Given the importance of RNA splicing to many cancers, we are continuing to work on identification of a discrete molecular target for SBLs to explain these data. Stay tuned!

Picture of Elvis saying thank you very much.

Congrats to Katie et al and special thanks to our collaborators in the Plate lab at Vanderbilt (TPP), and Irish Lab at U Colorado (flow)

TPP Volcano plot showing proteins stabilized and destabilized by induction with SBLs

We then used thermal proteome profiling to measure the perturbation of protein stability on induction with SBLs. Interestingly, we found perturbation in RNA processing and splicing effected on brief incubation SBLs. This was confirmed by mRNASeq and alternative splicing analysis.

Cell signaling pathway related to nutrient sensing, highlighting markers assayed by flow cytometry

We used a flow cytometry to investigate potential mechanistic possibilities of SBLs e induction showing inhibition of phospho-signaling related to nutrient signaling.

Structures of SBLs isolated from a single Streptomyces

This facilitated structure-activity relationship, verifying their potency against leukemia cell lines and the importance of the beta-lactone for cytotoxicity.

A close up of Multiplexed Activity Metabolomics showing debarcoded flow cytometry generated "Rothko" plots

Natural products containing beta-lactones are covalent protein modifiers, & their unknown mechanism and electrophilic nature got us curious about their mechanism.

Using genome mining, and our single cell Multiplexed Activity Metabolomics, we identified a new producer and isolated a family of SBLs.

Structures various showing SBLs from bacteria

Spirocyclic beta-lactones (SBLs) are a beautiful class of molecules produced by bacteria with a beta-lactone fused to a gamma-lactam with potent and selective activity against various cancer cell lines

We are pleased to share our new work in @pnas.org investigating the discovery, biosynthesis, and mechanistic investigation of a class of molecules we call spirocyclic beta-lactones! Skeetorial to follow. #natprod 🧪
www.pnas.org/doi/full/10....

Measuring the effect of complex metabolite mixtures directly in primary patient cells bridges a long-standing gap between product discovery and clinical relevance

Single-cell resolution adds mechanistic clarity that’s often missing in bulk

Thanks for sharing @brianobachmann.bsky.social

Special thanks to lab coworker champions Joe Balsamo and Hannah Thirman, and all of the outstanding contributors to this work. We hope you enjoy! pubs.rsc.org/en/content/a...

Multiplexed Activity Metabolomic Overview showing the generation and fractionation of extracts into well plates, the addition of patient derived cells from a biopsy.

We believe this method provides a way to start to link genotypes of cancer to their responses to small molecules, in this case secondary metabolites!

Deep thanks to my partner in crime, cytometric wizard @jonathanirish.bsky.social at U Colorado, and P. Brent Ferrell, MD and Vanderbilt.

tSNE plot of single cell data from compound exposure.

Mass cytometry overview

This is determined again at the single cell level, this time even more multiplexed, using mass cytometry, which can observe dozens of markers per cell using monoisotopic metal tagged antibodies and ICP-MS detection. We see striking differences between patients.

Schematic showing patients on the left, molecules tested against bone marrow in the middle, and a cartoon of bone marrow cells.

After isolation, we then test the pure compounds against biopsied bone marrow cell mixtures from 4  genotyped patients diagnosed with cancer, and healthy PBMCs, to see how purified compounds affect both wild cancer cells and normal hematopoetic cells.

MAM plot for identification of isoquinocyclin

MAM plot for identification of siderochelin

After determining which metabolites in an metabolomic array (fraction library of 8 - 96 wells) in a single flow fun, we prioritize them for  isolation and structure elucidation. We identified two knows, siderochelin and isoquinocycline with very interesting cellular perturbations.

Painting by Mark Rothko, which looks like our data format for debarcoded cells.

Scatter plots of single cell data in red and black resembling a Rothko painting.

We first use our platforms Multiplexed Activity Profiling & Multiplexed Activity Metabolomics for this workflow in extract and metabolite arrays. The output for activity in wells comes in the form of "Rothko plots" after debarcoding, which records the effects of metabolites in wells in single cells.

Pictures of cave walls, isolated microbes, and genomic analysis

This dovetails with 'genome mining' workflows which can derisk discovery. In these case studies, we use gifted actinomycetes isolated from caves as a source material, but this can be applied to extracts and metabolomic arrays from any source. www.micropublication.org/journals/bio...

Discovery of human cell selective effector molecules using single cell multiplexed activity metabolomics - Nature Communications Bioactive metabolites from plant and microbial extracts hold therapeutic potential. Here, the authors combine untargeted metabolomic arrays with flow cytometry-based single cell response profiling and...

This is accomplished using flow cytometry, which permits barcoding of metabolomic arrays and assays of multiplexed cell functional signaling markers per cell.

Multiplexed cytometry for single cell chemical biology Flow cytometry has great potential for screening in translational research areas due to its deep quantification of cellular features, ability to colle…

As described by graduate coworker Schares et al,the idea is to measure the effect of natural products in extracts and metabolomes against primary cells biopsied from patients diagnosed with cancer at single cell (ex vivo) resolution. This is accomplished using flow cytometry.

Overview of the paper in cartoon form

We are delighted to share our vision in #RSCChemBioy of how to incorporate natural product discovery (and any drug discovery) into the framework of personalized medicine - drug discovery using patients, but not in patients! #natprod #secmet pubs.rsc.org/en/content/a...

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Congrats, great stuff! What do you think the pKa of that enol is in your γ-lactone ? I'd guess <5. Seems relevant to its biological function at physiological pH...

Thrilled to be able to finally post my first adventure into secondary metabolism!:

Flavoaffinins, Elusive Cellulose-Binding Natural Products from an Anaerobic Bacterium

pubs.acs.org/doi/full/10....

Co-authors: Ruocheng Yu, f. undergrad Jessie Lee, @katherinem.bsky.social , and Emily Balskus!

Intratumoural vaccination via checkpoint degradation-coupled antigen presentation - Nature An intratumoural vaccination chimera reprograms tumour cells into an antigen-presenting state with restored anti-tumour immunity.

Clever combo idea. PD1 antibody degrader immuno drug conjugate.

www.nature.com/articles/s41...

Dean Stark Martini

Artistic notions of molecules are often formally wrong. I was gifted chemistry socks for fathers day by my 8 year old daughter many years ago &, in one of many parenting fails, could not control my facial reactions to the pentavalent. And look at the helicity handedness in most DNA molecule art...

TOC image of a large polyol polyketide coming from a cultured streptomyces and a large polyketide synthase biosynthetic gene cluster, with compounds showing activity in glowing fluorescent cells.

Cool compounds, Leshmania activitiesw/ good therapeutic index, and nice structure work. Hard work: "A culture (322 × 10 mL, 3.2 L) of the strain was obtained in the FR23 medium... 0.6 - 3.2 mg isolated of several analogs". #secmet
pubs.acs.org/doi/10.1021/...

Great news in the fight against antimicrobial resistance!

Two oral antibiotics have been approved by the FDA for uncomplicated urogenital gonorrhea. As Neisseria gonorrhoeae is becoming resistant to all known antibiotics, such breakthroughs were urgently needed. (1/4)
www.fda.gov/news-events/...

Isolation and genomic analysis of secondary metabolism in cave Actinomycetota from biofilms and Ceuthophilus https://pubmed.ncbi.nlm.nih.gov/41278891/

NRPStransformer, an Accurate Adenylation Domain Specificity Prediction Algorithm for Genome Mining of Nonribosomal Peptides Nonribosomal peptides serve as pivotal sources for drug discovery. Accurate prediction of the substrate specificity of adenylation domains in nonribosomal peptide synthetases is crucial for genome mining of nonribosomal peptides, yet current prediction methods fall short in accuracy. In this work, we analyzed 4,100 adenylation domains from documented nonribosomal peptide synthetases and found that the flavodoxin-like subdomain universally governs substrate specificity in all bacterial adenylation domains and that its phylogenetic analysis can correlate the sequences of adenylation domains and their substrate specificity. Leveraging the sequences within the flavodoxin-like subdomain, we developed a substrate specificity prediction algorithm using a protein language model, achieving 92% overall prediction accuracy for 43 frequently observed amino acids, significantly improving the prediction reliability. The efficacy of our prediction tool was validated through targeted genome mining, which led to the discovery of novel antimicrobial peptides. Our work lays a foundation to understand the sequence-to-function relationship of the bacterial adenylation domain and will facilitate the exploitation of nonribosomal peptides. NRPStransformer is available at http://www.nrpstransformer.cn.

A new entry in ML prediction of adenylation domain selectivity in nonribosomal peptide synthetases using a protein language model. Interestingly, it boasts 92% accuracy. Compare to the 25 year old Stachelhaus code (a type of homology modeling) at 89% accuracy. #secmet

Stanford’s Khosla Lab is pioneering a fluorescent probe that “lights up” active TG2 in the gut, enabling clearer detection and monitoring of celiac disease, as well as a potential biomarker for future therapies. Learn more: brnw.ch/21wXIdT