Modular synthesis of functional libraries by accelerated SuFEx click chemistry.

Accelerated SuFEx Click Chemistry (ASCC) is a powerful method for coupling aryl and alkyl alcohols with SuFEx-compatible functional groups. With its hallmark favorable kinetics and exceptional product yields, ASCC streamlines the synthetic workflow, simplifies the purification process, and is ideally suited for discovering functional molecules. We showcase the versatility and practicality of the ASCC reaction as a tool for the late-stage derivatization of bioactive molecules and in the array synthesis of sulfonate-linked, high-potency, microtubule targeting agents (MTAs) that exhibit nanomolar anticancer activity against multidrug-resistant cancer cell lines. These findings underscore ASCC's promise as a robust platform for drug discovery.

Designed novel nuclear localizing anticancer peptide targets p53 negative regulator MDM2 protein.

Intracellular protein-protein interactions provide a major therapeutic target for the development of peptide-based anticancer therapeutic agents. MDM2 is the 491-residue protein encoded by the MDM2 oncogene. Being a ubiquitin-protein ligase, MDM2 represses the transcription ability of the tumor suppressor p53 by proteasome-mediated degradation. Under typical cellular circumstances, a sustained p53 expression level is maintained by negative regulation of MDM2, whereas under stress conditions, this is alleviated to increase the p53 level. Modulation of MDM2-p53 interaction via fabrication of an MDM2-interacting peptide could be a useful strategy to inhibit subsequent proteasomal degradation of p53 and initiation of p53 signaling leading to the initiation of p53-mediated apoptosis of tumor cells. Here, in this research work, a novel anticancer peptide mPNC-NLS targeting the nucleus and the MDM2 protein (p53 negative regulator) was designed to promote the p53 protein activity for the prevention of cancer. It induces effective apoptosis in both A549 and U87 cells and remains non-cytotoxic to normal lung fibroblast cells (WI38). Further, immunocytochemistry and Western blot results confirm that the designed mPNC-NLS peptide induces the apoptotic death of lung cancer cells via activation of p53 and p21 proteins and remarkably stifled the in vitro growth of 3D multicellular spheroids composed of A549 cells.

Synthesis of Bifacial Peptide Nucleic Acids with Diketopiperazine Backbones.

We report a synthesis of bifacial peptide nucleic acids (bPNAs) with novel diketopiperazine (DKP) backbones that display unnatural melamine (M) bases, as well as native bases. To examine the structure-function scope of DKP bPNAs, we synthesized a set of bPNAs by using diaminopropionic acid, diaminobutyric acid, ornithine, and lysine derivatives to display the base-tripling motifs, which result in one, two, three, or four carbons linking the alpha carbon to the side-chain amine. Thermal denaturation of DNA hybrids with these bPNAs revealed that the optimal side-chain linkage was four carbons, corresponding to the lysine derivative. Accordingly, monomers displaying two bases per side-chain were prepared through double reductive alkylation of the ε-amine of Fmoc-lysine with acetaldehyde derivatives of adenine, cytidine, uridine, and melamine. With these building blocks in hand, DKP bPNAs were prepared to display a combination of native and synthetic (melamine) bases. Preliminary melting studies indicate binding signatures of cytidine- and melamine-displaying bPNAs to T-rich DNAs of noncanonical structure, though full characterization of this behavior is ongoing. The convenient and potentially scalable method described enables rapid access to DNA-binding scaffolds of low (<1 kD) molecular weight and previously established cell permeability. We expect that this straightforward and efficient approach to nucleic acid binders will enable studies on noncanonical nucleic acid hybridization.

Designed hybrid anticancer nuclear-localized peptide inhibits aggressive cancer cell proliferation.

Cell proliferation is a crucial step that might promote cancer if deregulated. Therefore, this vital segment is critically controlled by a complicated cell-cycle process in normal cells that is regulated by some regulatory proteins. It has been observed that p16 protein, playing a crucial role in cell-cycle progression/regulation, remains inactivated in different cancer cells. This inactivity of p16 protein leads to the enhancement of cancer cell proliferation by allowing uncontrolled cancer cell division. Hence, the activity of p16 protein needs to be restored using new viral vectors, small molecules as well as peptides to control/suppress this type of abnormal cell proliferation. In this work, we have taken an interesting approach to increase the efficiency and bio-availability of p16 peptide (functional part of p16 protein) to be an aggressive anti-leukemia therapeutic agent by conjugating a nuclear-localized signal (NLS) sequence and a short peptide (AVPI) with it. Moreover, this newly designed NLS attached hybrid peptide greatly affects XIAP expressing but p16 lower expressing human chronic myelogenous leukemia (CML) cell proliferation by targeting both nuclear (CDK4/cyclin D) and cellular factors (XIAP) and promoting the caspase-3 dependent apoptosis pathway.

Bifacial PNAs Destabilize MALAT1 by 3' A-Tail Displacement from the U-Rich Internal Loop.

We report herein a new class of synthetic reagents for targeting the element for nuclear expression (ENE) in MALAT1, a long noncoding RNA upregulated in many cancers. The cis-acting ENE contains a U-rich internal loop (URIL) that forms an 11 base UAU-rich triplex stem with the truncated 3' oligo-A tail of MALAT1, protecting the terminus from exonuclease digestion and greatly extending transcript lifetime. Bifacial peptide nucleic acids (bPNAs) similarly bind URILs via base triple formation between two uracil bases and a synthetic base, melamine. We synthesized a set of low molecular weight bPNAs composed of α-linked peptide, isodipeptide, and diketopiperazine backbones and evaluated their ENE binding efficacy in vitro via oligo-A strand displacement and consequent exonuclease sensitivity. Degradation was greatly enhanced by bPNA treatment in the presence of exonucleases, with ENE half-life plunging to 6 min from >24 h. RNA digestion kinetics could clearly distinguish between bPNAs with similar URIL affinities, highlighting the utility of functional assays for evaluating synthetic RNA binders. In vitro activity was mirrored by a 50% knockdown of MALAT1 expression in pancreatic cancer (PANC-1) cells upon treatment with bPNAs, consistent with intracellular digestion triggered by a similar ENE A-tail displacement mechanism. Pulldown from PANC-1 total RNA with biotinylated bPNA enriched MALAT1 > 4000× , supportive of bPNA-URIL selectivity. Together, these experiments establish the feasibility of native transcript targeting by bPNA in both in vitro and intracellular contexts. Reagents such as bPNAs may be useful tools for the investigation of transcripts stabilized by cis-acting poly(A) binding RNA elements.

A Small Molecule with Bridged Carbonyl and Tri-fluoro-aceto-phenone Groups Impedes Microtubule Dynamics and Subsequently Triggers Cancer Cell Apoptosis.

We identified a new microtubule targeted small molecule, which showed significant anticancer activity and induced apoptotic death of cancer cells. Precisely the central bridged carbonyl group and trifluoro-acetophenone group of a bis-benzothiazole molecule (BBT) interacts with tubulin close to the curcumin site and perturbs microtubule dynamics as well as causes microtubule depolymerization. We observed a significant enhancement of fluorescence while BBT interacts with the tubulin through bridged carbonyl moiety, a similar phenomenon to colchicine. Further, BBT activates tumor-suppressing bim and p53-puma axes to inhibit cancer survival. It also shows promising results against a tumor spheroid model. BBT is also capable of tumor regression, which shows that this molecule can serve as a potential template for the design of next-generation microtubule targeted anticancer drugs.

Unnatural bases for recognition of noncoding nucleic acid interfaces.

The notion of using synthetic heterocycles instead of the native bases to interface with DNA and RNA has been explored for nearly 60 years. Unnatural bases compatible with the DNA/RNA coding interface have the potential to expand the genetic code and co-opt the machinery of biology to access new macromolecular function; accordingly, this body of research is core to synthetic biology. While much of the literature on artificial bases focuses on code expansion, there is a significant and growing effort on docking synthetic heterocycles to noncoding nucleic acid interfaces; this approach seeks to illuminate major processes of nucleic acids, including regulation of transcription, translation, transport, and transcript lifetimes. These major avenues of research at the coding and noncoding interfaces have in common fundamental principles in molecular recognition. Herein, we provide an overview of foundational literature in biophysics of base recognition and unnatural bases in coding to provide context for the developing area of targeting noncoding nucleic acid interfaces with synthetic bases, with a focus on systems developed through iterative design and biophysical study.

Elevated level of circulatory sTLT1 induces inflammation through SYK/MEK/ERK signalling in coronary artery disease.

The role of inflammation in all phases of atherosclerotic process is well established and soluble TREM-like transcript 1 (sTLT1) is reported to be associated with chronic inflammation. Yet, no information is available about the involvement of sTLT1 in atherosclerotic cardiovascular disease. Present study was undertaken to determine the pathophysiological significance of sTLT1 in atherosclerosis by employing an observational study on human subjects (n=117) followed by experiments in human macrophages and atherosclerotic apolipoprotein E (apoE)-/- mice. Plasma level of sTLT1 was found to be significantly (P<0.05) higher in clinical (2342 ± 184 pg/ml) and subclinical cases (1773 ± 118 pg/ml) than healthy controls (461 ± 57 pg/ml). Moreover, statistical analyses further indicated that sTLT1 was not only associated with common risk factors for Coronary Artery Disease (CAD) in both clinical and subclinical groups but also strongly correlated with disease severity. Ex vivo studies on macrophages showed that sTLT1 interacts with FcÉ£ receptor I (FcÉ£RI) to activate spleen tyrosine kinase (SYK)-mediated downstream MAP kinase signalling cascade to activate nuclear factor-κ B (NF-kB). Activation of NF-kB induces secretion of tumour necrosis factor-α (TNF-α) from macrophage cells that plays pivotal role in governing the persistence of chronic inflammation. Atherosclerotic apoE-/- mice also showed high levels of sTLT1 and TNF-α in nearly occluded aortic stage indicating the contribution of sTLT1 in inflammation. Our results clearly demonstrate that sTLT1 is clinically related to the risk factors of CAD. We also showed that binding of sTLT1 with macrophage membrane receptor, FcÉ£R1 initiates inflammatory signals in macrophages suggesting its critical role in thrombus development and atherosclerosis.

Dual-Arm Nanocapsule Targets Neuropilin-1 Receptor and Microtubule: A Potential Nanomedicine Platform.

A multiarm nanomedicine template has been designed following bottom-up approach, which target neuropilin-1 (Nrp-1) receptor of cancer cells. Through this venture, we discovered that cucurbit [6] uril (CB [6]) binds with tubulin close to binding pocket of vinblastine site and perturbs tubulin polymerization. To increase the specificity of gold nanoparticle (GNP) toward Nrp-1-rich cancer cells, we further modified this GNP with Nrp-1 receptor-specific short peptide (CGNKRTR). Remarkably, we found an interesting self-assembly process upon addition of curcumin into the CB [6] and peptide-functionalized GNP, leading to the formation of a spherical nanocapsule (CGNP·Cur). It can deliver and release significantly higher amounts of anticancer drug curcumin in Nrp-1-rich cancer cells. It causes microtubule depolymerization and significant tumor regression in Nrp-1 overexpressed mice melanoma model. These interesting findings show that nanocapsule has high potential to develop a powerful anticancer nanomedicine and help in its preclinical validation.

Tripodal molecular propellers perturb microtubule dynamics: indole acts as a blade and plays a crucial role in anticancer activity.

An indole-rich tripodal microtubule inhibitor is designed, which binds at the DCVJ site of tubulin and inhibits its polymerization. It causes apoptotic death of cancer cells without affecting normal cells and inhibits the growth of tumors. Finally, STD-NMR and TR-NOESY experiments reveal that the indole appendages play a crucial role in interacting with tubulin.

Power of Tyrosine Assembly in Microtubule Stabilization and Neuroprotection Fueled by Phenol Appendages.

Microtubules play a crucial role in maintenance of structure, function, axonal extensions, cargo transport, and polarity of neurons. During neurodegenerative diseases, microtubule structure and function get severely damaged due to destabilization of its major structural proteins. Therefore, design and development of molecules that stabilize these microtubule networks have always been an important strategy for development of potential neurotherapeutic candidates. Toward this venture, we designed and developed a tyrosine rich trisubstituted triazine molecule (TY3) that stabilizes microtubules through close interaction with the taxol binding site. Detailed structural investigations revealed that the phenolic protons are the key interacting partners of tubulin. Interestingly, we found that this molecule is noncytotoxic in PC12 derived neurons, stabilizes microtubules against nocodazole induced depolymerization, and increases expression of acetylated tubulin (Ac-K40), an important marker of tubulin stability. Further, results show that TY3 significantly induces neurite sprouting as compared to the untreated control as well as the two other analogues (TS3 and TF3). It also possesses anti-Aß fibrillation properties as confirmed by ThT assay, which leads to its neuroprotective effect against amyloidogenic induced toxicity caused through nerve growth factor (NGF) deprivation in PC12 derived neurons. Remarkably, our results reveal that it reduces the expression of TrkA (pY490) associated with NGF deprived amyloidogenesis, which further proves that it is a potent amyloid ß inhibitor. Moreover, it promoted the health of the rat primary cortical neurons through higher expression of key neuronal markers such as MAP2 and Tuj1. Finally, we observed that it has good serum stability and has the ability to cross the blood-brain barrier (BBB). Overall, our work indicates the importance of phenolic -OH in promoting neuroprotection and its importance could be implemented in the development of future neurotherapeutics.

Matrix metalloproteinase targeted peptide vesicles for delivering anticancer drugs.

A matrix metalloproteinase (MMP) targeted tetrapeptide vesicle has been designed and developed, which strongly binds at a MMP9 enzymatic site. Interestingly, it has a propensity to encapsulate and deliver the doxorubicin drug specifically to the cancer cell, induces superior apoptotic death, and inhibits the metastatic cancer cell migration and growth of multicellular 3D spheroids.

Spatial Position Regulates Power of Tryptophan: Discovery of a Major-Groove-Specific Nuclear-Localizing, Cell-Penetrating Tetrapeptide.

Identification of key amino acids is required for development of efficient cell-penetrating peptides (CPPs) and has tremendous implications in medicine. Extensive research work has enlightened us about the importance of two amino acids, arginine and tryptophan, in cell penetration. Here, we present a top-down approach to show how spatial positions of two tryptophans regulate the cellular entry and nuclear localization. This enables us to develop short, non-toxic tetrapeptides with excellent potential for cell penetration and nuclear localization. Among them, Glu-Thr-Trp-Trp (ETWW) emerges as the most promising. Results suggest that it enters into cancer cells following an endocytic pathway and binds at the major groove of nuclear DNA, where successive tryptophan plays major role. We subsequently show that it is not a P-glycoprotein substrate and is non-toxic to PC12-derived neurons, suggesting its excellent potential as a CPP. Furthermore, its potential as a CPP is validated in multi-cellular 3D cell culture (spheroid) and in in vivo mice model. This study provides major fundamental insights about the positional importance of tryptophan and opens new avenues toward the development of next-generation CPPs and major-groove-specific anticancer drugs.

Amphiphilic Peptide-Based Supramolecular, Noncytotoxic, Stimuli-Responsive Hydrogels with Antibacterial Activity.

A series of peptides with a long fatty acyl chain covalently attached to the C-terminal part and a free amine (-NH2) group at the N-terminus have been designed so that these molecules can be assembled in aqueous medium by using various noncovalent interactions. Five different peptide amphiphiles with a general chemical formula [H2N-(CH2)nCONH-Phe-CONHC12 (n = 1-5, C12 = dodecylamine)] have been synthesized, characterized, and examined for self-assembly and hydrogelation. All of these molecules [P1 (n = 1), P2 (n = 2), P3 (n = 3), P4 (n = 4), P5 (n = 5)] form thermoresponsive hydrogels in water (pH 6.6) with a nanofibrillar network structure. Interestingly, the hydrogels obtained from compounds P4 and P5 exhibit potential antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and Gram-negative bacteria (Escherichia coli). Dose-dependent cell-viability studies using MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by taking human lung carcinoma (A549) cells vividly demonstrates the noncytotoxic nature of these gelator molecules in vitro. Hemolytic studies show nonsignificant or little hemolysis of human erythrocyte cells at the minimum inhibitory concentration (MIC) of these tested bacteria. Interestingly, it has been found that these antibacterial noncytotoxic hydrogels exhibit proteolytic resistance toward the enzymes proteinase K and chymotrypsin. Moreover, the gel strength and gel recovery time have been successfully modulated by varying the alkyl chain length of the N-terminally located amino acid residues. Similarly, the thermal stability of these hydrogels has been nicely tuned by altering the alkyl chain length of the N-terminally located amino acid residues. In the era of antibiotic-resistant strains of bacteria, the discovery of this new class of peptide-based antibacterial, proteolytically stable, injectable, and noncytotoxic soft materials holds future promise for the development of new antibiotics.

Biodegradable Neuro-Compatible Peptide Hydrogel Promotes Neurite Outgrowth, Shows Significant Neuroprotection, and Delivers Anti-Alzheimer Drug.

A novel neuro-compatible peptide-based hydrogel has been designed and developed, which contains microtubule stabilizing and neuroprotective short peptide. This hydrogel shows strong three-dimensional cross-linked fibrillary networks, which can capture water molecules. Interestingly, this hydrogel serves as excellent biocompatible soft material for 2D and 3D (neurosphere) neuron cell culture and provides stability of key cytoskeleton filaments such as microtubule and actin. Remarkably, it was observed that this hydrogel slowly enzymatically degrades and releases neuroprotective peptide, which promotes neurite outgrowth of neuron cell as well as exhibits excellent neuroprotection against anti-NGF-induced toxicity in neuron cells. Further, it can encapsulate anti-Alzheimer and anticancer hydrophobic drug curcumin, releases slowly, and inhibits significantly the growth of a 3D spheroid of neuron cancer cells. Thus, this novel neuroprotective hydrogel can be used for both neuronal cell transplantation for repairing brain damage as well as a delivery vehicle for neuroprotective agents, anti-Alzheimer, and anticancer molecules.

Cancer Cell Specific Delivery of Photosystem I Through Integrin Targeted Liposome Shows Significant Anticancer Activity.

Many anticancer drugs are developed for the treatment of cancer from natural sources. Photosystem I (PSI), a protein complex present in the chloroplast, is involved in photosynthesis and generates reactive oxygen species (ROS) in plant. Here, we used the ROS generation property of PSI for cancer therapy. We show that PSI can enter into different kinds of cancer cell like human lung carcinoma (A549) and mouse melanoma (B16F10) cell lines and generate ROS inside the cells. It inhibits the proliferation of cancer cell and causes apoptotic death of cancer cells. We also show that PSI induces apoptosis through mitochondria-dependent internal pathway, induces caspase3, causes DNA fragmentation, and arrests cell cycle at SubG0 phase. We also prepared, using C16-LDV lipopeptide [C16 long chain attached on the N-terminal of the tripeptide containing amino acids leucine (L), aspartic acid (D), and valine (V) abbreviated as NH2-LDV-COOH], α4ß1 integrin targeted liposomal formulation of PSI, which specifically kills the cancer cell without affecting normal cells, and it is found to be more potent compared to clinically used drug doxorubicin. Finally, we found that LDV liposomal formulation of PSI inhibits the growth of tumor in C57BL/6J mice model.

Apoferritin Nanocage Delivers Combination of Microtubule and Nucleus Targeting Anticancer Drugs.

An ideal nano drug delivery agent must be potent enough to carry high dose of therapeutics and competent enough in targeting specific cell of interest, having adequate optimized physiochemical properties and biocompatibility. Carrying differentially polar therapeutics simultaneously will make them superior in their class. However, it is of enormous challenge to the researchers to find such a unique nanocarrier and to engineer all of the above-mentioned features into it. In this manuscript, we have shown for the first time that apoferritin (Apf) can carry and deliver high dose of doxorubicin (Dox), docetaxel (Doc), and combination of both Dox and Doc specifically into the cancer cell and enhances killing compared to free drug without any functionalization or property modulation. In addition, we have shown that Apf alone is noncytotoxic in nature and interacts with intracellular tubulin/microtubule. Drug loaded Apf specifically bound and consequently internalized into the human breast cancer cell line (MCF7) and human cervical cancer cell line (HeLa) through receptor mediated endocytosis process and releases either single or combination of drugs in the endosome. We have also checked the binding efficacy of both drugs using molecular docking. Further, using fluorescence microscopy, we have shown that Apf can deliver combination of drugs inside cancer cells and the drugs exerts their effect thereof. Finally, we have studied the efficacy of Apf complexes with individual drugs and in combination compared to free drugs in a tumor mimicking 3D multicellular spheroid model of HeLa cell.

Novel tubulin-targeted cell penetrating antimitotic octapeptide.

An antimitotic cell penetrating octapeptide containing single Arg amino acid is discovered, which strongly binds with the exchangeable GTP/GDP binding site of tubulin, inhibits tubulin polymerization, reduces kinesin driven microtubule motility, activates apoptotic and mitotic check point proteins, induces apoptotic death and significantly inhibits the multicellular tumor spheroid growth of HeLa cells.

Peptide based hydrogels for cancer drug release: modulation of stiffness, drug release and proteolytic stability of hydrogels by incorporating d-amino acid residue(s).

Synthetic tripeptide based noncytotoxic hydrogelators have been discovered for releasing an anticancer drug at physiological pH and temparature. Interestingly, gel stiffness, drug release capacity and proteolytic stability of these hydrogels have been successfully modulated by incorporating d-amino acid residues, indicating their potential use for drug delivery in the future.