Identification of highly selective SIK1/2 inhibitors that modulate innate immune activation and suppress intestinal inflammation.

The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site. This effort resulted in SIK1/2-selective probes that inhibit key intracellular proximal signaling events including reducing phosphorylation of the SIK substrate cAMP response element binding protein (CREB) regulated transcription coactivator 3 (CRTC3) as detected with an internally generated phospho-Ser329-CRTC3-specific antibody. These inhibitors also suppress production of pro-inflammatory cytokines while inducing anti-inflammatory interleukin-10 in activated human and murine myeloid cells and in mice following a lipopolysaccharide challenge. Oral dosing of these compounds ameliorates disease in a murine colitis model. These findings define an approach to generate highly selective SIK1/2 inhibitors and establish that targeting these isoforms may be a useful strategy to suppress pathological inflammation.

Total synthesis and isolation of citrinalin and cyclopiamine congeners.

Many natural products that contain basic nitrogen atoms--for example alkaloids like morphine and quinine-have the potential to treat a broad range of human diseases. However, the presence of a nitrogen atom in a target molecule can complicate its chemical synthesis because of the basicity of nitrogen atoms and their susceptibility to oxidation. Obtaining such compounds by chemical synthesis can be further complicated by the presence of multiple nitrogen atoms, but it can be done by the selective introduction and removal of functional groups that mitigate basicity. Here we use such a strategy to complete the chemical syntheses of citrinalin B and cyclopiamine B. The chemical connections that have been realized as a result of these syntheses, in addition to the isolation of both 17-hydroxycitrinalin B and citrinalin C (which contains a bicyclo[2.2.2]diazaoctane structural unit) through carbon-13 feeding studies, support the existence of a common bicyclo[2.2.2]diazaoctane-containing biogenetic precursor to these compounds, as has been proposed previously.

Function and Structure of MalA/MalA', Iterative Halogenases for Late-Stage C-H Functionalization of Indole Alkaloids.

Malbrancheamide is a dichlorinated fungal indole alkaloid isolated from both Malbranchea aurantiaca and Malbranchea graminicola that belongs to a family of natural products containing a characteristic bicyclo[2.2.2]diazaoctane core. The introduction of chlorine atoms on the indole ring of malbrancheamide differentiates it from other members of this family and contributes significantly to its biological activity. In this study, we characterized the two flavin-dependent halogenases involved in the late-stage halogenation of malbrancheamide in two different fungal strains. MalA and MalA' catalyze the iterative dichlorination and monobromination of the free substrate premalbrancheamide as the final steps in the malbrancheamide biosynthetic pathway. Two unnatural bromo-chloro-malbrancheamide analogues were generated through MalA-mediated chemoenzymatic synthesis. Structural analysis and computational studies of MalA' in complex with three substrates revealed that the enzyme represents a new class of zinc-binding flavin-dependent halogenases and provides new insights into a potentially unique reaction mechanism.

Novel Reagent Space: Identifying Unorderable but Readily Synthesizable Building Blocks.

Drug discovery building blocks available commercially or within an internal inventory cover a diverse range of chemical space and yet describe only a tiny fraction of all chemically feasible reagents. Vendors will eagerly provide tools to search the former; there is no straightforward method of mining the latter. We describe a procedure and use case in assembling chemical structures not available for purchase but that could likely be synthesized in one robust chemical transformation starting from readily available building blocks. Accessing this vast virtual chemical space dramatically increases our curated collection of reagents available for medicinal chemistry exploration and novel hit generation, almost tripling the number of those with 10 or fewer atoms.

A unified strategy to reverse-prenylated indole alkaloids: total syntheses of preparaherquamide, premalbrancheamide, and (+)-VM-55599.

A full account of our studies toward reverse-prenylated indole alkaloids that contain a bicyclo[2.2.2]core is described. A divergent route is reported which has resulted in the synthesis of preparaherquamide, (+)-VM-55599, and premalbrancheamide. An intramolecular Dieckmann cyclization between an enolate and isocyanate was used to forge the bicyclo[2.2.2]diazaoctane core that is characteristic of these molecules. The pentacyclic indole scaffold was constructed through a one-pot Hofmann rearrangement followed by Fischer indole synthesis. The utilization of our previously reported indole peripheral functionalization strategy also led to natural products including malbrancheamides B, C, stephacidin A, notoamides F, I and R, aspergamide B, and waikialoid A. Ultimately, the divergent route that we devised provided access to a wide range of prenylated indole alkaloids that are differently substituted on the cyclic amine core.

Bioinspired chemical synthesis of monomeric and dimeric stephacidin A congeners.

Stephacidin A and its congeners are a collection of secondary metabolites that possess intriguing structural motifs. They stem from unusual biosynthetic sequences that lead to the incorporation of a prenyl or reverse-prenyl group into a bicyclo[2.2.2]diazaoctane framework, a chromene unit or the vestige thereof. To complement biosynthetic studies, which normally play a significant role in unveiling the biosynthetic pathways of natural products, here we demonstrate that chemical synthesis can provide important insights into biosynthesis. We identify a short total synthesis of congeners in the reverse-prenylated indole alkaloid family related to stephacidin A by taking advantage of a direct indole C6 halogenation of the related ketopremalbrancheamide. This novel strategic approach has now made possible the syntheses of several natural products, including malbrancheamides B and C, notoamides F, I and R, aspergamide B, and waikialoid A, which is a heterodimer of avrainvillamide and aspergamide B. Our approach to the preparation of these prenylated and reverse-prenylated indole alkaloids is bioinspired, and may also inform the as-yet undetermined biosynthesis of several congeners.

Unified Approach to Prenylated Indole Alkaloids: Total Syntheses of (-)-17-Hydroxy-Citrinalin B, (+)-Stephacidin A, and (+)-Notoamide I.

A unified strategy for the synthesis of congeners of the prenylated indole alkaloids is presented. This strategy has yielded the first synthesis of the natural product (-)-17-hydroxy-citrinalin B as well as syntheses of (+)-stephacidin A and (+)-notoamide I. An enolate addition to an in situ generated isocyanate was utilized in forging a key bicyclo[2.2.2]diazaoctane moiety, and in this way connected the two structural classes of the prenylated indole alkaloids through synthesis.