Biphasic modulation of tau liquid-liquid phase separation by polyphenols.

Molecular tools that modulate tau liquid-liquid phase separation (LLPS) promise to treat tauopathies. We screened a set of polyphenols and demonstrated concentration-dependent biphasic modulation of tau LLPS by gallic acid (GA), showcasing its ability to expedite the liquid-to-gel transition in tau condensates and effectively impede the formation of deleterious fibrillar aggregates.

G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer.

P38γ (MAPK12) is predominantly expressed in triple negative breast cancer cells (TNBC) and induces stem cell (CSC) expansion resulting in decreased survival of the patients due to metastasis. Abundance of G-rich sequences at MAPK12 promoter implied the functional probability to reverse tumorigenesis, though the formation of G-Quadruplex (G4) structures at MAPK12 promoter is elusive. Here, we identified two evolutionary consensus adjacent G4 motifs upstream of the MAPK12 promoter, forming parallel G4 structures. They exist in an equilibria between G4 and duplex, regulated by the binding turnover of Sp1 and Nucleolin that bind to these G4 motifs and regulate MAPK12 transcriptional homeostasis. To underscore the gene-regulatory functions of G4 motifs, we employed CRISPR-Cas9 system to eliminate G4s from TNBC cells and synthesized a naphthalene diimide (NDI) derivative (TGS24) which shows high-affinity binding to MAPK12-G4 and inhibits MAPK12 transcription. Deletion of G4 motifs and NDI compound interfere with the recruitment of the transcription factors, inhibiting MAPK12 expression in cancer cells. The molecular basis of NDI-induced G4 transcriptional regulation was analysed by RNA-seq analyses, which revealed that MAPK12-G4 inhibits oncogenic RAS transformation and trans-activation of NANOG. MAPK12-G4 also reduces CD44High/CD24Low population in TNBC cells and downregulates internal stem cell markers, arresting the stemness properties of cancer cells.

Intrinsically Disordered Ku Protein-Derived Cell-Penetrating Peptides.

Efficient delivery of bioactive ingredients into cells is a major challenge. Cell-penetrating peptides (CPPs) have emerged as promising vehicles for this purpose. We have developed novel CPPs derived from the flexible and disordered tail extensions of DNA-binding Ku proteins. Ku-P4, the lead CPP identified in this study, is biocompatible and displays high internalization efficacy. Biophysical studies show that the proline residue is crucial for preserving the intrinsically disordered state and biocompatibility. DNA binding studies showed effective DNA condensation to form a positively charged polyplex. The polyplex exhibited effective penetration through the cell membrane and delivered the plasmid DNA inside the cell. These novel CPPs have the potential to enhance the cellular uptake and therapeutic efficacy of peptide-drug or gene conjugates.

Small molecules and conjugates as theranostic agents.

Theranostics, the integration of therapy and diagnostics into a single entity for the purpose of monitoring disease progression and treatment response. Diagnostics involves identifying specific characteristics of a disease, while therapeutics refers to the treatment of the disease based on this identification. Advancements in medicinal chemistry and technology have led to the development of drug modalities that provide targeted therapeutic effects while also providing real-time updates on disease progression and treatment. The inclusion of imaging in therapy has significantly improved the prognosis of devastating diseases such as cancer and neurodegeneration. Currently, theranostic treatment approaches are based on nuclear medicine, while nanomedicine and a wide diversity of macromolecular systems such as gels, polymers, aptamers, and dendrimer-based agents are being developed for the purpose. Theranostic agents have significant roles to play in both early-stage drug development and clinical-stage therapeutic-containing drug candidates. This review will briefly outline the pros and cons of existing and evolving theranostic approaches before comprehensively discussing the role of small molecules and their conjugates.

A natural polyphenol activates and enhances GPX4 to mitigate amyloid-ß induced ferroptosis in Alzheimer's disease.

Ferroptosis, an iron-dependent cell death, plays a crucial role in the pathology of Alzheimer's disease (AD). Several characteristics of AD, including excessive iron accumulation, elevated lipid peroxide and reactive oxygen species (ROS) levels, and decreased glutathione peroxidase 4 (GPX4) levels, align with the features of ferroptosis. While traditional methods of inhibiting ferroptosis have centered on chelating Fe and trapping radicals, therapeutic strategies that modulate the GPX4 axis to mitigate ferroptosis in AD are yet to be explored. This report introduces naturally occurring polyphenols (PPs) as dual-acting therapeutic agents to synergistically alleviate ferroptosis and AD. The mechanisms of action encompass modulation of amyloid and tau cascade, reduction of oxidative stress, mitochondrial rescue, and inhibition of ferroptosis. For the first time, we show that a single multifunctional molecule, tannic acid (TA) binds at the activator site of GPX4, augmenting both its activity and cellular levels, providing a conceptually innovative and integrated approach for treating AD via the GPX4-ferroptosis axis. The ability of TA to enhance GPX4 levels under conditions of AD pathology opens up newer promising therapeutic avenues for combating the crosstalk between ferroptosis and AD.

Cyclic dipeptide-based small molecules modulate zinc-mediated liquid-liquid phase separation of tau.

Liquid-liquid phase separation (LLPS) is a complex physicochemical phenomenon mediated by multivalent transient weak interactions among macromolecules like polymers, proteins, and nucleic acids. It has implications in cellular physiology and disease conditions like cancer and neurodegenerative disorders. Many proteins associated with neurodegenerative disorders like RNA binding protein FUS (FUsed in Sarcoma), alpha-synuclein (α-Syn), TAR DNA binding protein 43 (TDP-43), and tau are shown to undergo LLPS. Recently, the tau protein responsible for Alzheimer's disease (AD) and other tauopathies is shown to phase separate into condensates in vitro and in vivo. The diverse noncovalent interactions among the biomolecules dictate the complex LLPS phenomenon. There are limited chemical tools to modulate protein LLPS which has therapeutic potential for neurodegenerative disorders. We have rationally designed cyclic dipeptide (CDP)-based small-molecule modulators (SMMs) by integrating multiple chemical groups that offer diverse chemical interactions to modulate tau LLPS. Among them, compound 1c effectively inhibits and dissolves Zn-mediated tau LLPS condensates. The SMM also inhibits tau condensate-to-fibril transition (tau aggregation through LLPS). This approach of designing SMMs of LLPS establishes a novel platform that has potential implication for the development of therapeutics for neurodegenerative disorders.

Rationally Designed Molecules Synergistically Modulate Multifaceted Aß Toxicity, Microglial Activation, and Neuroinflammation.

Synergistic modulation of multifaceted toxicity is the key to tackle multifactorial Alzheimer's disease (AD). The etiology of AD includes amyloid ß (Aß) amyloidosis, metal ion dyshomeostasis, reactive oxygen species (ROS), oxidative stress, mitochondrial damage, and neuroinflammation. We rationally designed multifunctional modulators by integrating pharmacophores for metal chelation, antioxidant and anti-inflammatory properties, and modulation of Aß42 aggregation on the naphthalene monoimide (NMI) scaffold. The in vitro and cellular studies of NMIs revealed that M3 synergistically modulates metal-independent and -dependent amyloid toxicity, scavenges ROS, alleviates oxidative stress, and emulates Nrf2-mediated stress response in neuronal cells. M3 effectively reduced structural and functional damage of mitochondria, reduced Cyt c levels, and rescued cells from apoptosis. The biological atomic force microscopy and Western blot analysis revealed the ability of M3 to suppress microglial activation and neuroinflammation through inhibition of the NF-κß pathway. The synergistic action of M3 is in agreement with our design strategy to develop a multifunctional therapeutic candidate by integrating multiple pharmacophores with distinct structural and functional elements to ameliorate the multifaceted toxicity of AD.

Multifunctional molecules with a bipyridyl core ameliorate multifaceted amyloid toxicity.

A series of 2,2'-bipyridine derivatives appended with structurally unique biomolecular auxiliaries were synthesized and investigated for their ability to ameliorate multifaceted amyloid toxicity. Our results highlight the roles of the metal-chelating bipyridine core and functional auxiliaries in effectively modulating metal-independent and -dependent amyloid toxicity, and oxidative stress observed in Alzheimer's disease (AD).

Targeting Oncogene Promoters and Ribosomal RNA Biogenesis by G-Quadruplex Binding Ligands Translate to Anticancer Activity.

G-Quadruplex (GQ) nucleic acids are promising therapeutic targets in anticancer research due to their structural robustness, polymorphism, and gene-regulatory functions. Here, we presented the structure-activity relationship of carbazole-based monocyanine ligands using region-specific functionalization with benzothiazole (TCA and TCZ), lepidine (LCA and LCZ), and quinaldine (QCA and QCZ) acceptor moieties and evaluated their binding profiles with different oncogenic GQs. Their differential turn-on fluorescence emission upon GQ binding confirmed the GQ-to-duplex selectivity of all carbazole ligands, while the isothermal titration calorimetry results showed selective interactions of TCZ and TCA to c-MYC and BCL-2 GQs, respectively. The aldehyde group in TCA favors stacking interactions with the tetrad of BCL-2 GQ, whereas TCZ provides selective groove interactions with c-MYC GQ. Dual-luciferase assay and chromatin immunoprecipitation (ChIP) showed that these molecules interfere with the recruitment of specific transcription factors at c-MYC and BCL-2 promoters and stabilize the promoter GQ structures to inhibit their constitutive transcription in cancer cells. Their intrinsic turn-on fluorescence response with longer lifetimes upon GQ binding allowed real-time visualization of GQ structures at subcellular compartments. Confocal microscopy revealed the uptake of these ligands in the nucleoli, resulting in nucleolar stress. ChIP studies further confirmed the inhibition of Nucleolin occupancy at multiple GQ-enriched regions of ribosomal DNA (rDNA) promoters, which arrested rRNA biogenesis. Therefore, carbazole ligands act as the "double-edged swords" to arrest c-MYC and BCL-2 overexpression as well as rRNA biogenesis, triggering synergistic inhibition of multiple oncogenic pathways and apoptosis in cancer cells.

Cyclic Dipeptide: A Privileged Molecular Scaffold to Derive Structural Diversity and Functional Utility.

Cyclic dipeptides (CDPs) are the simplest form of cyclic peptides with a wide range of applications from therapeutics to biomaterials. CDP is a versatile molecular platform endowed with unique properties such as conformational rigidity, intermolecular interactions, structural diversification through chemical synthesis, bioavailability and biocompatibility. A variety of natural products with the CDP core exhibit anticancer, antifungal, antibacterial, and antiviral activities. The inherent bioactivities have inspired the development of synthetic analogues as drug candidates and drug delivery systems. CDP plays a crucial role as conformation and molecular assembly directing core in the design of molecular receptors, peptidomimetics and fabrication of functional material architectures. In recent years, CDP has rapidly become a privileged scaffold for the design of advanced drug candidates, drug delivery agents, bioimaging, and biomaterials to mitigate numerous disease conditions. This review describes the structural diversification and multifarious biomedical applications of the CDP scaffold, discusses challenges, and provides future directions for the emerging field.

Molecular Architectonics-guided Design of Biomaterials.

The quest for mastering the controlled engineering of dynamic molecular assemblies is the basis of molecular architectonics. The rational use of noncovalent interactions to programme the molecular assemblies allow the construction of diverse molecular and material architectures with novel functional properties and applications. Understanding and controlling the assembly of molecular systems are daunting tasks owing to the complex factors that govern at the molecular level. Molecular architectures depend on the design of functional molecular modules through the judicious selection of functional core and auxiliary units to guide the precise molecular assembly and co-assembly patterns. Biomolecules with built-in information for molecular recognition are the ultimate examples of evolutionary guided molecular recognition systems that define the structure and functions of living organisms. Explicit use of biomolecules as auxiliary units to command the molecular assemblies of functional molecules is an intriguing exercise in the scheme of molecular architectonics. In this minireview, we discuss the implementation of the principles of molecular architectonics for the development of novel biomaterials with functional properties and applications ranging from sensing, drug delivery to neurogeneration and tissue engineering. We present the molecular designs pioneered by our group owing to the requirement and scope of the article while acknowledging the designs pursued by several research groups that befit the concept.

A matrix targeted fluorescent probe to monitor mitochondrial dynamics.

Mitochondria are an indispensable organelle for energy production and regulation of cellular metabolism. The structural and functional alterations to mitochondria instigate pathological conditions of cancer, and aging-associated and neurodegenerative disorders. The normal functioning of mitochondria is maintained by quality control mechanisms involving dynamic fission, fusion, biogenesis and mitophagy. Under conditions of mitophagy and neurodegenerative diseases, mitochondria are exposed to different acidic environments and high levels of reactive oxygen species (ROS). Therefore stable molecular tools and methods are required to monitor the pathways linked to mitochondrial dysfunction and disease conditions. Herein, we report a far-red fluorescent probe (Mito-TG) with excellent biocompatibility, biostability, photostability, chemical stability and turn on emission for selective targeting of the mitochondrial matrix in different live cells. The probe was successfully employed for monitoring dynamic processes of mitophagy and amyloid beta (Aß) induced mitochondrial structural changes.

Dendrimer Architectonics to Treat Cancer and Neurodegenerative Diseases with Implications in Theranostics and Personalized Medicine.

Integration of diagnostic and therapeutic functions in a single platform namely theranostics has become a cornerstone for personalized medicine. Theranostics platform facilitates noninvasive detection and treatment while allowing the monitoring of disease progression and therapeutic efficacy in case of chronic conditions of cancer and Alzheimer's disease (AD). Theranostic tools function by themselves or with the aid of carrier, viz. liposomes, micelles, polymers, or dendrimers. The dendrimer architectures (DA) are well-characterized molecular nanoobjects with a large number of terminal functional groups to enhance solubility and offer multivalency and multifunctional properties. Various noninvasive diagnostic tools like magnetic resonance imaging (MRI), computed tomography (CT), gamma scintigraphy, and optical techniques have been accomplished utilizing DAs for simultaneous imaging and drug delivery. Obstacles in the formulation design, drug loading, payload delivery, biocompatibility, overcoming cellular membrane and blood-brain barrier (BBB), and systemic circulation remain a bottleneck in translational efforts. This review focuses on the diagnostic, therapeutic and theranostic potential of DA-based nanocarriers in treating cancer and neurodegenerative disorders like AD and Parkinson's disease (PD), among others. In view of the inverse relationship between cancer and AD, designing suitable DA-based theranostic nanodrug with high selectivity has tremendous implications in personalized medicine to treat cancer and neurodegenerative disorders.

Recognition of G-quadruplex topology through hybrid binding with implications in cancer theranostics.

The selective recognition and imaging of oncogene specific G-quadruplex (GQ) structures holds great promise in the development of diagnostic therapy (theranostics) for cancer and has been challenging due to their structural dynamics and diversity. We report selective recognition of GQ by a small molecule through unique hybrid loop stacking and groove binding mode with turn on far-red fluorescence response and anticancer activity demonstrating the potential implications for GQ-targeted cancer theranostics. Methods: Biophysical investigation reveal the turn on far-red emission property of TGP18 for selective recognition of GQ. In cellulo studies including DNA damage and oxidative stress evaluation guided us to perform in vitro (3D spheroid) and in vivo (xenograft mice model) anti-cancer activity, and tumor tissue imaging to assess the theranostic potential of TGP18. Results: Neocuproine-based far-red turn on fluorescence probe TGP18 shows GQ-to-duplex selectivity and specifically recognizes BCL-2 GQ with high affinity through a unique hybrid binding mode involving loop-stacking and groove interactions. Our study reveals that the selective recognition originating from the distinct loop structure of GQ that alters the overall probe interaction and binding affinity. TGP18 binding to anti-apoptotic BCL-2 GQ ablates the pro-survival function and elicit anti-cancer activity by inducing apoptosis in cancer cells. We deciphered that inhibition of BCL-2 transcription synergized with signaling cascade of nucleolar stress, DNA damage and oxidative stress in triggering apoptosis signaling pathway. Conclusion: Intervention of GQ mediated lethality by TGP18 has translated into anti-cancer activity in both in vitro 3D spheroid culture and in vivo xenograft models of lung and breast cancer with superior efficacy for the former. In vivo therapeutic efficacy supplemented with tumor 3D spheroid and tissue imaging potential define the role of TGP18 in GQ-targeted cancer theranostics.

Polyampholyte-Based Synthetic Chaperone Modulate Amyloid Aggregation and Lithium Delivery.

Protein misfolding and aggregation is the pathological hallmark of Alzheimer's disease (AD). The etiopathogenesis of AD involves the accumulation of amyloid-ß (Aß) plaques in the brain, which disrupt the neuronal network and communication, causing neuronal death and severe cognitive impairment. Modulation of Aß aggregation by exogenous therapeutic agents is considered an effective strategy to treat AD. Frequent failure of drug candidates in various phases of clinical trials reiterates the need for alternative therapeutic strategies for AD treatment. Polyampholytes with cationic and anionic segments are considered as artificial protein mimics capable of modulating the protein misfolding and aggregation. We report a diblock copolymer of tryptophan-functionalized methacrylic acid (PTMA) polyampholyte synthesized through reversible addition-fragmentation chain transfer (RAFT) polymerization. Investigation revealed that PTMA acts as a synthetic chaperone to protect the native structure of the lysozyme under heat-induced aggregation conditions. PTMA effectively modulates Aß aggregation and rescues neuronal cells. Lithium has been shown to exhibit therapeutic efficacy in chronic neurological diseases including AD. PTMA sequesters and releases lithium ions in response to neuropathological pH stimuli, making it a promising candidate for lithium transport and delivery. The detailed studies demonstrate PTMA as aggregation modulator and lithium carrier with implications for combinational therapy to treat AD.

Antioxidant Berberine-Derivative Inhibits Multifaceted Amyloid Toxicity.

Multiple lines of evidence indicate that amyloid beta (Aß) peptide is responsible for the pathological devastation caused in Alzheimer's disease (AD). Aß aggregation species predominantly contribute to multifaceted toxicity observed in neuronal cells including generation of reactive oxygen species (ROS), mitochondrial dysfunction, interfering with synaptic signaling, and activation of premature apoptosis. Herein, we report a natural product berberine-derived (Ber-D) multifunctional inhibitor to ameliorate in cellulo multifaceted toxicity of AD. The structural attributes of polyphenolic Ber-D have contributed to its efficient Cu chelation and arresting the redox cycle to prevent the generation of ROS and rescue biomacromolecules from oxidative damage. Ber-D inhibits metal-dependent and -independent Aß aggregation, which is supported by in silico studies. Ber-D treatment averts mitochondrial dysfunction and corresponding neuronal toxicity contributing to premature apoptosis. These key multifunctional attributes make Ber-D a potential therapeutic candidate to ameliorate multifaceted Aß toxicity in AD.

Injectable Silk Fibroin-Based Hydrogel for Sustained Insulin Delivery in Diabetic Rats.

Diabetes is a chronic disease affecting over 400 million people worldwide. Inadequate production of insulin due to loss of beta cells or insulin resistance within the body imbalances the glucose homeostasis, resulting in an abrupt increase of blood glucose level. The conventional and last resort of treatment involves repeated subcutaneous insulin injections to maintain the physiological glucose homeostasis. However, continuous and multiple subcutaneous injections are associated with poor patient compliance and local amyloidosis of insulin, which can be overcome with controlled and sustained insulin delivery. In this context, we have designed and formulated an injectable silk fibroin hydrogel (iSFH) to realize sustained insulin delivery over a prolonged period under diabetic conditions. The specific composition of glycol additives (ethylene glycol and triethylene glycol) allowed the silk fibroin protein to form an injectable hydrogel within 50 min. The detailed characterization of iSFH by a field-emission scanning electron microscope displayed the desired mesoporous structures, which are appropriate for drug (insulin) encapsulation in its active form. Interestingly, the subcutaneous injection of iSFH-encapsulated insulin (insulin-iSFH) in diabetic T1DM Wistar rats showed controlled release of insulin and restored physiological glucose homeostasis up to 4 days. The biocompatible and biodegradable nature of iSFH makes it a potential drug delivery system for active storage, and controlled and sustained delivery of insulin in diabetic conditions to maintain the physiological glucose level.

Molecular Architectonics of Cyclic Dipeptide Amphiphiles and Their Application in Drug Delivery.

Assembly and co-assemblies of peptide amphiphiles through specific noncovalent forces expand the space of molecular architectonics-driven construction of diverse nanoarchitectures with potential biological applications. In this work, cyclic dipeptide amphiphiles (CDPAs) of cyclo(Gly-Asp) with varying lengths of alkyl chains (C8-C18) were synthesized, and their molecular organization was studied. The noncovalent interactions of the components, CDP and alkyl chain, drive the molecular self-assembly of CDPAs into well-defined and diverse nanoarchitectures such as nanotubes, nanospheres, nano/microsheets, and flowers. The co-assembly of CDPAs with biological molecules such as nucleosides was studied to ascertain their utility as potential drug delivery vehicles. Mechanical properties of these nanoarchitectures in nanoindentation study established them as robust in nature. A temperature-dependent NMR study confirmed the formation of stable co-assembly of CDPAs, primarily driven by the intermolecular hydrogen bonding interactions. Computational modeling of oligomers of CDPAs and their co-assembly with nucleosides/nucleotides reveal the molecular level interactions and driving force behind such assemblies. CDPAs exhibit good biocompatibility and cytocompatibility, as revealed by the cellular studies which substantiated their suitability for drug delivery applications. The co-assembly of CDPA with an anticancer drug 5-bromo-2'-deoxyuridine (BrdU) was studied as a drug delivery platform and cytotoxicity was successfully assessed in HeLa cells. Computational modeling of the oligomers of CDPAs and their co-assembly with the drug molecule was performed to understand the molecular level interactions and driving force behind the assemblies. Our findings reveal the design strategy to construct diverse structural architectures using CDP as the modular building unit and specific molecular interactions driven co-assembly for potential application as drug delivery carrier.

Biomolecules-derived biomaterials.

Human civilization has witnessed the use of materials-derived from biomolecules of plants and animal origin for biomedical applications since ancient era. In recent years, precision design principles have been adopted to develop novel biomaterials derived from biomolecules. The biomolecules-derived biomaterials fabrication is dependent on chemical, biochemical and mechanical parameters of biomolecules and their bulk materials. Thus, structural variations and weak noncovalent interactions present within the basic building blocks greatly influence the functional features and applications. This comprehensive review provides one-stop information on recent innovations of various biomaterial-types derived from a diverse class of biomolecules through selected and representative examples with potential biomedical applications ranging from diagnosis, biosensing, antimicrobial efficacy, anticancer therapeutics, drug delivery, bioprinting, bioimaging, tissue engineering and regenerative medicine. The discussion systematically follows the top-down approach in the order of molecular complexity viz., biomacromolecules, oligomers and monomers of all classes of biomolecules (proteins, nucleic acids, carbohydrates and lipids) including a special section on biohybrid materials derived from molecular systems integrated with more than one class of biomolecules. In addition to providing overview of impressive advancements in the area, synergistic integration of biomolecules with synthetic materials to develop smart biomaterials is emphasized to improve the chemical, mechanical, stimuli-responsiveness, immunogenicity and biocompatibility features.

Peptide-modulated self-assembly as a versatile strategy for tumor supramolecular nanotheranostics.

Advances in supramolecular self-assembly have promoted the development of theranostics, the combination of both therapeutic and diagnostic functions in a single nanoplatform, which is closely associated with antitumor applications and has shown promising potential in personalized medicine. Peptide-modulated self-assembly serves as a versatile strategy for tumor supramolecular nanotheranostics possessing controllability, programmability, functionality and biosafety, thus promoting the translation of nanotheranostics from bench to bedside. In this review, we will focus on the self-assembly of peptide-photosensitizers and peptide-drugs as well as multicomponent cooperative self-assembly for the fabrication of nanotheranostics that integrate diagnosis and therapeutics for antitumor applications. Emphasis will be placed on building block design, interaction strategies and the potential relationships between their structures and properties, aiming to increase understanding of the critical role of peptide-modulated self-assembly in advancing antitumor supramolecular nanotheranostics.