Design and synthesis of nucleic acid nano-environment interactome-targeting small molecule PROTACs and their anticancer activity.

Targeted protein degradation through PROteolysis TArgeting Chimeras (PROTACs) is a relatively new modality in cellular interventions. The minimum requirement for PROTACs to function is forming a tertiary complex of the protein of interest (POI), E3 ligase, and the molecular glue PROTAC. Here, we propose a new approach to modulate the nano-environment interactome of a non-protein target through a plausible quaternary complex of interactome-biomolecule of interest (BOI)-PROTAC and E3 ligase. We report nucleic acid-targeting PROTAC (NA-TAC) molecules by conjugating DNA-binding and E3 ligase ligands. We demonstrate that NA-TACs can target the G-quadruplex DNA and induce elevated DNA damage and cytotoxicity compared to the conventional G-quadruplex binding ligands. Our new class of NA-TACs lays the foundation for small molecule-based non-protein targeting PROTACs for interactome and nanoenvironment mapping and nucleic acid-targeted precision medicines.

Cysteine-independent CRISPR-Associated Protein Labeling for Presentation and Co-delivery of Molecules Toward Genetic and Epigenetic Regulations.

Labeling of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins (Cas) remains an immense challenge for their genome engineering applications. To date, cysteine-mediated bioconjugation is the most efficient strategy for labeling Cas proteins. However, introducing a cysteine residue in the protein at the right place might be challenging without perturbing the enzymatic activity. We report a method that does not require cysteine residues for small molecule presentation on the CRISPR-associated protein SpCas9 for in vitro protein detection, probing cellular protein expression, and nuclear co-delivery of molecules in mammalian cells. We repurposed a simple protein purification tag His6 peptide for non-covalent labeling of molecules on the CRISPR enzyme SpCas9. The small molecule labeling enabled us to rapidly detect SpCas9 in a biochemical assay. We demonstrate that small molecule labeling can be utilized for probing bacterial protein expression in realtime. Furthermore, we coupled SpCas9's nuclear-targeting ability in co-delivering the presenting small molecules to the mammalian cell nucleus for prospective genome engineering applications. Furthermore, we demonstrate that the method can be generalized to label oligonucleotides for multiplexing CRISPR-based genome editing and template-mediated DNA repair applications. This work paves the way for genomic loci-specific bioactive small molecule and oligonucleotide co-delivery toward genetic and epigenetic regulations.

CRISPR-Based Precision Molecular Diagnostics for Disease Detection and Surveillance.

Sensitive, rapid, and portable molecular diagnostics is the future of disease surveillance, containment, and therapy. The recent SARS-CoV-2 pandemic has reminded us of the vulnerability of lives from ever-evolving pathogens. At the same time, it has provided opportunities to bridge the gap by translating basic molecular biology into therapeutic tools. One such molecular biology technique is CRISPR (clustered regularly interspaced short palindromic repeat) which has revolutionized the field of molecular diagnostics at the need of the hour. The use of CRISPR-Cas systems has been widespread in biology research due to the ease of performing genetic manipulations. In 2012, CRISPR-Cas systems were, for the first time, shown to be reprogrammable, i.e., capable of performing sequence-specific gene editing. This discovery catapulted the field of CRISPR-Cas research and opened many unexplored avenues in the field of gene editing, from basic research to therapeutics. One such field that benefitted greatly from this discovery was molecular diagnostics, as using CRISPR-Cas technologies enabled existing diagnostic methods to become more sensitive, accurate, and portable, a necessity in disease control. This Review aims to capture some of the trajectories and advances made in this arena and provides a comprehensive understanding of the methods and their potential use as point-of-care diagnostics.

A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules.

The need to control the activity and fidelity of CRISPR-associated nucleases has resulted in a demand for inhibitory anti-CRISPR molecules. The small-molecule inhibitor discovery platforms available at present are not generalizable to multiple nuclease classes, only target the initial step in the catalytic activity and require high concentrations of nuclease, resulting in inhibitors with suboptimal attributes, including poor potency. Here we report a high-throughput discovery pipeline consisting of a fluorescence resonance energy transfer-based assay that is generalizable to contemporary and emerging nucleases, operates at low nuclease concentrations and targets all catalytic steps. We applied this pipeline to identify BRD7586, a cell-permeable small-molecule inhibitor of SpCas9 that is twofold more potent than other inhibitors identified to date. Furthermore, unlike the reported inhibitors, BRD7586 enhanced SpCas9 specificity and its activity was independent of the genomic loci, DNA-repair pathway or mode of nuclease delivery. Overall, these studies describe a general pipeline to identify inhibitors of contemporary and emerging CRISPR-associated nucleases.

Rational Design of Silicon-Based Zinc Ionophores.

Ionophores transport ions across biological membranes and have wide-ranging applications, but a platform for their rapid development does not exist. We report a platform for developing ionophores from metal-ion chelators, which are readily available with wide-ranging affinities and specificities, and structural data that can aid rational design. Specifically, we fine-tuned the binding affinity and lipophilicity of a ZnII -chelating ligand by introducing silyl groups proximal to the ZnII -binding pocket, which generated ionophores that performed better than most of the currently known ZnII ionophores. Furthermore, these silicon-based ionophores were specific for ZnII over other metals and exhibited better antibacterial activity and less toxicity to mammalian cells than several known ZnII ionophores, including pyrithione. These studies establish rational design principles for the rapid development of potent and specific ionophores and a new class of antibacterial agents.

Harnessing reaction-based probes to preferentially target pancreatic ß-cells and ß-like cells.

Highly sensitive approaches to target insulin-expressing cells would allow more effective imaging, sorting, and analysis of pancreatic ß-cells. Here, we introduce the use of a reaction-based probe, diacetylated Zinpyr1 (DA-ZP1), to image pancreatic ß-cells and ß-like cells derived from human pluripotent stem cells. We harness the high intracellular zinc concentration of ß-cells to induce a fluorescence signal in cells after administration of DA-ZP1. Given its specificity and rapid uptake by cells, we used DA-ZP1 to purify live stem cell-derived ß-like cells as confirmed by immunostaining analysis. We tested the ability of DA-ZP1 to image transplanted human islet grafts and endogenous mouse pancreatic islets in vivo after its systemic administration into mice. Thus, DA-ZP1 enables purification of insulin-secreting ß-like cells for downstream applications, such as functional studies, gene-expression, and cell-cell interaction analyses and can be used to label engrafted human islets and endogenous mouse islets in vivo.

Native Zinc Catalyzes Selective and Traceless Release of Small Molecules in ß-Cells.

The loss of insulin-producing ß-cells is the central pathological event in type 1 and 2 diabetes, which has led to efforts to identify molecules to promote ß-cell proliferation, protection, and imaging. However, the lack of ß-cell specificity of these molecules jeopardizes their therapeutic potential. A general platform for selective release of small-molecule cargoes in ß-cells over other islet cells ex vivo or other cell-types in an organismal context will be immensely valuable in advancing diabetes research and therapeutic development. Here, we leverage the unusually high Zn(II) concentration in ß-cells to develop a Zn(II)-based prodrug system to selectively and tracelessly deliver bioactive small molecules and fluorophores to ß-cells. The Zn(II)-targeting mechanism enriches the inactive cargo in ß-cells as compared to other pancreatic cells; importantly, Zn(II)-mediated hydrolysis triggers cargo activation. This prodrug system, with modular components that allow for fine-tuning selectivity, should enable the safer and more effective targeting of ß-cells.

A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9.

The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh <500 Da and are cell permeable, reversible, and stable under physiological conditions. We developed a suite of high-throughput assays for SpCas9 functions, including a primary screening assay for SpCas9 binding to the protospacer adjacent motif, and used these assays to screen a structurally diverse collection of natural-product-like small molecules to ultimately identify compounds that disrupt the SpCas9-DNA interaction. Using these synthetic anti-CRISPR small molecules, we demonstrated dose and temporal control of SpCas9 and catalytically impaired SpCas9 technologies, including transcription activation, and identified a pharmacophore for SpCas9 inhibition using structure-activity relationships. These studies establish a platform for rapidly identifying synthetic, miniature, cell-permeable, and reversible inhibitors against both SpCas9 and next-generation CRISPR-associated nucleases.

A Singular System with Precise Dosing and Spatiotemporal Control of CRISPR-Cas9.

Several genome engineering applications of CRISPR-Cas9, an RNA-guided DNA endonuclease, require precision control of Cas9 activity over dosage, timing, and targeted site in an organism. While some control of Cas9 activity over dose and time have been achieved using small molecules, and spatial control using light, no singular system with control over all the three attributes exists. Furthermore, the reported small-molecule systems lack wide dynamic range, have background activity in the absence of the small-molecule controller, and are not biologically inert, while the optogenetic systems require prolonged exposure to high-intensity light. We previously reported a small-molecule-controlled Cas9 system with some dosage and temporal control. By photocaging this Cas9 activator to render it biologically inert and photoactivatable, and employing next-generation protein engineering approaches, we have built a system with a wide dynamic range, low background, and fast photoactivation using a low-intensity light while rendering the small-molecule activator biologically inert. We anticipate these precision controls will propel the development of practical applications of Cas9.

Precision Control of CRISPR-Cas9 Using Small Molecules and Light.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas system is an adaptive immune system of bacteria that has furnished several RNA-guided DNA endonucleases (e.g., Cas9) that are revolutionizing the field of genome engineering. Cas9 is being used to effect genomic alterations as well as in gene drives, where a particular trait may be propagated through a targeted species population over several generations. The ease of targeting catalytically impaired Cas9 to any genomic loci has led to development of technologies for base editing, chromatin imaging and modeling, epigenetic editing, and gene regulation. Unsurprisingly, Cas9 is being developed for numerous applications in biotechnology and biomedical research and as a gene therapy agent for multiple pathologies. There is a need for precise control of Cas9 activity over several dimensions, including those of dose, time, and space in these applications. Such precision controls, which are required of therapeutic agents, are particularly important for Cas9 as off-target effects, chromosomal translocations, immunogenic response, genotoxicity, and embryonic mosaicism are observed at elevated levels and with prolonged activity of Cas9. Here, we provide a perspective on advances in the precision control of Cas9 over aforementioned dimensions using external stimuli (e.g., small molecules or light) for controlled activation, inhibition, or degradation of Cas9.

Targeting G-quadruplex DNA structures in the telomere and oncogene promoter regions by benzimidazole‒carbazole ligands.

Recent studies support the idea that G-quadruplex structures in the promoter regions of oncogenes and telomere DNA can serve as potential therapeutic targets in the treatment of cancer. Accordingly, several different types of organic small molecules that stabilize G-quadruplex structures and inhibit telomerase activity have been discerned. Here, we describe the binding of benzimidazole-carbazole ligands to G-quadruplex structures formed in G-rich DNA sequences containing the promoter regions of human c-MYC, c-KIT1, c-KIT2, VEGF and BCL2 proto-oncogenes. The fluorescence spectroscopic data indicate that benzimidazole-carbazole ligands bind and stabilize the G-quadruplexes in the promoter region of oncogenes. The molecular docking studies provide insights into the mode and extent of binding of this class of ligands to the G-quadruplexes formed in oncogene promoters. The high stability of these G-quadruplex structures was validated by thermal denaturation and telomerase-catalyzed extension of the 3' end. Notably, benzimidazole-carbazole ligands suppress the expression of oncogenes in cancer cells in a dose-dependent manner. We anticipate that benzimidazole-carbazole ligands, by virtue of their ability to stabilize G-quadruplex structures in the promoter regions of oncogenes, might reduce the risk of cancer through the loss of function in the proteins encoded by these genes.

Motion-Induced Changes in Emission as an Effective Strategy for the Ratiometric Probing of Human Serum Albumin and Trypsin in Biological Fluids.

Herein, we report the formation of a highly luminescent, pH-sensitive, thermoreversible nanoaggregate in pure aqueous medium through the self-agglomeration of carbazole-based amphiphiles. The self-assembly process restricted the intramolecular motion of the molecules and induced a change in its emission signal from blue to cyan, owing to an aggregation-induced emission (AIE) effect. A similar type of ratiometric response was also observed in the presence of human serum albumin (HSA). However, in this case, the molecular motion of the flexible fluorescent probe was restricted by its embedded microenvironment, owing to a motion-induced change in emission (MICE) effect, not by aggregation. Moreover, the probe showed quite high selectivity for HSA over other serum albumin proteins. Our carbazole-based fluorescent probes are a unique example of the ratiometric sensing of HSA through the sole involvement of reversible noncovalent interactions. Considering the important of HSA in clinical diagnosis, a wide range of biological fluids, such as human urine, saliva, and plasma, were screened to analyze their HSA content. In addition, this system was also employed for the detection of trypsin at subnanomolar concentrations through the digestion of HSA.

A Versatile Probe for Caffeine Detection in Real-Life Samples via Excitation-Triggered Alteration in the Sensing Behavior of Fluorescent Organic Nanoaggregates.

Excitation triggered alteration in the sensing behavior of fluorescent nanoaggregates was explored in water, considering caffeine as the "target analyte". Merely by changing the excitation wavelength, we could specifically excite either the monomeric species or the fluorescent nanoaggregates. The monomer showed highly sensitive interaction with caffeine despite poor selectivity, while the "strongly associated" fluorescent nanoaggregates displayed relatively high selectivity with low sensitivity. In addition, the extent of self-aggregation was also found to be influenced by the micropolarity of the local surroundings and the electronics of the core carbazole unit. Furthermore, the present protocol was utilized for the estimation of caffeine in different beverages and biological fluids with reasonably high accuracy. Inexpensive, portable paper strips were designed for a rapid, on-site detection of caffeine without involving sophisticated instruments or trained technicians.

Novel ruthenium azo-quinoline complexes with enhanced photonuclease activity in human cancer cells.

Coordinatively saturated ruthenium complexes with a variable net charge are currently under intense investigation for their anticancer potential. These complexes, possessing long wavelength metal-to-ligand charge transfer with DNA photonuclease activity, have shown promising cytotoxic profiles. Although most of the ruthenium complexes exhibit significant photochemotherapeutic activity, their poor entry into cells hinder their development as potential drug molecules. Here, we report the synthesis and characterization of four new ruthenium (II) azo-8-hydroxyquinoline complexes, their mode of in vitro DNA binding and antiproliferative properties against cultured human cancer cell lines. The activity of these compounds prior to photoirradiation is minimal. However, they could induce DNA photonuclease activity through the generation of reactive oxygen species upon exposure to light. The activities exhibited by these complexes were found to be more efficient (>5-fold) than cisplatin, emphasizing their therapeutic potential. Collectively, these results support the idea that ruthenium (II) azo-8-hydroxyquinoline complexes can serve as potential agents in photodynamic anticancer therapy.

Transcription regulation of CDKN1A (p21/CIP1/WAF1) by TRF2 is epigenetically controlled through the REST repressor complex.

We observed extra-telomeric binding of the telomere repeat binding factor TRF2 within the promoter of the cyclin-dependent kinase CDKNIA (p21/CIP1/WAF1). This result in TRF2 induced transcription repression of p21. Interestingly, p21 repression was through engagement of the REST-coREST-LSD1-repressor complex and altered histone marks at the p21 promoter in a TRF2-dependent fashion. Furthermore, mutational analysis shows p21 repression requires interaction of TRF2 with a p21 promoter G-quadruplex. Physiologically, TRF2-mediated p21 repression attenuated drug-induced activation of cellular DNA damage response by evading G2/M arrest in cancer cells. Together these reveal for the first time role of TRF2 in REST- repressor complex mediated transcription repression.

Multidimensional chemical control of CRISPR-Cas9.

Cas9-based technologies have transformed genome engineering and the interrogation of genomic functions, but methods to control such technologies across numerous dimensions-including dose, time, specificity, and mutually exclusive modulation of multiple genes-are still lacking. We conferred such multidimensional controls to diverse Cas9 systems by leveraging small-molecule-regulated protein degron domains. Application of our strategy to both Cas9-mediated genome editing and transcriptional activities opens new avenues for systematic genome interrogation.

Discovery and Structural Characterization of G-quadruplex DNA in Human Acetyl-CoA Carboxylase Gene Promoters: Its Role in Transcriptional Regulation and as a Therapeutic Target for Human Disease.

Accumulating evidence suggests that G-quadruplexes play vital roles in gene expression, DNA replication, and recombination. Three distinct promoters (PI, PII, and PIII) regulate human acetyl-CoA carboxylase 1 (ACC1) gene expression. In this study, we asked whether the G-rich sequences within the human ACC1 (PI and PII) promoters can form G-quadruplex structures and regulate normal DNA transactions. Using multiple complementary methods, we show that G-rich sequences of PI and PII promoters form intramolecular G-quadruplex structures and then establish unambiguously the topologies of these structures. Importantly, G-quadruplex formation in ACC1 gene promoter region blocks DNA replication and suppresses transcription, and this effect was further augmented by G-quadruplex stabilizing ligands. Altogether, these results are consistent with the notion that G-quadruplex structures exist within the human ACC1 gene promoter region, whose activity can be suppressed by G-quadruplex stabilizing ligands, thereby revealing a novel regulatory mechanism of ACC1 gene expression and as a possible therapeutic target.

New dimeric carbazole-benzimidazole mixed ligands for the stabilization of human telomeric G-quadruplex DNA and as telomerase inhibitors. A remarkable influence of the spacer.

The development of G-quadruplex (G4) DNA binding small molecules has become an important strategy for selectively targeting cancer cells. Herein, we report the design and evolution of a new kind of carbazole-based benzimidazole dimers for their efficient telomerase inhibition activity. Spectroscopic titrations reveal the ligands high affinity toward the G4 DNA with significantly higher selectivity over duplex-DNA. The electrophoretic mobility shift assay shows that the ligands efficiently promote the formation of G4 DNA even at a lower concentration of the stabilizing K(+) ions. The TRAP-LIG assay demonstrates the ligand's potential telomerase inhibition activity and also establishes that the activity proceeds via G4 DNA stabilization. An efficient nuclear internalization of the ligands in several common cancer cells (HeLa, HT1080, and A549) also enabled differentiation between normal HFF cells in co-cultures of cancer and normal ones. The ligands induce significant apoptotic response and antiproliferative activity toward cancer cells selectively when compared to the normal cells.

Design and synthesis of new benzimidazole-carbazole conjugates for the stabilization of human telomeric DNA, telomerase inhibition, and their selective action on cancer cells.

Cell-permeable small molecules that enhance the stability of the G-quadruplex (G4) DNA structures are currently among the most intensively pursued ligands for inhibition of the telomerase activity. Herein we report the design and syntheses of four novel benzimidazole-carbazole conjugates and demonstrate their high binding affinity to G4 DNA. S1 nuclease assay confirmed the ligand mediated G-quadruplex DNA protection. Additional evidence from Telomeric Repeat Amplification Protocol (TRAP-LIG) assay demonstrated efficient telomerase inhibition activity by the ligands. Two of the ligands showed IC50 values in the sub-micromolar range in the TRAP-LIG assay, which are the best among the benzimidazole derivatives reported so far. The ligands also exhibited cancer cell selective nuclear internalization, nuclear condensation, fragmentation, and eventually antiproliferative activity in long-term cell viability assays. Annexin V-FITC/PI staining assays confirm that the cell death induced by the ligands follows an apoptotic pathway. An insight into the mode of ligand binding was obtained from the molecular dynamics simulations.

Advances in the molecular design of potential anticancer agents via targeting of human telomeric DNA.

Telomerases are an attractive drug target to develop new generation drugs against cancer. A telomere appears from the chromosomal termini and protects it from double-stranded DNA degradation. A short telomere promotes genomic instability, like end-to-end fusion and regulates the over-expression of the telomere repairing enzyme, telomerase. The telomerase maintains the telomere length, which may lead to genetically abnormal situations, leading to cancer. Thus, the design and synthesis of an efficient telomerase inhibitor is a viable strategy toward anticancer drugs development. Accordingly, small molecule induced stabilization of the G-quadruplex structure, formed by the human telomeric DNA, is an area of contemporary scientific art. Several such compounds efficiently stabilize the G-quadruplex forms of nucleic acids, which often leads to telomerase inhibition. This Feature article presents the discovery and development of the telomere structure, function and evolution in telomere targeted anticancer drug design and incorporates the recent advances in this area, in addition to discussing the advantages and disadvantages in the methods, and prospects for the future.