N1-Benzyl Tryptamine Pan-SHIP1/2 Inhibitors: Synthesis and Preliminary Biological Evaluation as Anti-Tumor Agents.

Inhibition of phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase (SHIP) with small molecule inhibitors leads to apoptosis in tumor cells. Inhibitors that target both SHIP1 and SHIP2 (pan-SHIP1/2 inhibitors) may have benefits in these areas since paralog compensation is not possible when both SHIP paralogs are being inhibited. A series of tryptamine-based pan-SHIP1/2 inhibitors have been synthesized and evaluated for their ability to inhibit the SHIP paralogs. The most active compounds were also evaluated for their effects on cancer cell lines.

A biosynthetic pathway for the selective sulfonation of steroidal metabolites by human gut bacteria.

Members of the human gut microbiome enzymatically process many bioactive molecules in the gastrointestinal tract. Most gut bacterial modifications characterized so far are hydrolytic or reductive in nature. Here we report that abundant human gut bacteria from the phylum Bacteroidetes perform conjugative modifications by selectively sulfonating steroidal metabolites. While sulfonation is a ubiquitous biochemical modification, this activity has not yet been characterized in gut microbes. Using genetic and biochemical approaches, we identify a widespread biosynthetic gene cluster that encodes both a sulfotransferase (BtSULT, BT0416) and enzymes that synthesize the sulfonate donor adenosine 3'-phosphate-5'-phosphosulfate (PAPS), including an APS kinase (CysC, BT0413) and an ATP sulfurylase (CysD and CysN, BT0414-BT0415). BtSULT selectively sulfonates steroidal metabolites with a flat A/B ring fusion, including cholesterol. Germ-free mice monocolonized with Bacteroides thetaiotaomicron ΔBT0416 exhibited reduced gastrointestinal levels of cholesterol sulfate (Ch-S) compared with wild-type B. thetaiotaomicron-colonized mice. The presence of BtSULT and BtSULT homologues in bacteria inhibited leucocyte migration in vitro and in vivo, and abundances of cluster genes were significantly reduced in patients with inflammatory bowel disease. Together, these data provide a mechanism by which gut bacteria sulfonate steroidal metabolites and suggest that these compounds can modulate immune cell trafficking in the host.

Inhibition of microbial deconjugation of micellar bile acids protects against intestinal permeability and liver injury.

Altered host-microbe interactions and increased intestinal permeability have been implicated in disease pathogenesis. However, the mechanisms by which intestinal microbes affect epithelial barrier integrity remain unclear. Here, we investigate the impact of bacterial metabolism of host-produced bile acid (BA) metabolites on epithelial barrier integrity. We observe that rats fed a choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD) exhibit reduced intestinal abundance of host-produced conjugated BAs at early time points, coinciding with increased gut permeability. We show that in vitro, conjugated BAs protect gut epithelial monolayers from damage caused by bacterially produced unconjugated BAs through micelle formation. We then demonstrate that inhibition of bacterial BA deconjugation with a small-molecule inhibitor prevents the development of pathologic intestinal permeability and hepatic inflammation in CDAHFD-fed rats. Our study identifies a signaling-independent, physicochemical mechanism for conjugated BA-mediated protection of epithelial barrier function and suggests that rational manipulation of microbial BA metabolism could be leveraged to regulate gut barrier integrity.

A Gut-Restricted Lithocholic Acid Analog as an Inhibitor of Gut Bacterial Bile Salt Hydrolases.

Bile acids play crucial roles in host physiology by acting both as detergents that aid in digestion and as signaling molecules that bind to host receptors. Gut bacterial bile salt hydrolase (BSH) enzymes perform the gateway reaction leading to the conversion of host-produced primary bile acids into bacterially modified secondary bile acids. Small molecule probes that target BSHs will help elucidate the causal roles of these metabolites in host physiology. We previously reported the development of a covalent BSH inhibitor with low gut permeability. Here, we build on our previous findings and describe the development of a second-generation gut-restricted BSH inhibitor with enhanced potency, reduced off-target effects, and durable in vivo efficacy. Structure-activity relationship (SAR) studies focused on the bile acid core identified a compound, AAA-10, containing a C3-sulfonated lithocholic acid scaffold and an alpha-fluoromethyl ketone warhead as a potent pan-BSH inhibitor. This compound inhibits BSH activity in mouse and human fecal slurry, bacterial cultures, and purified BSH proteins and displays reduced toxicity against mammalian cells compared to first generation compounds. Oral administration of AAA-10 to wild-type mice for 5 days resulted in a decrease in the abundance of the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) in the mouse GI tract with low systemic exposure of AAA-10, demonstrating that AAA-10 is an effective tool for inhibiting BSH activity and modulating bile acid pool composition in vivo.

A microbial metabolite remodels the gut-liver axis following bariatric surgery.

Bariatric surgery is the most effective treatment for type 2 diabetes and is associated with changes in gut metabolites. Previous work uncovered a gut-restricted TGR5 agonist with anti-diabetic properties-cholic acid-7-sulfate (CA7S)-that is elevated following sleeve gastrectomy (SG). Here, we elucidate a microbiome-dependent pathway by which SG increases CA7S production. We show that a microbial metabolite, lithocholic acid (LCA), is increased in murine portal veins post-SG and by activating the vitamin D receptor, induces hepatic mSult2A1/hSULT2A expression to drive CA7S production. An SG-induced shift in the microbiome increases gut expression of the bile acid transporters Asbt and Ostα, which in turn facilitate selective transport of LCA across the gut epithelium. Cecal microbiota transplant from SG animals is sufficient to recreate the pathway in germ-free (GF) animals. Activation of this gut-liver pathway leads to CA7S synthesis and GLP-1 secretion, causally connecting a microbial metabolite with the improvement of diabetic phenotypes.

Development of a covalent inhibitor of gut bacterial bile salt hydrolases.

Bile salt hydrolase (BSH) enzymes are widely expressed by human gut bacteria and catalyze the gateway reaction leading to secondary bile acid formation. Bile acids regulate key metabolic and immune processes by binding to host receptors. There is an unmet need for a potent tool to inhibit BSHs across all gut bacteria to study the effects of bile acids on host physiology. Here, we report the development of a covalent pan-inhibitor of gut bacterial BSHs. From a rationally designed candidate library, we identified a lead compound bearing an alpha-fluoromethyl ketone warhead that modifies BSH at the catalytic cysteine residue. This inhibitor abolished BSH activity in conventional mouse feces. Mice gavaged with a single dose of this compound displayed decreased BSH activity and decreased deconjugated bile acid levels in feces. Our studies demonstrate the potential of a covalent BSH inhibitor to modulate bile acid composition in vivo.

Pan-SHIP1/2 inhibitors promote microglia effector functions essential for CNS homeostasis.

We show here that both SHIP1 (Inpp5d) and its paralog SHIP2 (Inppl1) are expressed at protein level in microglia. To examine whether targeting of SHIP paralogs might influence microglial physiology and function, we tested the capacity of SHIP1-selective, SHIP2-selective and pan-SHIP1/2 inhibitors for their ability to impact on microglia proliferation, lysosomal compartment size and phagocytic function. We find that highly potent pan-SHIP1/2 inhibitors can significantly increase lysosomal compartment size, and phagocytosis of dead neurons and amyloid beta (Aß)1-42 by microglia in vitro We show that one of the more-potent and water-soluble pan-SHIP1/2 inhibitors, K161, can penetrate the blood-brain barrier. Consistent with this, K161 increases the capacity of CNS-resident microglia to phagocytose Aß and apoptotic neurons following systemic administration. These findings provide the first demonstration that small molecule modulation of microglia function in vivo is feasible, and suggest that dual inhibition of the SHIP1 and 2 paralogs can provide a novel means to enhance basal microglial homeostatic functions for therapeutic purposes in Alzheimer's disease and, possibly, other types of dementia where increased microglial function could be beneficial.

Dialkylation of Indoles with Trichloroacetimidates to Access 3,3-Disubstituted Indolenines.

2-Substituted indoles may be directly transformed to 3,3-dialkyl indolenines with trichloroacetimidate electrophiles and the Lewis acid TMSOTf. These reactions provide rapid access to complex indolenines which are present in a variety of complex natural products and medicinally relevant small molecule structures. This method provides an alternative to the use of transition metal catalysis. The indolenines are readily transformed into spiroindoline systems which are privileged scaffolds in medicinal chemistry.

Ester Formation via Symbiotic Activation Utilizing Trichloroacetimidate Electrophiles.

Trichloroacetimidates are useful reagents for the synthesis of esters under mild conditions that do not require an exogenous promoter. These conditions avoid the undesired decomposition of substrates with sensitive functional groups that are often observed with the use of strong Lewis or Brønsted acids. With heating, these reactions have been extended to benzyl esters without electron-donating groups. These inexpensive and convenient methods should find application in the formation of esters in complex substrates.

A selective gut bacterial bile salt hydrolase alters host metabolism.

The human gut microbiota impacts host metabolism and has been implicated in the pathophysiology of obesity and metabolic syndromes. However, defining the roles of specific microbial activities and metabolites on host phenotypes has proven challenging due to the complexity of the microbiome-host ecosystem. Here, we identify strains from the abundant gut bacterial phylum Bacteroidetes that display selective bile salt hydrolase (BSH) activity. Using isogenic strains of wild-type and BSH-deleted Bacteroides thetaiotaomicron, we selectively modulated the levels of the bile acid tauro-ß-muricholic acid in monocolonized gnotobiotic mice. B. thetaiotaomicron BSH mutant-colonized mice displayed altered metabolism, including reduced weight gain and respiratory exchange ratios, as well as transcriptional changes in metabolic, circadian rhythm, and immune pathways in the gut and liver. Our results demonstrate that metabolites generated by a single microbial gene and enzymatic activity can profoundly alter host metabolism and gene expression at local and organism-level scales.

Synthesis of 3,3'-Disubstituted Indolenines Utilizing the Lewis Acid Catalyzed Alkylation of 2,3-Disubstituted Indoles with Trichloroacetimidates.

Trichloroacetimidates function as effective electrophiles for the selective C3-alkylation of 2,3-disubstituted indoles to provide 3,3'-disubstituted indolenines. These indolenines are common synthetic intermediates that are often utilized in the synthesis of complex molecules. Effective reaction conditions utilizing Lewis acid catalysts have been determined, and the scope of the reaction with respect to indole and imidate reaction partner has been investigated. This chemistry provides an alternative to base promoted and transition metal catalyzed methods that are more commonly utilized to access similar indolenines.

Rearrangement of Benzylic Trichloroacetimidates to Benzylic Trichloroacetamides.

The rearrangement of allylic trichloroacetimidates is a well-known transformation, but the corresponding rearrangement of benzylic trichloroacetimidates has not been explored as a method for the synthesis of benzylic amines. Conditions that provide the trichloroacetamide product from a benzylic trichloroacetimidate in high yield have been developed. Methods were also investigated to transform the trichloroacetamide product directly into the corresponding amine, carbamate, and urea. A cationic mechanism for the rearrangement is implicated by the available data.

Lewis Acid Catalyzed Displacement of Trichloroacetimidates in the Synthesis of Functionalized Pyrroloindolines.

The pyrroloindoline core is found in many natural products. These structures often differ at the C3a position, which may be substituted with an oxygen, nitrogen, or sp(3)- or sp(2)-hybridized carbon. Utilizing a trichloroacetimidate leaving group, a diversity-oriented approach to these structures has been developed. The trichloroacetimidate intermediate allows for the rapid incorporation of anilines, alcohols, thiols, and carbon nucleophiles. This method was applied in the synthesis of arundinine and a formal synthesis of psychotriasine.

Synthesis and initial evaluation of quinoline-based inhibitors of the SH2-containing inositol 5'-phosphatase (SHIP).

Recently, inhibition of the SH2-containing inositol 5'-phosphatase 1 (SHIP1) has become an attractive strategy for facilitating engraftment of MHC-I mismatched bone marrow grafts, increasing the number of adult stem cells in vivo, and inducing mobilization of hematopoietic stem cells. Utilizing high-throughput screening, two quinoline small molecules (NSC13480 and NSC305787) that inhibit SHIP1 enzymatic activity were discovered. New syntheses of these inhibitors have been developed which verified the relative stereochemistry of these structures. Utilizing this synthetic route, some analogs of these quinolines have been prepared and tested for their ability to inhibit SHIP. These structure activity studies determined that an amine tethered to the quinoline core is required for SHIP inhibition. SHIP inhibition may explain the antitumor effects of similar quinoline amino alcohols and provides an impetus for further synthetic studies in this class of compounds.