Brian O. Bachmann

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

Introgression impacts the evolution of bacteria, but species borders are rarely fuzzy Nature Communications - It is commonly thought that bacterial species borders tend to be fuzzy, due to frequent exchange of DNA. Here, Diop et al. quantify the patterns of gene flow between core...

Our latest paper is out with @adiop.bsky.social and @gmdouglas.bsky.social. We analyzed the extent of homologous recombination between bacterial species (introgression) and how it affects species borders (it can vary a lot depending on the approach used to classify species!). rdcu.be/eQAMf

Discovery and Biosynthetic Interrogation of a Cobalt-Containing Terpene-Polyketide-Nonribosomal Peptide Tribrid with Anticoronavirus Activity The structural complexity of natural products arises from the complexity of their biosynthetic pathways and directly contributes to their biological activities. Guided by this principle, we screened 5...

"Cobaltribin", a cobalt containing complex isolated from a streptomyces (3.1 mg from 40 × 0.5 L). #secmet,
pubs.acs.org/doi/10.1021/...

Discovery and Heterologous Expression of the Soil Metagenome-Derived Lasso Peptide Metanodin with an Unprecedented Ring Structure Culture-independent metagenomic approaches have proven to be effective tools for identifying previously hidden biosynthetic gene clusters (BGCs) encoding novel natural products with potential medical relevance. However, producing these compounds remains challenging as metagenomic BGCs often originate from organisms phylogenetically distant from available heterologous hosts. Lasso peptides, a subclass of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products, exhibit diverse bioactivities, yet no lasso peptide has previously been discovered directly from a metagenome. Here, we report the discovery and heterologous expression of the first soil metagenome-derived lasso peptide. Expression of its biosynthetic gene cluster in Escherichia coli, followed by mass spectrometry analysis, strongly supported the predicted amino acid sequence and lasso structure of the peptide. Notably, this lasso peptide is the first to feature asparagine as the ring-forming residue at position one. Taxonomic analysis of the corresponding BGC identified an uncultivated member of the Steroidobacterales family (Gammaproteobacteria) as the closest known relative of the potential native host. These findings underscore the potential of metagenomic genome mining to reveal structurally novel RiPPs and to expand our understanding of the natural diversity of lasso peptides.

Happy to share our newest manuscript about the discovery and hererologous expression of metanodin, a new lassopeptide with unprecedented structural features directly from soil metagenomes. pubs.acs.org/doi/full/10....
#secmet #lassopeptides #syntheticbiology

Picture of white millipedes next to an HPLC chromatogram, with one blue peak and five red peaks above blue and red chemical structures describing scaffold of the molecules comprising the peaks. Andrognathanols (blue) are 6,6,6,5-tetracyclic bridged alkaloids and andrognathines are 5,6-fused bicyclic alkaloids.

The Discovery of Complex Heterocycles from Millipede Secretions, fantastic work from the Mevers group that I saw the full story from at SERMACS this week.

pubs.acs.org/doi/10.1021/...
#secmet

It is not an unusual methodology to entertain a counter intuitive hypothesis in science. Good scientists do this habitually.

Fantastic: 44 new pyrrolobenzodiazepine's via an engineered single module reductase terminated NRPS system #natprod #secmet

This gives the George Gershwin song “Let’s call the Whole Thing Off” a whole new spin.

You like potato and I like potato
You like tomato and I like tomato
Potato, potahto, tomato, tomahto
Let's call the whole thing off

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Honored to host Dr. Martin Burke (@chemistryIllinois) as keynote at the VICB Student Research Symposium! His talk, "Imagine a World Where Anyone Can Make Molecules," is inspiring the next generation of molecular makers.

@vanderbilt.edu @vubasicsciences.bsky.social