Hannah L. Thirman

Four previously unreported glycosylated linear polyene polyketides, kofumicins A−D (1−4), were isolated from Kutzneria kofuensisNRRL 24061 and were found to inhibit Staphylococcus aureus growth. The kofumicins were identified from the crude extract using a bacterial activity metabolomics-guided approach, which employs split-flow HPLC-UV/MS to generate microtiter plate arrays enabling dereplication and bioactivity assessment prior to isolation. The structures were determined using NMR data, HR-ESI-MS, and biosynthetic gene cluster inference, establishing the kofumicins as new members of a family of glycosylated amphoteric linear polyene polyketides. Kofumicins A−C (1−3) share a conserved polyketide chain but differ in disaccharide composition and glycosidic regiochemistry, while kofumicin D (4) exhibits a truncated polyketide chain with a monosaccharide. Distance-constrained molecular dynamics calculations suggest that the kofumicins adopt a bent bolaamphiphilic conformation. Kofumicin C (3), bearing 6-deoxyglucose as the polyketide-linked sugar, was the most potent glycovariant tested, with MICs of 2−4 μg/mL against S. aureus strains including MRSA, while kofumicin B (2), bearing a glucose at the same position, exhibited MICs of 16−32 μg/mL. These results identify the C-6 substituent of the polyketide-linked sugar as an important determinant of antibacterial potency in this compound class.

Spirocyclic β-lactones (SβLs) are ring-strain activated natural products produced by actinomycetota, possessing potent and selective cytotoxicity, and with an unknown pharmacological mechanism. To further the understanding of their mechanism of action and the discovery of new SβLs, we obtained a series of known and previously not isolated SβLs from Streptomyces platensis , which contained a cryptic SβL-producing gene cluster. Utilizing single cell Multiplexed Activity Metabolomics to assess a panel of regulated cell injury and cell death markers in metabolomic arrays tested against MV-4-11 cells, phosphoprotein-S6 phosphorylation inhibition was observed for several metabolomic features, prompting us to prioritize these metabolites for structure elucidation. Several compounds were isolated including one new variant, permitting correlation of a cryptic biosynthetic gene cluster entry for oxazolomycin D (OxD) family metabolites. Cytotoxicity and flow cytometric structure–activity relationships for isolated analogs were determined using an expanded phospho-flow metabolism marker panel suggesting that SβLs possess two pharmacophores: a mixed polyketide component, involved in target engagement and inhibition, and a nonribosomal peptide synthetase-derived covalent β-lactone warhead, contributing to cytotoxicity. Global cellular thermal proteome profiling analysis of OxD treated cells implicated potent interaction with and modulation of spliceosome associated proteins. Spliceosome-associated functional changes were validated by alternate splicing analysis upon induction by OxD. As aberrant RNA splicing occurs in nearly all cancer types, the spliceosome-associated cellular response profiles of OxD define an unexplored opportunity for therapeutic development in cancers dependent upon alternative splicing.

Natural products derived from biosynthetically apt microorganisms represent an important source for chemotherapeutic agents founded first via non-specific, cytotoxicity-guided isolation, then investigated for translational potential. Analyzing biosynthetic gene clusters within microbes predicts significant untapped potential of undiscovered therapeutically relevant natural products; however, stimulating biosynthesis of such compounds in sufficient quantities for isolation, structure determination, and biological assessment remains a gap. We address this with a bioactivity discovery pipeline that first utilizes Multiplexed Activity Profiling to identify stimulus-dependent induction of bioactive secondary metabolites via functional responses in human leukemia cell lines measured with fluorescence flow cytometry. Next, active extracts are analyzed via Multiplexed Activity Metabolomics, which correlates single cell assays with spectrally defined chromatographic arrays to identify bioactive metabolomic features prior to isolation. Two experimental case studies using this workflow with cave-microbe extracts identified new molecular phenotypes of the pyridine-pyrrolidine alkaloid siderochelin and the pyrrolopyrrole-functionalized anthracycline isoquinocycline B. Finally, we investigated the effects of anthracycline functionalization on human primary cells using single cell mass cytometry to understand if changes in anthracycline aglycon structure and glycosylation impacted cell selectivity. Despite sharing an anthracyclinone pharmacophore core, variants differentially impacted responses of leukemia cell populations within and among acute myeloid leukemia (AML) patient samples. This suggests pharmacophore assumptions may not guide assessment of therapeutic potential for anthracyclines and that modest structural differences can elicit marked changes in cellular function in different patients. Taken together, the depth of information afforded by single cell molecular phenotype-based discovery and mass cytometry deep cell profiling provides new patient-level insight into biological mechanisms of new and previously discovered molecules that may find expanded use in the clinic.

A challenge in chemical biology is to study structure-activity relationships (SAR) in vivo in cells. Multiplexed activity profiling (MAP), developed for natural product discovery, is well-suited to address this challenge as it is high throughput, singlecell, and measures multiple hallmark cellular functions. Applying MAP while systematically varying molecular structure (SAR-MAP) could reveal previously unappreciated activity within chemical families and prioritize candidate molecules for further characterization. Here we use SAR-MAP to identify structural features responsible for specific bioactivities of the natural product family, rocaglates. MV411 leukemia cells and healthy human leukocytes are selected for proof-of-concept screening. Testing 600 representative molecules using MAP classifies roughly half of tested rocaglates (9 of 19) as bioactive. SAR-MAP elucidates a methoxy substituent on select rocaglate pyrimidinones as responsible for a desirable anti-leukemia activity. Thus, SAR-MAP can be immediately applied to identify structural variations driving natural product activity in cell lines and primary human cells.

The role of dynamics in enzymatic function is a highly debated topic. Dihydrofolate reductase (DHFR), due to its universality and the depth with which it has been studied, is a model system in this debate. Myriad previous works have identified networks of residues in positions near to and remote from the active site that are involved in dynamics and others that are important for catalysis. For example, specific mutations on the Met20 loop in E. coli DHFR (N23PP/S148A) are known to disrupt millisecond-timescale motions and reduce catalytic activity. However, how and if networks of dynamically coupled residues influence the evolution of DHFR is still an unanswered question. In this study, we first identify, by statistical coupling analysis and molecular dynamic simulations, a network of coevolving residues, which possess increased correlated motions. We then go on to show that allosteric communication in this network is selectively knocked down in N23PP/S148A mutant E. coli DHFR. Finally, we identify two sites in the human DHFR sector which may accommodate the Met20 loop double proline mutation while preserving dynamics. These findings strongly implicate protein dynamics as a driving force for evolution.

A central challenge in chemical biology is to distinguish molecular families in which small structural changes trigger large changes in cell biology. Such families might be ideal scaffolds for developing cell-selective chemical effectors – for example, molecules that activate DNA damage responses in malignant cells while sparing healthy cells. Across closely related structural variants, subtle structural changes have the potential to result in contrasting bioactivity patterns across different cell types. Here, we tested a 600-compound Diversity Set of screening molecules from the Boston University Center for Molecular Discovery (BU-CMD) in a novel phospho-flow assay that tracked fundamental cell biological processes, including DNA damage response, apoptosis, M-phase cell cycle, and protein synthesis in MV411 leukemia cells. Among the chemotypes screened, synthetic congeners of the rocaglate family were especially bioactive. In follow-up studies, 37 rocaglates were selected and deeply characterized using 12 million additional cellular measurements across MV411 leukemia cells and healthy peripheral blood mononuclear cells. Of the selected rocaglates, 92% displayed significant bioactivity in human cells, and 65% selectively induced DNA damage responses in leukemia and not healthy human blood cells. Furthermore, the signaling and cell-type selectivity were connected to structural features of rocaglate subfamilies. In particular, three rocaglates from the rocaglate pyrimidinone (RP) structural subclass were the only molecules that activated exceptional DNA damage responses in leukemia cells without activating a detectable DNA damage response in healthy cells. These results indicate that the RP subset should be extensively characterized for anticancer therapeutic potential as it relates to the DNA damage response. This single cell profiling approach advances a chemical biology platform to dissect how systematic variations in chemical structure can profoundly and differentially impact basic functions of healthy and diseased cells.