Self-Assembly and Antimicrobial Activity of Lipopeptides Containing Lysine-Rich Tripeptides.

The conformation and self-assembly of two pairs of model lipidated tripeptides in aqueous solution are probed using a combination of spectroscopic methods along with cryogenic-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS). The palmitoylated lipopeptides comprise C16-YKK or C16-WKK (with two l-lysine residues) or their respective derivatives containing d-lysine (k), i.e., C16-Ykk and C16-Wkk. All four molecules self-assemble into spherical micelles which show structure factor effects in SAXS profiles due to intermicellar packing in aqueous solution. Consistent with micellar structures, the tripeptides in the coronas have a largely unordered conformation, as probed using spectroscopic methods. The molecules are found to have good cytocompatibility with fibroblasts at sufficiently low concentrations, although some loss of cell viability is noted at the highest concentrations examined (above the critical aggregation concentration of the lipopeptides, determined from fluorescence dye probe measurements). Preliminary tests also showed antimicrobial activity against both Gram-negative and Gram-positive bacteria.

Evolution of Potential Antimitotic Stapled Peptides from Multiple Helical Peptide Stretches of the Tubulin Heterodimer Interface: Helix-Mimicking Stapled Peptide Tubulin Inhibitors.

Protein-protein interactions play a crucial role in microtubule dynamics. Microtubules are considered as a key target for the design and development of anticancer therapeutics, where inhibition of tubulin-tubulin interactions plays a crucial role. Here, we focused on a few key helical stretches at the interface of α,ß-tubulin heterodimers and developed a structural mimic of these helical peptides, which can serve as potent inhibitors of microtubule polymerization. To induce helicity, we have made stapled analogues of these sequences. Thereafter, we modified the lead sequences of the antimitotic stapled peptides with halo derivatives. It is observed that halo-substituted stapled peptides follow an interesting trend for the electronegativity of halogen atoms in interaction patterns with tubulin and a correlation in the toxicity profile. Remarkably, we found that para-fluorophenylalanine-modified stapled peptide is the most potent inhibitors, which perturbs microtubule dynamics, induces apoptotic death, and inhibits the growth of melanoma.

Fluorine Substituted Proline Enhances the Tubulin Binding Potential of a Tetrapeptide at the GTP Binding Pocket Causing the Inhibition of Microtubule Motility and an Antimitotic Effect.

The microtubule is regarded as the key target for designing anticancer and neurotherapeutic drugs due to its functional importance in eukaryotic cells including neurons. The microtubule is a dynamic hollow polymer tube consisting of α,ß-tubulin heterodimer. Polymerization of α,ß-tubulin heterodimer resulted in microtubule formation. GTP plays a crucial role in microtubule polymerization. It binds at the exchangeable binding site of the ß-tubulin heterodimer, and it is one of the most crucial therapeutic hot spots for designing anticancer therapeutics. In this manuscript, we have shown using an in silico strategy and various in vitro and cellular experiments that the binding affinity to the tubulin and cancer therapeutic potential of an exchangeable GTP/GDP binding antimitotic tetrapeptide (SP: Ser-Leu-Arg-Pro) is increased through changing proline with the multifluorine substituted proline. This study showcases the importance of the proline amino acid and its pyrrolidine ring in the regulation of binding with tubulin at the GTP binding pocket.

Recent trends in the development of peptide and protein-based hydrogel therapeutics for the healing of CNS injury.

Traumatic brain injury (TBI) and spinal cord injury (SCI) cause millions of deaths and permanent or prolonged physical disabilities around the globe every year. It generally happens due to various incidents, such as accidents during sports, war, physical assault, and strokes which result in severe damage to brain and spinal cord. If this remains untreated, traumatic CNS injuries may lead to early development of several neurodegenerative diseases like Alzheimer's, Parkinson, multiple sclerosis, and other mental illnesses. The initial physical reaction, which is also termed as the primary phase, includes swelling, followed by inflammation as a result of internal haemorrhage causing damage to indigenous tissue, i.e., axonal shear injury, rupture of blood vessels, and partial impaired supply of oxygen and essential nutrients in the neurons, thereby initiating a cascade of events causing secondary injuries such as hypoxia, hypotension, cognitive impairment, seizures, imbalanced calcium homeostasis and glutamate-induced excitotoxicity resulting in concomitant neuronal cell death and cumulative permanent tissue damage. In the modern era of advanced biomedical technology, we are still living with scarcity of the clinically applicable comparative non-invasive therapeutic strategies for regeneration or functional recovery of neurons or neural networks after a massive CNS injury. One of the key reasons for this scarcity is the limited regenerative ability of neurons in CNS. Growth-impermissive glial scar and the lack of a synthetic biocompatible platform for proper neural tissue engineering and controlled supply of drugs further retard the healing process. Injectable or implantable hydrogel materials, consisting majorly of water in its porous three-dimensional (3D) structure, can serve as an excellent drug delivery platform as well as a transplanted cell-supporting scaffold medium. Among the various neuro-compatible bioinspired materials, we are limiting our discussion to the recent advancement of engineered biomaterials comprising mainly of peptides and proteins due to their growing demand, low immunogenicity and versatility in the fabrication of neuro regenerative medicine. In this article, we try to explore all the recent scientific avenues that are developing gradually to make peptide and peptide-conjugated biomaterial hydrogels as a therapeutic and supporting scaffold for treating CNS injuries.

Extracellular Matrix (ECM)-Mimicking Neuroprotective Injectable Sulfo-Functionalized Peptide Hydrogel for Repairing Brain Injury.

Brain injury can lead to the loss of neuronal functions and connections, along with the damage of the extracellular matrix (ECM). Thus, it ultimately results in devastating long-term damage, and recovery from this damage is a challenging task. To address this issue, we have designed a sulfo-group-functionalized injectable biocompatible peptide hydrogel, which not only mimics the ECM and supports the damaged neurons but also releases a neurotrophic factor around the injured sites of the brain in the presence of the matrix metalloproteinase 9 (MMP9) enzyme. It has also been observed that the driving force of hydrogel formation is a ß-sheet secondary structure and π-π stacking interactions between Phe-Phe moieties. The hydrogel is able not only to promote neurite outgrowth of PC12-derived neurons and primary neurons cultured in its presence but also to nullify the toxic effects of anti-nerve growth factor (Anti-NGF)-induced neurons. It also promotes the expression of vital neuronal markers in rat cortical primary neurons, displays substantial potential in neuroregeneration, and also promotes fast recovery of the sham injured mice brain. Increased expression of reactive astrocytes in the hippocampal dentate gyrus region of the sham injured brain clearly suggests its tremendous ability in the neural repair of the damaged brain. Thus, we can convincingly state that our hydrogel is capable of repairing brain injury by mimicking an ECM-like environment and providing neuroprotection to the damaged neurons.

Biocompatible Lipopeptide-Based Antibacterial Hydrogel.

A biocompatible hydrogel containing a hexapeptide as a key unit has been designed and fabricated. Our design construct comprises a ß-sheet-rich short hexapeptide in the center with a hydrophobic long chain and hydrophilic triple lysine unit attached at the N- and C-terminals, respectively. Thus, it is this amphiphilic nature of the molecule that facilitates gelation. It can capture solvent molecules in the three-dimensional cross-linked fibrillar networks. The amphiphilic character of the construct has been modulated to produce an excellent biocompatible soft material for the inhibition of bacterial growth by rupturing the bacterial cell membrane. This hydrogel is also stable against enzymatic degradation (proteinase K) and, most importantly, offers a biocompatible environment for the growth of normal mammalian cells due to its noncytotoxic nature as observed through the cell viability assay. From the hemolytic assay, the morphology of the human red blood cells is found to be almost intact, which suggests that the hydrogel can be used in biomedical applications. Thus, this newly designed antibacterial hydrogel can be used as both an antibacterial biomaterial and a biocompatible scaffold for mammalian cell culture.

Neuro-Regenerative Choline-Functionalized Injectable Graphene Oxide Hydrogel Repairs Focal Brain Injury.

Brain damage is associated with spatial imbalance of cholinergic system, which makes severe impact in recovery of damaged neurons of brain. Therefore, maintenance of cholinergic system is extremely important. Here, we fabricated an injectable hydrogel with acetylcholine-functionalized graphene oxide and poly(acrylic acid). Results revealed that this hydrogel is non-cytotoxic, promotes neurite outgrowth, stabilizes microtubule networks, and enhances the expression of some key neural markers in rat cortical primary neurons. Further, this hydrogel exhibits significant potential in neuro-regeneration and also promotes fast recovery of the sham injured mice brain. Moreover, we found significant enhancement of reactive astrocytes in the hippocampal dentate gyrus region of the sham injured brain, indicating its excellent potential in neural repair of the damaged brain. Finally, above results clearly indicate that this neuro-regenerative hydrogel is highly capable of maintaining the cholinergic balance through local release of acetylcholine in the injured brain, which is crucial for brain repair.

Designed Tetrapeptide Interacts with Tubulin and Microtubule.

Microtubules regulate eukaryotic cell functions, which have tremendous implication in tumor progression. Thus, the design of novel approaches for controlling microtubule function is extremely important. In this manuscript, a novel tetrapeptide Ser-Leu-Arg-Pro (SLRP) has been designed and synthesized from a small peptide library consisting of 14 tetrapeptides, which perturbs microtubule function through interaction in the "anchor region". We have studied the role of peptides on microtubule function on a chemically functionalized 2D platform. Interestingly, we have found that SLRP binds with tubulin and inhibits the kinesin-driven microtubule motility on a kinesin-immobilized chemically functionalized 2D platform. Further, this peptide modulator interacts with intracellular tubulin/microtubule and depolymerizes the microtubule networks. These interesting findings of perturbation of microtubule function both on engineered platforms and inside the cell by this small peptide modulator inspired us to study the effect of this tetrapeptide on cancer cell proliferation. We found that the novel tetrapeptide modulator causes moderate cytotoxicity to the human breast cancer cell (MCF-7 cell), induces the apoptotic death of MCF-7 cell, and activates the tumor suppressor proteins p53 and cyclin-dependent kinase inhibitor 1 (p21). To the best of our knowledge, this is the shortest peptide discovered, which perturbs microtubule function both on an engineered 2D platform and inside the cell.

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.

Design of a novel microtubule targeted peptide vesicle for delivering different anticancer drugs.

A microtubule targeted peptide-based delivery vehicle has been designed using two oppositely charged peptides, which targets tubulin/microtubules, delivers both hydrophilic and hydrophobic drugs into their target site through lysosome at acidic pH. Drug loaded vesicles show a significant anticancer effect compared to control drugs in a 2D monolayer and a 3D spheroid cell.

Novel hexapeptide interacts with tubulin and microtubules, inhibits Aß fibrillation, and shows significant neuroprotection.

Herein, we report a novel hexapeptide, derived from activity dependent neuroprotective protein (ADNP), that spontaneously self-assembles to form antiparallel ß-sheet structure and produces nanovesicles under physiological conditions. This peptide not only strongly binds with ß-tubulin in the taxol binding site but also binds with the microtubule lattice in vitro as well as in intracellular microtubule networks. Interestingly, it shows inhibition of amyloid fibril formation upon co-incubation with Aß peptide following an interesting mechanistic pathway and excellent neuroprotection in PC12 cells treated with anti-nerve growth factor (NGF). The potential of this hexapeptide opens up a new paradigm in design and development of novel therapeutics for AD.

A short GC rich DNA derived from microbial origin targets tubulin/microtubules and induces apoptotic death of cancer cells.

A short GC rich DNA derived from microbial origin interacts with tubulin/microtubules activates p53 over expression and induces apoptotic death of human breast cancer (MCF-7) cells.