Mincle, a member of the C-type lectin receptor (CLR) family, detects various glycolipids and glycerolipids such as trehalose dimycolate (TDM) from Mycobacterium tuberculosis, leading to the activation of the innate immune system. In this study, we developed new fluorescence-labeled molecular probes, TDE-Fluor-Ligand and TDE-Reactive-Probe, based on the structure of trehalose diester to elucidate the intracellular behavior of Mincle and its ligands. TDE-Fluor-Ligand was prepared for the ligand analysis, and TDE-Reactive-Probe was specifically designed to label Mincle by turn-on fluorescent affinity labeling. Live-cell imaging analysis using these probes revealed that TDE-Fluor-Ligand internalizes into the cell in a Mincle-dependent manner. Furthermore, imaging analysis using TDE-Reactive-Probe successfully detected Mincle in cells in a Mincle expression-dependent manner.

Hydrogen sulfide (H2S) is an important gasotransmitter that has shown many physiological effects, ranging from anti-inflammation to antioxidation. To advance research on H2S, donor compounds that can slowly release H2S in biological conditions while producing minimal bioactive byproducts are essential. Herein, we report the evaluation of thiosaccharides as hydrolysis-based H2S donors. These compounds were found to slowly produce H2S over days, in aqueous buffers and in cells. Their H2S release rates could be affected by the hydroxyl protection groups of thiosaccharides, with faster release by electron-donating groups and slower release by electron-withdrawing groups. We also demonstrated the vasodilatory effect of 1-thioglucose using arterial rings isolated from adult ewes, which is likely due to H2S release. Altogether, thiosaccharides might be suitable slow-releasing H2S donors for biological applications.

A mechanistic basis for luciferase bioluminescence provides a glimpse into its evolutionary role for organism survival, as it provides a blueprint to engineer luciferase enzymes for advanced technological applications. Gaussia Luciferase is among the brightest natural luciferases, but (1) the evolutionary development of its luminescence behavior remains unclear, (2) recent fundamental studies utilized Escherichia coli expression systems instead of eukaryotic expression systems, and (3) notable mutants have been discovered but not integrated into a comprehensive mechanistic analysis. We describe new mechanistic observations from GLuc by addressing these gaps. We monitored the fluorescent coelenterazine-to-coelenteramide conversion to study turnover kinetics of mammalian-derived GLuc; this assay characterized the positive cooperativity kinetics of GLuc. The nonluminescent mutants, R76A and R147A, still turn over the substrate with high efficiency, each demonstrating sustained positive cooperativity. Through mass spectrometry, mutational analysis, and analytical liquid chromatography, we demonstrate that GLuc undergoes methionine oxidation during substrate turnover and that this impacts the luciferase's flash-type luminescence; we did not observe indications of covalent attachment with the substrate, product, or their intermediates. Chromatography of luciferases derived from ancestral sequence reconstruction highlighted that the extent of methionine-induced surface changes was greater for earlier ancestral luciferases. Ancestral sequence reconstruction also revealed that earlier ancestral copepod luciferases produced less light when compared to GLuc.

Autophagy inhibition represents a promising therapeutic approach for the management of various cancers including nonsmall cell lung cancer (NSCLC). We previously reported SBP-7455, a dual inhibitor of unc-51-like kinase 1 (ULK1) and its homologue ULK2 and described its effects on triple-negative breast cancer (TNBC) cells. Herein we report the design, synthesis, and characterization of SBP-5147 and SBP-7501, two new dual ULK1/2 inhibitors that are cytotoxic against NSCLC cells, inhibit autophagic flux in A549 cells, and present greater oral exposure than SBP-7455 at a lower dose. In addition, SBP-5147 effectively modulates autophagy and increases the expression of major histocompatibility complex (MHC) class I in NSCLC cells, which may support the rationale for ULK1/2 inhibition as a strategy to overcome resistance to immunotherapy. Together these data support the use of ULK inhibitors as part of a cancer treatment strategy, either as a single agent or in combination with current therapies.

Fungal secondary metabolites have historically provided important applications in a variety of industries. Penicillium camemberti, a fungus with a role in cheese production, was domesticated to food use partly due to its metabolically depleted characteristic, minimizing the risk of toxic compound formation. However, antiSMASH analysis of the genome reveals that strains of the species do contain various cryptic biosynthetic gene clusters and, thus, have the potential capability of producing multiple secondary metabolites despite its limited compound production under normal laboratory conditions. Here, we genetically engineered Penicillium camemberti strain IMV00769, which is genetically similar to cheese-making isolates, by deleting negative global regulator, mcrA. This deletion resulted in the production of secondary metabolites not previously produced by this strain, including fumigermin, a compound patented for cosmetic applications for the reduction of skin wrinkles, enhancement of skin elasticity, and skin whitening. Our findings highlight the power of global regulator manipulation to activate cryptic biosynthetic pathways and expand the range of natural products accessible from domesticated fungal strains.

Efficient delivery remains a major challenge for therapeutic genome editing because many widely used CRISPR nucleases are large and leave limited space for regulatory elements or additional payloads in a single adeno-associated virus (AAV) vector. Miniature Cas12 nucleases are particularly appealing, as their reduced size alleviates packaging constraints while preserving RNA-guided DNA cleavage. Here, we outline a workflow that links large-scale sequence mining with phylogenetic and structural filtering, followed by PAM profiling, in vitro cleavage, bacterial genome interference, and genome-editing assays in human cells to confirm activity. This protocol is intended to distill broad sequence collections into a small set of compact Cas12 nucleases with demonstrated functions that can serve as starting points for further engineering in delivery-limited settings.

Quantitative live cell monitoring of catalytic activity is essential for advancing chemical biology, yet designing substrate probes that combine broad applicability with finely tunable kinetics remains a significant challenge. While glyco-bisacetal-based substrates (BABS) have proven applicable to several enzymes, their alkyl-hemiacetal core can limit turnover rates for certain enzymes. Herein, we report a novel one-pot, three-component glycosylation strategy to synthesize Aryl-BABS through the trapping of transient aryl-hemiacetals. This approach enables rapid diversification of the bisacetal scaffold using various phenols, yielding a library of aryl-bisacetal substrates. Kinetic evaluation of catalytic hydrolysis with a model glycosidase demonstrated that these Aryl-BABS are efficiently processed, with turnover rates up to 2 orders of magnitude faster than analogous alkyl glycosides and approaching those seen for activated p-nitrophenyl glycosides. Simple substitutions to phenol lead to a 20-fold range of kinetic tunability. Crucially, stopped-flow studies combined with kinetic simulations revealed that the breakdown of the enzymatically released aryl-hemiacetal is extremely rapid, at least 100-fold faster than that of alkyl-hemiacetals. This synthetic and kinetic tunability offers a powerful roadmap for developing advanced substrate probes of biocatalysts, eventually enabling quantitative measurement of previously intractable enzymes in living systems.

Riboglow probes are small molecules where a synthetic fluorophore is connected to an RNA-binding moiety via a chemical linker. Upon binding a short RNA sequence, probe fluorescence intensity and lifetime increase. The fluorescence change is modulated by the architecture of the chemical linker. Here, we systematically interrogated the linker composition in a series of Riboglow probes and assessed fluorescence properties. We found that glycine linkers result in higher fluorescence turn-on compared to a polyethylene glycol linker of similar length. When varying the length of the polyglycine linker, we found that increasing the number of glycine residues led to more substantial fluorescence turn-on upon RNA-ligand binding. Surprisingly, the composition of the Riboglow chemical linker influences fluorescence lifetime contrast when comparing probe binding to two different RNA ligands, a quality necessary for RNA multiplexing. Finally, evaluating probe fluorescence lifetimes in live mammalian cells demonstrated the ability of new Riboglow probes to visualize RNAs live. Insights gained from the systematic assessment of the linker's architecture will dictate the rational design of future fluorophore-quencher probe designs.

Actinobacteria are a rich source of bioactive compounds and unique biosynthetic chemistry. Micromonospora echinospora subsp. challisensis NRRL 12255 produces the aromatic polyketide TLN-05220, which exhibits nanomolar activity against antibiotic-resistant human pathogens, including vancomycin-resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus. The pentangular polyphenol core of TLN-05220 is decorated with a piperazinone moiety; yet, the enzymes responsible for the construction of this uncommon modification from amino acid precursors are unknown. Synthetic piperazinone-containing molecules have diverse antimicrobial, antiviral, anticancer, and anti-inflammatory bioactivity profiles, and determining biosynthetic routes for the assembly of this heterocycle may enhance drug discovery and medicinal chemistry efforts. We identified a putative TLN-05220 biosynthetic gene cluster (BGC) in the commercially available strain M. echinospora ATCC 15837 that contains both type-I and type-II polyketide synthases, two predicted asparagine synthetase-like enzymes, and two genes (tln1 and tln5) that putatively encode pyridoxal 5'-phosphate (PLP)-dependent amino acid synthases. Stable isotopic feeding studies coupled with liquid chromatography-mass spectrometry (LC-MS) identified l-alanine, l-serine, and glycine as metabolic precursors of TLN-05220. Subsequent in vitro enzymology established that Tln1 is a PLP-dependent alanine racemase, while Tln5 performs a stereoselective β-substitution reaction of O-phospho-l-serine with a preferential d-alanine nucleophile. Alanine racemization and pseudodipeptide l-serine-Cβ-N-d-alanine (d,l-PDP) incorporation into TLN-05220 were further supported using deuterated intermediates and mass spectrometry techniques. Establishing the enzymes that catalyze amino acid installation within TLN-05220 inspires further biosynthetic discovery and engineering while enabling the biocatalytic syntheses of novel amino acid-containing polyketide antibiotics.

Bacteria use a process of chemical communication called quorum sensing to regulate group behaviors. Quorum sensing relies on the synthesis, release, and detection of signal molecules called autoinducers that accumulate with increasing cell density. The pathogen Vibrio cholerae makes and detects three autoinducers which together, regulate genes required for group behaviors including virulence and biofilm formation. Two autoinducers are produced by dedicated autoinducer synthases that employ S-adenosyl methionine as a substrate. The third autoinducer, 3,5-dimethylpyrazin-2-ol (DPO), is produced from threonine and alanine. The threonine dehydrogenase (Tdh) enzyme oxidizes l-threonine to 2-amino-3-ketobutyric acid, which spontaneously decarboxylates to aminoacetone. Here, we define the steps required to convert aminoacetone and alanine into DPO. We show that diverse adenylate-forming enzymes can condense ATP and d- or l-alanine to form alanyl-adenylate, the necessary intermediate in DPO biosynthesis. Upon release, alanyl-adenylate spontaneously condenses with aminoacetone to form N-alanyl-aminoacetone, which cyclizes to form DPO. We propose that DPO is distinct from other autoinducers in that there is apparently no dedicated synthase. Rather, a collection of enzymes contribute to the production of this quorum-sensing autoinducer.