Search

The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.

Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells. [ABSTRACT FROM AUTHOR]

Background: Investigation of the Alzheimer's Disease (AD) brain proteome, along with weighted co‐expression network analysis, revealed a module enriched with proteins involved in inflammation (PMID: 27989508). This module progressively increases in brains from Controls to Asymptomatic AD to those with Symptomatic AD. Moesin (MSN) and CD44 emerged as key inflammation module hub proteins. MSN is a cytoskeletal protein with roles in focal adhesion‐mediated cell motility; CD44 is a transmembrane receptor that interacts with MSN. This project aims to develop chemical tools to disrupt this pathway. Method: To investigate the AD‐relevant distribution of MSN, tissue sections and cells were stained with MSN‐specific antibodies as well as with Iba1, 4G8 and pHF‐Tau antibodies. Purified FERM domains of MSN and EPB41L3 were produced in E. coli and co‐crystallized or soaked with ligands after crystallization. The CD44 ectodomain was expressed in bacteria and refolded or secreted from HEK293 cells. DNA‐encoded library screens were performed as described (http://www.x‐chemrx.com/our‐science/). Result: MSN‐staining in cortical human brain sections localised to endothelial cells and microglia‐like cells. MSN‐positive cells also stained for microglia‐specific marker, Iba1. In AD brains, MSN‐positive microglia‐like cells were present around amyloid plaques and tangle‐like structures. These immunohistochemical studies confirmed the association of MSN with AD pathology. Co‐crystal structures of Moesin and the related FERM protein EPB41L3 revealed the binding mode of the intracellular tail of CD44 to Moesin. This binding interaction was then used to develop HTRF‐based assays for high‐throughput screening for molecules that disrupt the CD44‐MSN interaction. In parallel, a crystal‐based fragment screen using EPB41L3 identified binding pockets and initial hits. Hits and chemical staring points will be further developed with the Open‐AD Medicinal Chemistry core at UNC. To identify inhibitors of CD44 ectodomain binding to hyaluronic acid we screened DNA‐encoded libraries. The screens identified micromolar hits which are being further developed, aiming to obtain potent brain‐penetrant inhibitors of CD44. Conclusion: (1) Moesin expression is associated with AD pathology. (2) The CD44‐Moesin signalling axis was targeted for potential points of intervention. (3) We have developed the assays, structural biology and chemical starting points to develop small‐molecule inhibitors of the CD44 ectodomain and the CD44‐MSN interaction. [ABSTRACT FROM AUTHOR]

Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape, and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains, or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcriptional activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

The recent global COVID-19 pandemic has highlighted treatments for coronavirus infection as an unmet medical need. The main protease (M pro ) has been an important target for the development of SARS-CoV-2 direct-acting antivirals. Nirmatrelvir as a covalent M pro inhibitor was the first such approved therapy. Although M pro inhibitors of various chemical classes have been reported, they are generally less active against nirmatrelvir-resistant variants and have limited pan-coronavirus potential, presenting a significant human health risk upon future outbreaks. We here present a novel approach and utilized DNA-encoded chemical library screening to identify the noncovalent M pro inhibitor 5 , which demonstrated a distinct binding mode to nirmatrelvir. A macrocyclization strategy designed to lock the active conformation resulted in lactone 12 with significantly improved antiviral activity. Further optimization led to the potent lactam 26 , which demonstrated exceptional potency against nirmatrelvir-resistant variants as well as against a panel of viral main proteases from other coronaviruses.

Bfl-1 is overexpressed in both hematological and solid tumors; therefore, inhibitors of Bfl-1 are highly desirable. A DNA-encoded chemical library (DEL) screen against Bfl-1 identified the first known reversible covalent small-molecule ligand for Bfl-1. The binding was validated through biophysical and biochemical techniques, which confirmed the reversible covalent mechanism of action and pointed to binding through Cys55. This represented the first identification of a cyano-acrylamide reversible covalent compound from a DEL screen and highlights further opportunities for covalent drug discovery through DEL screening. A 10-fold improvement in potency was achieved through a systematic SAR exploration of the hit. The more potent analogue compound 13 was successfully cocrystallized in Bfl-1, revealing the binding mode and providing further evidence of a covalent interaction with Cys55., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

DNA-encoded chemical library (DEL) technology provides a time- and cost-efficient method to simultaneously screen billions of compounds for their affinity to a protein target of interest. Here we report its use to identify a novel chemical series of inhibitors of the thioesterase activity of polyketide synthase 13 (Pks13) from Mycobacterium tuberculosis (Mtb). We present three chemically distinct series of inhibitors along with their enzymatic and Mtb whole cell potency, the measure of on-target activity in cells, and the crystal structures of inhibitor-enzyme complexes illuminating their interactions with the active site of the enzyme. One of these inhibitors showed a favorable pharmacokinetic profile and demonstrated efficacy in an acute mouse model of tuberculosis (TB) infection. These findings and assay developments will aid in the advancement of TB drug discovery.

The rapid development of programmable site-specific endonucleases has led to a dramatic increase in genome engineering activities for research and therapeutic purposes. Specific loci of interest in the genomes of a wide range of organisms including mammals can now be modified using zinc-finger nucleases, transcription activator-like effectornucleases, and CRISPR-associated Cas9 endonucleases in a site-specific manner, in some cases requiring relatively modest effort for endonuclease design, construction, and application. While these technologies have made genome engineering widely accessible, the ability of programmable nucleases to cleave off-target sequences can limit their applicability and raise concerns about therapeutic safety. In this chapter, we review methods to evaluate and improve the DNA cleavage activity of programmable site-specific endonucleases and describe a procedure for a comprehensive off-target profiling method based on the in vitro selection of very large (~10(12)-membered) libraries of potential nuclease substrates.