinics for facilitating efficient blood clotting to handle traumatic coagulopathy and impaired blood clotting.

The effects of inserting unsubstituted -amino acids into the strand segments of model -hairpin peptides was investigated by using four synthetic decapeptides, Boc-Leu-Val-Xxx-Val-D-Pro-Gly-Leu-Xxx-Val-Val-OMe: peptide 1 (Xxx=Gly), peptide 2 (Xxx=$\beta$Gly=$\beta$ahGly=homoglycine, -$\beta$glycine), peptide 3 (Xxx=$\gamma$Abu=-$\gamma$aminobutyric acid), peptide 4 (Xxx=$\delta$Ava=-$\delta$aminovaleric acid). $^1H\hspace{2mm}NMR$ studies (500 MHz, methanol) reveal several critical cross-strand NOEs, providing evidence for -$\beta$hairpin conformations in peptides 2-4. In peptide 3, the NMR results support the formation of the nucleating turn, however, evidence for cross-strand registry is not detected. Single-crystal X-ray diffraction studies of peptide 3 reveal a -$\beta$hairpin conformation for both molecules in the crystallographic asymmetric unit, stabilized by four cross-strand hydrogen bonds, with the $\gamma$Abu residues accommodated within the strands. The D-Pro-Gly segment in both molecules (A,B) adopts a type II -$\beta$turn conformation. The circular dichroism spectrum for peptide 3 is characterized by a negative CD band at 229 nm, whereas for peptides 2 and 4, the negative band is centered at 225 nm, suggesting a correlation between the orientation of the amide units in the strand segments and the observed CD pattern.

The incporation of beta-amino acid residues into the strand segments of designed beta-hairpin leads to the formation of polar sheets, since in the case of beta-peptide strands, all adjacent carbonyl groups point in one direction and the amide groups orient in the opposite direction. The conformational analysis of two designed peptide hairpins composed of alpha/beta-hybrid segments are described: Boc-Leu-betaPhe-Val-(D)-Pro-Gly-Leu-betaPhe-Val-OMe (1) and Boc-betaLeu-Phe-betaVal-D-Pro-Gly-betaLeu-Phe-betaVal-OMe (2). A 500-MHz 1H-NMR (nuclear magnetic resonance) analysis in methanol supports a significant population of hairpin conformations in both peptides. Diagnostic nuclear Overhauser effects (NOEs) are observed in both cases. X-ray diffraction studies on single crystals of peptide 1 reveal a beta-hairpin conformation in both the molecules, which constitute the crystallographic asymmetric unit. Three cross-strand hydrogen bonds and a nucleating type II' beta-turn at the D-Pro-Gly segment are observed in the two independent molecules. In peptide 1, the betaPhe residues at positions 2 and 7 occur at the nonhydrogen-bonding position, with the benzyl side chains pointing on opposite faces of the beta-sheet. The observed aromatic centroid-to-centroid distances are 8.92 A (molecule A) and 8.94 A (molecule B). In peptide 2, the aromatic rings must occupy facing positions in antiparallel strands, in the NMR-derived structure. Peptide 1 yields a normal "hairpin-like" CD spectrum in methanol with a minimum at 224 nm. The CD spectrum of peptide 2 reveals a negative band at 234 nm and a positive band at 221 nm, suggestive of an exciton split doublet. Modeling of the facing Phe side chains at the hydrogen-bonding position of a canonical beta-hairpin suggests that interring separation is approximately 4.78 A for the gauche+ gauche- (g+ g-) rotamer. A previously reported peptide beta-hairpin composed of only alpha-amino acids, Boc-Leu-Phe-Val-D-Pro-Gly-Leu-Phe-Val-OMe also exhibited an anomalous far-UV (ultraviolet) CD (circular dichroism) spectrum, which was interpreted in terms of interactions between facing aromatic chromophores, Phe 2 and Phe 7 (C. Zhao, P. L. Polavarapu, C. Das, and P. Balaram, Journal of the American Chemical Society, 2000, Vol 122, pp. 8228-8231).

We designed β-strand peptides that stabilize integral membrane proteins (IMPs). β-strand peptides self-assemble in solution as filaments and become restructured upon association with IMPs; resulting IMP-β-strand peptide complexes resisted aggregation when diluted in detergent-free buffer and were visible as stable, single particles with low detergent background in electron micrographs. β-strand peptides enabled clear visualization of flexible conformations in the highly dynamic ATP-binding cassette (ABC) transporter MsbA.

Conformational studies on the synthetic 11-aa peptide t -butoxycarbonyl (Boc)-Val-Ala-Phe-α-aminoisobutyric acid (Aib)-( R )-β 3 -homovaline (βVal)-( S )-β 3 -homophenylalanine (βPhe)-Aib-Val-Ala-Phe-Aib-methyl ester (OMe) (peptide 1; βVal and βPhe are β amino acids generated by homologation of the corresponding l -residues) establish that insertion of two consecutive β residues into a polypeptide helix can be accomplished without significant structural distortion. Crystal-structure analysis reveals a continuous helical conformation encompassing the segment of residues 2–10 of peptide 1. At the site of insertion of the ββ segment, helical hydrogen-bonded rings are expanded. A C 15 hydrogen bond for the αββ segment and two C 14 hydrogen bonds for the ααβ or βαα segments have been characterized. The following conformational angles were determined from the crystal structure for the β residues: βVal-5 (φ = –126°, θ = 76°, and ψ = –124) and βPhe-6 (φ =–88°, θ = 80°, and ψ =–118). The N terminus of the peptide is partially unfolded in crystals. The 500-MHz 1 H-NMR studies establish a continuous helix over the entire length of the peptide in CDCl 3 solution, as evidenced by diagnostic nuclear Overhauser effects. The presence of seven intramolecular hydrogen bonds is also established by using solvent dependence of NH chemical shifts.

The incorporation of the -amino acid residues into specific positions in the strands and -turn segments of peptide hairpins is being systematically explored. The presence of an additional torsion variable about the C()C() bond () enhances the conformational repertoire in -residues. The conformational analysis of three designed peptide hairpins composed of /-hybrid segments is described: Boc-Leu-Val-Val-DPro-Phe- Leu-Val-Val-OMe (1), Boc-Leu-Val-Val-DPro-Gly-Leu-Val-Val-OMe (2), and Boc-Leu-Val-Phe-Val-DPro- Gly-Leu-Phe-Val-Val-OMe (3). 500-MHz 1H-NMR Analysis supports a preponderance of -hairpin conformation in solution for all three peptides, with critical cross-strand NOEs providing evidence for the proposed structures. The crystal structure of peptide 2 reveals a -hairpin conformation with two -residues occupying facing, non-H-bonded positions in antiparallel -strands. Notably, Val(3 ) adopts a gauche conformation about the C()C() bond (65) without disturbing cross-strand H-bonding. The crystal structure of 2, together with previously published crystal structures of peptides 3 and Boc-Phe-Phe-DPro-Gly- Phe-Phe-OMe, provide an opportunity to visualize the packing of peptide sheets with local −polar segments× formed as a consequence of reversal peptide-bond orientation. The available structural evidence for hairpins suggests that -residues can be accommodated into nucleating turn segments and into both the H-bonding and non-H-bonding positions on the strands.

Half a century has passed since the hydrogen-bonded secondary structures of polypeptides and proteins were first recognized. An extraordinary wealth of conformational information is now available on peptides and proteins, which are formed of alpha-amino acid residues. More recently, the discovery of well-folded structures in oligopeptides containing beta-amino acids has focused a great deal of current interest on the conformational properties of peptides constructed from higher homologues (omega) of alpha-amino acids. This review examines the nature of intramolecularly hydrogen-bonded conformations of hybrid peptides formed by amino acid residues, with a varying number of backbone atoms. The beta-turn, a ubiquitous structural feature formed by two residue (alphaalpha) segments in proteins and peptides, is stabilized by a 10-atom (C10) intramolecular 4--1 hydrogen bond. Hybrid turns may be classified by comparison with their alphaalpha counterparts. The available crystallographic information on hydrogen-bonded hybrid turns is surveyed in this review. Several recent examples demonstrate that individual omega-amino acid residues and hybrid dipeptide segments may be incorporated into the regular structures of alpha-peptides. Examples of both peptide helices and hairpins are presented. The present review explores the relationships between folded conformations in hybrid sequences and their counterparts in all alpha-residue sequences. The use of stereochemically constrained omega-residues promises to expand the range of peptide design strategies to include omega-amino acids. This approach is exemplified by well-folded structures like the C12 (alphagamma) and C14 (gammagamma) helices formed in short peptides containing multiply substituted gamma-residues. The achiral gamma-residue gabapentin is a readily accessible building block in the design of peptides containing gamma-amino acids. The construction of globular polypeptide structures using diverse hybrid sequences appears to be a realistic possibility.