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SHP2 is a protein tyrosine phosphatase that plays a critical role in the full activation of the Ras/MAPK pathway upon stimulation of receptor tyrosine kinases (RTKs), which are frequently amplified or mutationally activated in human cancer. In addition, activating mutations in SHP2 result in developmental disorders and hematologic malignancies. Several allosteric inhibitors have been developed for SHP2 and are currently in clinical trials. Here, we report the development and evaluation of a SHP2 PROTAC created by conjugating RMC-4550 with pomalidomide using a PEG linker. This molecule is highly selective for SHP2, induces degradation of SHP2 in leukemic cells at sub-micromolar concentration, inhibits MAPK signaling, and suppresses cancer cell growth. SHP2 PROTACs serve as an alternative strategy for targeting ERK-dependent cancers and are useful tools alongside allosteric inhibitors for dissecting the mechanisms by which SHP2 exerts its oncogenic activity.

Bariatric surgery, the most effective treatment for obesity and type 2 diabetes, is associated with increased levels of the incretin hormone glucagon-like peptide-1 (GLP-1) and changes in levels of circulating bile acids. The levels of individual bile acids in the gastrointestinal (GI) tract after surgery have, however, remained largely unstudied. Using ultra-high performance liquid chromatography-mass spectrometry-based quantification, we observed an increase in an endogenous bile acid, cholic acid-7-sulfate (CA7S), in the GI tract of both mice and humans after sleeve gastrectomy. We show that CA7S is a Takeda G-protein receptor 5 (TGR5) agonist that increases Tgr5 expression and induces GLP-1 secretion. Furthermore, CA7S administration increases glucose tolerance in insulin-resistant mice in a TGR5-dependent manner. CA7S remains gut restricted, minimizing off-target effects previously observed for TGR5 agonists absorbed into the circulation. By studying changes in individual metabolites after surgery, the present study has revealed a naturally occurring TGR5 agonist that exerts systemic glucoregulatory effects while remaining confined to the gut.

Over the past 70 years, the search for small molecules from nature has transformed biomedical research: natural products are the basis for half of all pharmaceuticals; the quest for total synthesis of natural products fueled development of methodologies for organic synthesis; and their biosynthesis presented unprecedented biochemical transformations, expanding our chemo-enzymatic toolkit. Initially, the discovery of small molecules was driven by bioactivity-guided fractionation. However, this approach yielded the frequent rediscovery of already known metabolites. As a result, focus shifted to identifying novel scaffolds through either structure-first methods or genome mining, relegating function as a secondary concern. Over the past two decades, the laboratory of Jon Clardy has taken an alternative route and focused on an ecology-driven, function-first approach in pursuit of uncovering bacterial small molecules with biological activity. In this review, we highlight several examples that showcase this ecology-first approach. Though the highlighted systems are diverse, unifying themes are (1) to understand how microbes interact with their host or environment, (2) to gain insights into the environmental roles of microbial metabolites, and (3) to explore pharmaceutical potential from these ecologically relevant metabolites.

Bacterial symbionts frequently provide chemical defenses for their hosts, and such systems can provide discovery pathways to new antifungals and structurally intriguing metabolites. This report describes a small family of naturally occurring small molecules with chimeric structures and a mixed biosynthesis that features an unexpected but key nonenzymatic step. An insect-associated Pseudomonas protegens strain's activity in an in vivo murine candidiasis assay led to the discovery of a family of highly hydrogen-deficient metabolites. Bioactivity- and mass-guided fractionation led to the pyonitrins, highly complex aromatic metabolites in which 10 of the 20 carbons are quaternary, and 7 of them are contiguous. The P. protegens genome revealed that the production of the pyonitrins is the result of a spontaneous reaction between biosynthetic intermediates of two well-studied Pseudomonas metabolites, pyochelin and pyrrolnitrin. The combined discovery of the pyonitrins and identification of the responsible biosynthetic gene clusters revealed an unexpected biosynthetic route that would have prevented the discovery of these metabolites by bioinformatic analysis alone.

Significance The bacteria that live within the human gut play crucial roles in regulating our primary metabolism, protecting us from pathogens, and developing our immune system. Imbalances in bacterial community structure have been implicated in many diseases, such as Crohn’s disease, an inflammatory bowel disease. We found and characterized an inflammatory polysaccharide produced by the gut bacterium Ruminococcus gnavus, populations of which bloom during flares of symptoms in patients with Crohn’s disease. This molecule induces the production of inflammatory cytokines like TNFα by dendritic cells and may contribute to the association between R. gnavus and Crohn’s disease. This work establishes a plausible molecular mechanism that may explain the association between a member of the gut microbiome and an inflammatory disease., A substantial and increasing number of human diseases are associated with changes in the gut microbiota, and discovering the molecules and mechanisms underlying these associations represents a major research goal. Multiple studies associate Ruminococcus gnavus, a prevalent gut microbe, with Crohn’s disease, a major type of inflammatory bowel disease. We have found that R. gnavus synthesizes and secretes a complex glucorhamnan polysaccharide with a rhamnose backbone and glucose sidechains. Chemical and spectroscopic studies indicated that the glucorhamnan was largely a repeating unit of five sugars with a linear backbone formed from three rhamnose units and a short sidechain composed of two glucose units. The rhamnose backbone is made from 1,2- and 1,3-linked rhamnose units, and the sidechain has a terminal glucose linked to a 1,6-glucose. This glucorhamnan potently induces inflammatory cytokine (TNFα) secretion by dendritic cells, and TNFα secretion is dependent on toll-like receptor 4 (TLR4). We also identify a putative biosynthetic gene cluster for this molecule, which has the four biosynthetic genes needed to convert glucose to rhamnose and the five glycosyl transferases needed to build the repeating pentasaccharide unit of the inflammatory glucorhamnan.

SHP2 is a protein tyrosine phosphatase that plays a critical role in the full activation of the Ras/MAPK pathway upon stimulation of receptor tyrosine kinases (RTKs), which are frequently amplified or mutationally activated in human cancer. In addition, activating mutations in SHP2 result in developmental disorders and hematologic malignancies. Several allosteric inhibitors have been developed for SHP2 and are currently in clinical trials. Here, we report the development and evaluation of a SHP2 PROTAC created by conjugating RMC-4550 with pomalidomide using a PEG linker. This molecule is highly selective for SHP2, induces degradation of SHP2 in leukemic cells at sub-micromolar concentration, inhibits MAPK signaling, and suppresses cancer cell growth. SHP2 PROTACs serve as an alternative strategy for targeting ERK-dependent cancers and are useful tools alongside allosteric inhibitors for dissecting the mechanisms by which SHP2 exerts its oncogenic activity.

Significance Microbes shape human health and disease. Today, there are increasing correlations of gut microbes with specific diseases, but the underlying molecules and mechanism linking the two are not known. This stifles both the understanding of disease causation and the development of suitable treatments. We investigated a dozen patient isolates of Ruminococcus gnavus, a prevalent gut microbe linked to inflammatory bowel disease (IBD). We found that some isolates possess a protective capsule that encourages a symbiotic relationship with the host immune system, while others lack this protective capsule and elicit a robust inflammatory immune response. This work reveals a path by which R. gnavus could be connected to IBD and potential therapeutic interventions., Active inflammatory bowel disease (IBD) often coincides with increases of Ruminococcus gnavus, a gut microbe found in nearly everyone. It was not known how, or if, this correlation contributed to disease. We investigated clinical isolates of R. gnavus to identify molecular mechanisms that would link R. gnavus to inflammation. Here, we show that only some isolates of R. gnavus produce a capsular polysaccharide that promotes a tolerogenic immune response, whereas isolates lacking functional capsule biosynthetic genes elicit robust proinflammatory responses in vitro. Germ-free mice colonized with an isolate of R. gnavus lacking a capsule show increased measures of gut inflammation compared to those colonized with an encapsulated isolate in vivo. These observations in the context of our earlier identification of an inflammatory cell-wall polysaccharide reveal how some strains of R. gnavus could drive the inflammatory responses that characterize IBD.

Active inflammatory bowel disease (IBD) often coincides with increases of Ruminococcus gnavus, a gut microbe found in nearly everyone. It was not known how, or if, this correlation contributed to disease. We investigated clinical isolates of R. gnavus to identify molecular mechanisms that would link R. gnavus to inflammation. Here, we show that only some isolates of R. gnavus produce a capsular polysaccharide that promotes a tolerogenic immune response, whereas isolates lacking functional capsule biosynthetic genes elicit robust proinflammatory responses in vitro. Germ-free mice colonized with an isolate of R. gnavus lacking a capsule show increased measures of gut inflammation compared to those colonized with an encapsulated isolate in vivo. These observations in the context of our earlier identification of an inflammatory cell-wall polysaccharide reveal how some strains of R. gnavus could drive the inflammatory responses that characterize IBD.

Bariatric surgery, the most effective treatment for obesity and type 2 diabetes, is consistently associated with increased levels of the incretin hormone GLP-1 and changes in overall levels of circulating bile acids. The levels of individual bile acids in the GI tract following surgery, however, have remained largely unstudied. Using UPLC-MS-based quantification, we observed an increase in an endogenous bile acid, cholic acid-7-sulfate (CA7S), in the GI tract of both mice and humans after sleeve gastrectomy. We show that CA7S is a TGR5 agonist that induces GLP-1 secretion in vitro and in vivo and that CA7S administration increases glucose tolerance in insulin-resistant mice in a GLP-1 receptor-dependent manner. CA7S remains gut-restricted, minimizing off-target effects previously observed for TGR5 agonists absorbed into circulation. By studying changes in individual metabolites following surgery, this study has revealed a naturally occurring TGR5 agonist that exerts systemic glucoregulatory effects while remaining confined to the gut.

The gut microbiota produce hundreds of molecules that are present at high concentrations in the host circulation. Unraveling the contribution of each molecule to host biology remains difficult. We developed a system for constructing clean deletions in Clostridium spp., the source of many molecules from the gut microbiome. By applying this method to the model commensal organism Clostridium sporogenes, we knocked out genes for 10 C. sporogenes–derived molecules that accumulate in host tissues. In mice colonized by a C. sporogenes for which the production of branched short-chain fatty acids was knocked out, we discovered that these microbial products have immunoglobulin A–modulatory activity.

Bariatric surgery, the most effective treatment for obesity and type 2 diabetes, is associated with increased levels of the incretin hormone glucagon-like peptide-1 (GLP-1) and changes in levels of circulating bile acids. The levels of individual bile acids in the gastrointestinal (GI) tract after surgery have, however, remained largely unstudied. Using ultra-high performance liquid chromatography-mass spectrometry-based quantification, we observed an increase in an endogenous bile acid, cholic acid-7-sulfate (CA7S), in the GI tract of both mice and humans after sleeve gastrectomy. We show that CA7S is a Takeda G-protein receptor 5 (TGR5) agonist that increases Tgr5 expression and induces GLP-1 secretion. Furthermore, CA7S administration increases glucose tolerance in insulin-resistant mice in a TGR5-dependent manner. CA7S remains gut restricted, minimizing off-target effects previously observed for TGR5 agonists absorbed into the circulation. By studying changes in individual metabolites after surgery, the present study has revealed a naturally occurring TGR5 agonist that exerts systemic glucoregulatory effects while remaining confined to the gut.

Staphylococcus aureus and Streptococcus pneumoniae infections cause significant morbidity and mortality in humans. For both, nasal colonization is a risk factor for infection. Studies of nasal microbiota identify Dolosigranulum pigrum as a benign bacterium present when adults are free of S. aureus or when children are free of S. pneumoniae. Here, we validated these in vivo associations with functional assays. We found that D. pigrum inhibited S. aureus in vitro and, together with a specific nasal Corynebacterium species, also inhibited S. pneumoniae. Furthermore, genomic analysis of D. pigrum indicated that it must obtain key nutrients from other nasal bacteria or from humans. These phenotypic interactions support the idea of a role for microbe-microbe interactions in shaping the composition of human nasal microbiota and implicate D. pigrum as a mutualist of humans. These findings support the feasibility of future development of microbe-targeted interventions to reshape nasal microbiota composition to exclude S. aureus and/or S. pneumoniae., Multiple epidemiological studies identify Dolosigranulum pigrum as a candidate beneficial bacterium based on its positive association with health, including negative associations with nasal/nasopharyngeal colonization by the pathogenic species Staphylococcus aureus and Streptococcus pneumoniae. Using a multipronged approach to gain new insights into D. pigrum function, we observed phenotypic interactions and predictions of genomic capacity that support the idea of a role for microbe-microbe interactions involving D. pigrum in shaping the composition of human nasal microbiota. We identified in vivo community-level and in vitro phenotypic cooperation by specific nasal Corynebacterium species. Also, D. pigrum inhibited S. aureus growth in vitro, whereas robust inhibition of S. pneumoniae required both D. pigrum and a nasal Corynebacterium together. D. pigrum l-lactic acid production was insufficient to account for these inhibitions. Genomic analysis of 11 strains revealed that D. pigrum has a small genome (average 1.86 Mb) and multiple predicted auxotrophies consistent with D. pigrum relying on its human host and on cocolonizing bacteria for key nutrients. Further, the accessory genome of D. pigrum harbored a diverse repertoire of biosynthetic gene clusters, some of which may have a role in microbe-microbe interactions. These new insights into D. pigrum’s functions advance the field from compositional analysis to genomic and phenotypic experimentation on a potentially beneficial bacterial resident of the human upper respiratory tract and lay the foundation for future animal and clinical experiments. IMPORTANCE Staphylococcus aureus and Streptococcus pneumoniae infections cause significant morbidity and mortality in humans. For both, nasal colonization is a risk factor for infection. Studies of nasal microbiota identify Dolosigranulum pigrum as a benign bacterium present when adults are free of S. aureus or when children are free of S. pneumoniae. Here, we validated these in vivo associations with functional assays. We found that D. pigrum inhibited S. aureus in vitro and, together with a specific nasal Corynebacterium species, also inhibited S. pneumoniae. Furthermore, genomic analysis of D. pigrum indicated that it must obtain key nutrients from other nasal bacteria or from humans. These phenotypic interactions support the idea of a role for microbe-microbe interactions in shaping the composition of human nasal microbiota and implicate D. pigrum as a mutualist of humans. These findings support the feasibility of future development of microbe-targeted interventions to reshape nasal microbiota composition to exclude S. aureus and/or S. pneumoniae.

Covering: up to 2016In this highlight, we describe the current landscape for dereplication and discovery of natural products based on the measurement of the intact mass by LC-MS. Often it is assumed that because better mass accuracy (provided by higher resolution mass spectrometers) is necessary for absolute chemical formula determination (≤1 part-per-million), that it is also necessary for dereplication of natural products. However, the average ability to dereplicate tapers off at ∼10 ppm, with modest improvement gained from better mass accuracy when querying focused databases of natural products. We also highlight some recent examples of how these platforms are applied to synthetic biology, and recent methods for dereplication and correlation of substructures using tandem MS data. We also offer this highlight to serve as a brief primer for those entering the field of mass spectrometry-based natural products discovery.

The microbial world offers a rich source of bioactive compounds for those able to sift through it. Technologies capable of quantitatively detecting natural products while simultaneously identifying known compounds would expedite the search for new pharmaceutical leads. Prior efforts have targeted histone deacetylases in fungi to globally activate the production of new secondary metabolites, yet no study has directly assessed its effects with minimal bias at the metabolomic level. Using untargeted metabolomics, we monitored changes in >1000 small molecules secreted from the model fungus, Aspergillus nidulans, following genetic or chemical reductions in histone deacetylase activity (HDACi). Through quantitative, differential analyses, we found that nearly equal numbers of compounds were up- and down-regulated by >100 fold. We detected products from both known and unknown biosynthetic pathways and discovered that A. nidulans is capable of producing fellutamides, proteasome inhibitors whose expression was induced by ∼100 fold or greater upon HDACi. This work adds momentum to an "omics"-driven resurgence in natural products research, where direct detection replaces bioactivity as the primary screen for new pharmacophores.

Despite containing an α-amino acid, the versatile cofactor S-adenosylmethionine (SAM) is not a known building block for non-ribosomal peptide synthetase (NRPS) assembly lines. Here we report an unusual NRPS module from colibactin biosynthesis that uses SAM for amide bond formation and subsequent cyclopropanation. Our findings showcase a new use for SAM and reveal a novel biosynthetic route to a functional group that likely mediates colibactin’s genotoxicity.

Unlocking the biochemical stores of fungi is key for developing future pharmaceuticals. Through reduced expression of a critical histone deacetylase in Aspergillus nidulans, increases of up to 100-fold were observed in the levels of 15 new aspercryptins, recently described lipopeptides with two non-canonical amino acids derived from octanoic and dodecanoic acids. In addition to two NMR-verified structures, MS/MS networking helped uncover an additional 13 aspercryptins. The aspercryptins break the conventional structural orientation of lipopeptides and appear “backward” when compared to known compounds of this class. We have also confirmed the 14-gene aspercryptin biosynthetic gene cluster, which encodes two fatty acid synthases and several enzymes to convert saturated octanoic and dodecanoic acid to α-amino acids.