Moneesha Fernandes

Highly Efficient (R-X-R)-Type Carbamates as Molecular Transporters for Cellular Delivery

The (R-X-R) motif-containing arginine-rich peptides are among the most effective cell-penetrating peptides. The replacement of amide linkages in the (R-X-R) motif by carbamate linkages as in (r-ahx-r)(4) or (r-ahx-r-r-apr-r)(2) increases the efficacy of such oligomers several-fold. Internalization of these oligomers in mammalian cell lines occurs by an energy-independent process. These oligomers show efficient delivery of biologically active plasmid DNA into CHO-K1 cells.

N-(3-Aminoalkyl)proline derivatives with potent antigycation activity

The importance of amino acids in the therapy of conditions such as renal failure, neurological disorders and congenital defects has been documented. Some amino acids such as lysine and glycine have also been reported to have antiglycating activity. Herein we report the synthesis of a new series of N-(3-aminoalkyl)proline derivatives which are non-natural in nature. The compounds were unambiguously characterized by NMR, mass and IR spectroscopy. Their in vitro antiglycation activity was studied by circular dichroism and fluorescence spectrometry. The mechanism of action was also studied and found to take place by inhibition of Amadori product formation. The inhibition of AGE formation was further confirmed by western blot and LC-MS/MS analyses and the IC50 values of the potent compounds were determined. Compounds containing hydroxyl substituents at C4 were found to have superior antiglycation properties than those containing azide substituents at the same position. The compounds were additionally found to possess good anti-oxidant properties, which could lead to further reduction in AGE formation. Moreover, the title compounds were found to have low cytotoxicity in mammalian cells, another important attribute. Thus, the title compounds represent a novel promising class of antiglycating agents.

Efficient Cellular Entry of (r-x-r)-Type Carbamate–Plasmid DNA Complexes and Its Implication for Noninvasive Topical DNA Delivery to Skin

Arginine-rich cell penetrating peptides are powerful tools for in vitro as well as in vivo delivery of a wide plethora of biomolecules. However, presence of consecutive arginine residues leads to enhanced amenability for proteolytic degradation as well as steric hindrances for membrane interactions which compromise its bioavailability. In order to overcome these limitations we previously reported a safe and stable octaarginine based oligomer, i.e., (r-x-r)4-carbamate, where the backbone amide linkages were replaced by carbamate linkages and 6-aminohexanoic acid based spacer moieties were incorporated for better flexibility, hydrophobicity, optimal spacing of guanidinium groups, and protection against proteolytic cleavage; resulting in improved transfection efficiency over its amide counterpart. In the present work we have investigated the mechanism behind this enhanced transfection efficiency and, based on our observations, demonstrate how the synergistic effect of rationalized oligomer designing, complex characteristics, and cell type contributes to overall effective intracellular delivery. Our results indicate that the (r-x-r)4-carbamate-plasmid DNA complexes primarily utilize lipid raft dependent pathway of cellular entry more than other pathways, and this possibly facilitates their increased entry in the lipid raft rich milieu of skin cells. We also emphasize the utility of oligomer (r-x-r)4-carbamate as an efficient carrier for topical delivery of nucleic acids in skin tissue. This carrier can be utilized for safe, efficient, and noninvasive delivery of therapeutically relevant macromolecular hydrophilic cargo like nucleic acids to skin.

Superior HIV-1 TAR Binders with Conformationally Constrained R52 Arginine Mimics in the Tat(48-57) Peptide

We report a 100‐fold increase in binding affinity of the Tat(48–57) peptide to HIV‐1 transcriptional activator‐responsive element (TAR) RNA by replacing Arg52, an essential and critical residue for Tats specific binding, with (2S,4S)‐4‐guanidinoproline. The resulting αTat1M peptide is a far superior binder than γTat1M, a peptide containing another conformationally constrained arginine mimic, (2S,4S)‐4‐amino‐N‐(3‐guanidinopropyl)proline, or even the control Tat peptide (CtrlTat) itself. Our observations are supported by circular dichroism (CD), isothermal titration calorimetry (ITC), gel electrophoresis and UV spectroscopy studies. Molecular dynamics simulations suggest increased interactions between the more compact αTat1M and TAR RNA, relative to CtrlTat. The CD signature of the RNA itself remains largely unchanged upon binding of the peptides. The Tat mimetics further have better cell uptake properties than the control Tat peptide, thus increasing their potential application as specific TAR‐binding molecules.

(R-X-R)4-motif peptides containing conformationally constrained cyclohexane-derived spacers: Effect on cellular uptake

Arginine‐rich peptides having the (R‐X‐R)n motif are among the most effective cell‐penetrating peptides (CPPs). Herein we report a several‐fold increase in the efficacy of such CPPs if the linear flexible spacer (‐X‐) in the (R‐X‐R) motif is replaced by constrained cyclic 1,4‐substituted‐cyclohexane‐derived spacers. Internalization of these oligomers in mammalian cell lines was found to be an energy‐dependent process. Incorporation of these constrained, non‐proteinogenic amino acid spacers in the CPPs is shown to enhance their proteolytic stability.

4′-Epi-DNA: A DNA Mimic Containing 4′-hydroxymethyl-α-l-Xylo-Thymidine with Compact Backbone like RNA

ABSTRACT Synthesis of C4′-epi-DNA containing 3′→ 5″ linkages is reported for the first time. Crystal structure study of the monomer indicated that though the dihedral angle O3′-C3′-C4′-C5″ in this case would be like in RNA, the sugar conformation would remain like that in DNA. The study of the effect of this backbone configuration in DNA with respect to its binding to cDNA and RNA is reported in this note. GRAPHICAL ABSTRACT

159. RXR-Carbamate – A Novel Molecular Transporter for Skin

Cell penetrating peptides are powerful tools for delivery of various therapeutics. One of the critical intrinsic features of these carriers is the presence of minimum 7-9 arginine residues which allows electrostatic interaction with cargos like nucleic acids as well as negatively charged cell surface molecules for facile entry and cargo delivery. However cellular entry might be compromised if the guanidium groups are consecutive and not spaced because of compromised cellular interaction. Another limitation is the amenability of these homoarginine systems to proteolytic cleavage which leads to their low bio-availability. We previously reported a safe and stable homoarginine peptide-based system (R-X-R)-carbamate where amide linkages were replaced by stable carbamate linkages and arginine residues were separated by aminohexanoic acid spacers. This alteration imparted better flexibility and hydrophobicity to these systems in addition to protection against proteolytic cleavage and minimal steric hindrances. These molecules entered cells efficiently and showed high transfection efficiencies in comparison to their amide counterparts. We have now investigated the mechanism behind enhanced transfection efficiency and based on our observations, demonstrate the applicability of this system as an efficient carrier for topical delivery of biomolecules in skin. We demonstrated through flow cytometry studies using chemical inhibitors of different known cellular uptake pathways that (R-X-R)-carbamate- DNA complexes not only show much higher uptake than amide counterpart but the uptake was drastically decreased in presence of inhibitor Methyl-β-Cyclodextrin -a known blocker of lipid raft mediated entry pathway. We have further validated the entry of these carriers using colocalization studies with pathway specific markers. Further, retention, stability and transfection efficiency studies involving depletion of cholesterol (which is a major component of lipid raft structures) using extraction method is being carried out. Comparative transfection efficiency in lipid raft enriched cancerous cell lines is being used for further validation. Lipid rafts act as major mediators of viral infections and are also involved in entry of nanocomplexes in skin. Therefore we explored if the (R-X-R)-carbamate-DNA complexes that are more biased to enter the cells through lipid raft dependant pathways also exhibit efficient entry into skin cells and tissue. The uptake of (R-X-R)-carbamate-DNA complexes and the transfection efficiency were studied in HaCaT cells and human skin tissue. High levels of transfection was achieved in human skin tissue after topical application of these complexes. Further studies on mechanism of interaction of these complexes with skin and the impact on skin integrity is currently ongoing. Ultimately this study will help us in obtaining a carrier system which is safe, more stable and efficient to deliver multiple cargo types in skin in a non-invasive manner for therapeutic applications. Moreover we intend to gain insights into the exact mechanism that it follows during its trajectory inside the skin cells and tissue.