Integration of CRISPR/Cas9 with artificial intelligence for improved cancer therapeutics.

Gene editing has great potential in treating diseases caused by well-characterized molecular alterations. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based gene-editing tools has substantially improved the precision and efficiency of gene editing. The CRISPR/Cas9 system offers several advantages over the existing gene-editing approaches, such as its ability to target practically any genomic sequence, enabling the rapid development and deployment of novel CRISPR-mediated knock-out/knock-in methods. CRISPR/Cas9 has been widely used to develop cancer models, validate essential genes as druggable targets, study drug-resistance mechanisms, explore gene non-coding areas, and develop biomarkers. CRISPR gene editing can create more-effective chimeric antigen receptor (CAR)-T cells that are durable, cost-effective, and more readily available. However, further research is needed to define the CRISPR/Cas9 system's pros and cons, establish best practices, and determine social and ethical implications. This review summarizes recent CRISPR/Cas9 developments, particularly in cancer research and immunotherapy, and the potential of CRISPR/Cas9-based screening in developing cancer precision medicine and engineering models for targeted cancer therapy, highlighting the existing challenges and future directions. Lastly, we highlight the role of artificial intelligence in refining the CRISPR system's on-target and off-target effects, a critical factor for the broader application in cancer therapeutics.

Exo-Enzymatic Addition of Diazirine-Modified Sialic Acid to Cell Surfaces Enables Photocrosslinking of Glycoproteins.

Glycan binding often mediates extracellular macromolecular recognition events. Accurate characterization of these binding interactions can be difficult because of dissociation and scrambling that occur during purification and analysis steps. Use of photocrosslinking methods has been pursued to covalently capture glycan-dependent interactions in situ; however, use of metabolic glycan engineering methods to incorporate photocrosslinking sugar analogs is limited to certain cell types. Here, we report an exo-enzymatic labeling method to add a diazirine-modified sialic acid (SiaDAz) to cell surface glycoconjugates. The method involves the chemoenzymatic synthesis of diazirine-modified CMP-sialic acid (CMP-SiaDAz), followed by sialyltransferase-catalyzed addition of SiaDAz to desialylated cell surfaces. Cell surface SiaDAzylation is compatible with multiple cell types and is facilitated by endogenous extracellular sialyltransferase activity present in Daudi B cells. This method for extracellular addition of α2-6-linked SiaDAz enables UV-induced crosslinking of CD22, demonstrating the utility for covalent capture of glycan-mediated binding interactions.

Stereoselective fatty acylation is essential for the release of lipidated WNT proteins from the acyltransferase Porcupine (PORCN).

The maintenance of adult animal tissues depends upon highly conserved intercellular signaling molecules that include the secreted WNT proteins. Although it is generally accepted that lipidation of WNTs by the acyltransferase Porcupine (PORCN) and their subsequent recognition by the Wntless (WLS) protein is essential for their cellular secretion, the molecular understanding of this process remains limited. Using structurally diverse fatty acyl donor analogs and mouse embryonic fibroblasts expressing PORCN protein from different metazoan phyla, we demonstrate here that PORCN active-site features, which are conserved across the animal kingdom, enforce cis-Δ9 fatty acylation of WNTs. Aberrant acylation of a WNT with an exogenously supplied trans-Δ9 fatty acid induced the accumulation of WNT-PORCN complexes, suggesting that the fatty acyl species is critical for the extrication of lipidated WNTs from PORCN. Our findings reveal a previously unrecognized fatty acyl-selective checkpoint in the manufacturing of a lipoprotein that forms a basis for WNT signaling sensitivity to trans fats and to PORCN inhibitors in clinical development.

Regulation of tankyrase activity by a catalytic domain dimer interface.

Tankyrases (TNKS and TNKS2) are enzymes that catalyze poly-ADP-ribosylation (PARsylation) of their target proteins. Tankyrase-mediated PARsylation plays critical regulatory roles in cell signaling, particularly in the Wnt/ß-catenin pathway. The sterile alpha motif (SAM) domain in tankyrases mediates their oligomerization, which is essential for tankyrase function. The oligomerization regulates the catalytic activity of tankyrases, but the underlying mechanism is unclear. Our analyses of crystal structures of the tankyrase catalytic domain suggest that formation of a head-to-head dimer regulates the catalytic activity. Our activity assays show that residues in the catalytic domain dimer interface are important for the PARsylation activity of tankyrases both in solution and cells. The dimer is weak and may only form in the context of the SAM domain-mediated oligomers of tankyrases, consistent with the dependence of the tankyrase activity on the SAM domain.

Clickable photoaffinity ligands for the human serotonin transporter based on the selective serotonin reuptake inhibitor (S)-citalopram.

To date, the development of photoaffinity ligands targeting the human serotonin transporter (hSERT), a key protein involved in disease states such as depression and anxiety, have been radioisotope-based (i.e., 3H or 125I). This letter instead highlights three derivatives of the selective serotonin reuptake inhibitor (SSRI) (S)-citalopram that were rationally designed and synthesized to contain a photoreactive benzophenone or an aryl azide for protein target capture via photoaffinity labeling and a terminal alkyne or an aliphatic azide for click chemistry-based proteomics. Specifically, clickable benzophenone-based (S)-citalopram photoprobe 6 (hSERT Ki = 0.16 nM) displayed 11-fold higher binding affinity at hSERT when compared to (S)-citalopram (hSERT Ki = 1.77 nM), and was subsequently shown to successfully undergo tandem photoaffinity labeling-biorthogonal conjugation using purified hSERT. Given clickable photoprobes can be used for various applications depending on which reporter is attached by click chemistry subsequent to photoaffinity labeling, photoprobe 6 is expected to find value in structure-function studies and other research applications involving hSERT (e.g., imaging).

Development of a triazole class of highly potent Porcn inhibitors.

The acyltransferase Porcupine (Porcn) is essential for the secretion of Wnt proteins which contribute to embryonic development, tissue regeneration, and tumorigenesis. We have previously discovered four molecular scaffolds harboring Porcn-inhibitory activity. Comparison of their structures led to the identification of a general scaffold that can be readily assembled by modular synthesis. We report herein the development of a triazole version of this new class of Porcn inhibitors. This study yielded IWP-O1, a Porcn inhibitor with an EC50 value of 80pM in a cultured cell reporter assay of Wnt signaling. Additionally, IWP-O1 has significantly improved metabolic stability over our previously reported Porcn inhibitors.

4-Deoxy-4-fluoro-GalNAz (4FGalNAz) Is a Metabolic Chemical Reporter of O-GlcNAc Modifications, Highlighting the Notable Substrate Flexibility of O-GlcNAc Transferase.

Bio-orthogonal chemistries have revolutionized many fields. For example, metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain a bio-orthogonal functionality, such as azides or alkynes. MCRs are metabolically incorporated into glycoproteins by living systems, and bio-orthogonal reactions can be subsequently employed to install visualization and enrichment tags. Unfortunately, most MCRs are not selective for one class of glycosylation (e.g., N-linked vs O-linked), complicating the types of information that can be gleaned. We and others have successfully created MCRs that are selective for intracellular O-GlcNAc modification by altering the structure of the MCR and thus biasing it to certain metabolic pathways and/or O-GlcNAc transferase (OGT). Here, we attempt to do the same for the core GalNAc residue of mucin O-linked glycosylation. The most widely applied MCR for mucin O-linked glycosylation, GalNAz, can be enzymatically epimerized at the 4-hydroxyl to give GlcNAz. This results in a mixture of cell-surface and O-GlcNAc labeling. We reasoned that replacing the 4-hydroxyl of GalNAz with a fluorine would lock the stereochemistry of this position in place, causing the MCR to be more selective. After synthesis, we found that 4FGalNAz labels a variety of proteins in mammalian cells and does not perturb endogenous glycosylation pathways unlike 4FGalNAc. However, through subsequent proteomic and biochemical characterization, we found that 4FGalNAz does not widely label cell-surface glycoproteins but instead is primarily a substrate for OGT. Although these results are somewhat unexpected, they once again highlight the large substrate flexibility of OGT, with interesting and important implications for intracellular protein modification by a potential range of abiotic and native monosaccharides.

Azido-iodo-N-benzyl derivatives of threo-methylphenidate (Ritalin, Concerta): Rational design, synthesis, pharmacological evaluation, and dopamine transporter photoaffinity labeling.

In contrast to tropane-based compounds such as benztropine and cocaine, non-tropane-based photoaffinity ligands for the dopamine transporter (DAT) are relatively unexplored. Towards addressing this knowledge gap, ligands were synthesized in which the piperidine nitrogen of 3- and 4-iodomethylphenidate was substituted with a benzyl group bearing a photoreactive azide. Analog (±)-3a demonstrated modest DAT affinity and a radioiodinated version was shown to bind covalently to rat striatal DAT and hDAT expressed in cultured cells. Co-incubation of (±)-3a with nonradioactive d-(+)-methylphenidate or (-)-2-ß-carbomethoxy-3-ß-(4-fluorophenyl)tropane (ß-CFT, WIN-35,428, a cocaine analog) blocked DAT labeling. Compound (±)-3a represents the first successful example of a DAT photoaffinity ligand based on the methylphenidate scaffold. Such ligands are expected to assist in mapping non-tropane ligand-binding pockets within plasma membrane monoamine transporters.

Monitoring Wnt Protein Acylation Using an In Vitro Cyclo-Addition Reaction.

We describe here a technique for visualizing the lipidation status of Wnt proteins using azide-alkyne cycloaddition chemistry (click chemistry) and SDS-PAGE. This protocol incorporates in vivo labeling of a Wnt-IgG Fc fusion protein using an alkynylated palmitate probe but departs from a traditional approach by incorporating a secondary cycloaddition reaction performed on single-step purified Wnt protein immobilized on protein A resin. This approach mitigates experimental noise by decreasing the contribution of labeling from other palmitoylated proteins and by providing a robust method for normalizing labeling efficiency based on protein abundance.

Novel Azido-Iodo Photoaffinity Ligands for the Human Serotonin Transporter Based on the Selective Serotonin Reuptake Inhibitor (S)-Citalopram.

Three photoaffinity ligands (PALs) for the human serotonin transporter (hSERT) were synthesized based on the selective serotonin reuptake inhibitor (SSRI), (S)-citalopram (1). The classic 4-azido-3-iodo-phenyl group was appended to either the C-1 or C-5 position of the parent molecule, with variable-length linkers, to generate ligands 15, 22, and 26. These ligands retained high to moderate affinity binding (K(i) = 24-227 nM) for hSERT, as assessed by [(3)H]5-HT transport inhibition. When tested against Ser438Thr hSERT, all three PALs showed dramatic rightward shifts in inhibitory potency, with Ki values ranging from 3.8 to 9.9 µM, consistent with the role of Ser438 as a key residue for high-affinity binding of many SSRIs, including (S)-citalopram. Photoactivation studies demonstrated irreversible adduction to hSERT by all ligands, but the reduced (S)-citalopram inhibition of labeling by [(125)I]15 compared to that by [(125)I]22 and [(125)I]26 suggests differences in binding mode(s). These radioligands will be useful for characterizing the drug-protein binding interactions for (S)-citalopram at hSERT.

Evolution of a Compact Photoprobe for the Dopamine Transporter Based on (±)-threo-Methylphenidate.

The development of photoaffinity ligands for determining covalent points of attachment to the dopamine transporter (DAT) has predominantly focused on tropane-based compounds bearing variable-length linkers between the photoreactive group and inhibitor pharmacophore. In order to expand the array of photoprobes useful for mapping inhibitor-binding pockets within the DAT, a compact non-tropane ligand was synthesized featuring a photoreactive azide and iodine tag directly attached to the aromatic ring of (±)-threo-methylphenidate. (±)-threo-4-Azido-3-iodomethylphenidate ((±)-6); K(i) = 4.0 ± 0.8 nM) displayed high affinity for hDAT. Moreover, a radioiodinated analog of (±)-6 demonstrated covalent ligation to the DAT in cultured cells and rat striatal membranes, thus suggesting the potential utility of this photoprobe in DAT structure-function studies.