Development of substituted benzimidazoles as inhibitors of human aldehyde dehydrogenase 1A isoenzymes.

Aldehyde dehydrogenase 1A (ALDH1A) isoforms may be a useful target for overcoming chemotherapy resistance in high-grade serous ovarian cancer (HGSOC) and other solid tumor cancers. However, as different cancers express different ALDH1A isoforms, isoform selective inhibitors may have a limited therapeutic scope. Furthermore, resistance to an ALDH1A isoform selective inhibitor could arise via induction of expression of other ALDH1A isoforms. As such, we have focused on the development of pan-ALDH1A inhibitors, rather than on ALDH1A isoform selective compounds. Herein, we report the development of a new group of pan-ALDH1A inhibitors to assess whether broad spectrum ALDH1A inhibition is an effective adjunct to chemotherapy in HGSOC. Optimization of the CM10 scaffold, aided by ALDH1A1 crystal structures, led to improved biochemical potencies, improved cellular efficacy as demonstrated by reduction in ALDEFLUOR signal in HGSOC cells, and substantial improvements in liver microsomal stability. Based on this work we identified two compounds 17 and 25 suitable for future in vivo proof of concept experiments.

Structural diversification of hapalindole and fischerindole natural products via cascade biocatalysis.

Hapalindoles and related compounds (ambiguines, fischerindoles, welwitindolinones) are a diverse class of indole alkaloid natural products. They are typically isolated from the Stigonemataceae order of cyanobacteria and possess a broad scope of biological activities. Recently the biosynthetic pathway for assembly of these metabolites has been elucidated. In order to generate the core ring system, L-tryptophan is converted into the cis-indole isonitrile subunit before being prenylated with geranyl pyrophosphate at the C-3 position. A class of cyclases (Stig) catalyzes a three-step process including a Cope rearrangement, 6-exo-trig cyclization and electrophilic aromatic substitution to create a polycyclic core. Formation of the initial alkaloid is followed by diverse late-stage tailoring reactions mediated by additional biosynthetic enzymes to give rise to the wide array of structural variations observed in this compound class. Herein, we demonstrate the versatility and utility of the Fam prenyltransferase and Stig cyclases toward core structural diversification of this family of indole alkaloids. Through synthesis of cis-indole isonitrile subunit derivatives, and aided by protein engineering and computational analysis, we have employed cascade biocatalysis to generate a range of derivatives, and gained insights into the basis for substrate flexibility in this system.

Recent advances in hapalindole-type cyanobacterial alkaloids: biosynthesis, synthesis, and biological activity.

Covering: 1984 up to the end of 2020Hapalindoles, fischerindoles, ambiguines and welwitindolinones are all members of a class of indole alkaloid natural products that have been isolated from the Stigonematales order of cyanobacteria. These compounds possess a polycyclic ring system, unique functional groups and various stereo- and regiochemical isomers. Since their initial isolation in 1984, they have been explored as potential therapeutics due to their wide variety of biological activities. Although numerous groups have pursued total syntheses of these densely functionalized structures, hapalindole biosynthesis has only recently been unveiled. Several groups have uncovered a wide range of novel enzymes that catalyze formation and tailoring of the hapalindole-type metabolites. In this article, we provide an overview of these natural products, their biological activities, highlight general synthetic routes, and provide an extensive review on the surprising biosynthetic processes leading to these structurally diverse metabolites.

The Synthesis of Functionalized 3-Aryl- and 3-Heteroaryloxazolidin-2-ones and Tetrahydro-3-aryl-1,3-oxazin-2-ones via the Iodocyclocarbamation Reaction: Access to Privileged Chemical Structures and Scope and Limitations of the Method.

3-Aryl- and 3-heteroaryloxazolidin-2-ones, by virtue of the diverse pharmacologic activities exhibited by them after subtle changes to their appended substituents, are becoming increasingly important and should be considered privileged chemical structures. The iodocyclocarbamation reaction has been extensively used to make many 3-alkyl-5-(halomethyl)oxazolidin-2-ones, but the corresponding aromatic congeners have been relatively underexplored. We suggest that racemic 3-aryl- and 3-heteroaryl-5-(iodomethyl)oxazolidin-2-ones, readily prepared by the iodocyclocarbamation reaction of N-allylated N-aryl or N-heteroaryl carbamates, may be useful intermediates for the rapid preparation of potential lead compounds with biological activity. We exemplify this point by using this approach to prepare racemic linezolid, an antibacterial agent. Herein, we report the results of our systematic investigation into the scope and limitations of this process and have identified some distinguishing characteristics within the aryl/heteroaryl series. We also describe the first preparation of 3-aryloxazolidin-2-ones bearing new functionalized C-5 substituents derived from conjugated 1,3-dienyl and cumulated 1,2-dienyl carbamate precursors. Finally, we describe the utility of the iodocyclocarbamation reaction for making six-membered tetrahydro-3-aryl-1,3-oxazin-2-ones.

Multicomponent Microscale Biosynthesis of Unnatural Cyanobacterial Indole Alkaloids.

Genome sequencing and bioinformatics tools have facilitated the identification and expression of an increasing number of cryptic biosynthetic gene clusters (BGCs). However, functional analysis of all components of a metabolic pathway to precisely determine biocatalytic properties remains time-consuming and labor intensive. One way to speed this process involves microscale cell-free protein synthesis (CFPS) for direct gene to biochemical function analysis, which has rarely been applied to study multicomponent enzymatic systems in specialized metabolism. We sought to establish an in vitro transcription/translation (TT)-assay to assess assembly of cyanobacterial-derived hapalindole-type natural products (cNPs) because of their diverse bioactivity profiles and complex structural diversity. Using a CFPS system including a plasmid bearing famD2 prenyltransferase from Fischerella ambigua UTEX 1903, we showed production of the central prenylated intermediate (3GC) in the presence of exogenous geranyl-pyrophosphate (GPP) and cis-indole isonitrile. Further addition of a plasmid bearing the famC1 Stig cyclase resulted in synthesis of both FamD2 and FamC1 enzymes, which was confirmed by proteomics analysis, and catalyzed assembly of 12-epi-hapalindole U. Further combinations of Stig cyclases (FamC1-C4) produced hapalindole U and hapalindole H, while FisC identified from Fischerella sp. SAG46.79 generated 12-epi-fischerindole U. The CFPS system was further employed to screen six unnatural halogenated cis-indole isonitrile substrates using FamC1 and FisC, and the reactions were scaled-up using chemoenzymatic synthesis and identified as 5- and 6-fluoro-12-epi-hapalindole U, and 5- and 6-fluoro-12-epi-fischerindole U, respectively. This approach represents an effective, high throughput strategy to determine the functional role of biosynthetic enzymes from diverse natural product BGCs.

Control of Stereoselectivity in Diverse Hapalindole Metabolites is Mediated by Cofactor-Induced Combinatorial Pairing of Stig Cyclases.

Stereospecific polycyclic core formation of hapalindoles and fischerindoles is controlled by Stig cyclases through a three-step cascade involving Cope rearrangement, 6-exo-trig cyclization, and a final electrophilic aromatic substitution. Reported here is a comprehensive study of all currently annotated Stig cyclases, revealing that these proteins can assemble into heteromeric complexes, induced by Ca2+ , to cooperatively control the stereochemistry of hapalindole natural products.

Engineered Production of Hapalindole Alkaloids in the Cyanobacterium Synechococcus sp. UTEX 2973.

Cyanobacteria produce numerous valuable bioactive secondary metabolites (natural products) including alkaloids, isoprenoids, nonribosomal peptides, and polyketides. However, the genomic organization of the biosynthetic gene clusters, complex gene expression patterns, and low compound yields synthesized by the native producers currently limits access to the vast majority of these valuable molecules for detailed studies. Molecular cloning and expression of such clusters in heterotrophic hosts is often precarious owing to genetic and biochemical incompatibilities. Production of such biomolecules in photoautotrophic hosts analogous to the native producers is an attractive alternative that has been under-explored. Here, we describe engineering of the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce key compounds of the hapalindole family of indole-isonitrile alkaloids. Engineering of the 42-kbp "fam" hapalindole pathway from the cyanobacterium Fischerella ambigua UTEX 1903 into S2973 was accomplished by rationally reconstructing six to seven core biosynthetic genes into synthetic operons. The resulting Synechococcus strains afforded controllable production of indole-isonitrile biosynthetic intermediates and hapalindoles H and 12-epi-hapalindole U at a titer of 0.75-3 mg/L. Exchanging genes encoding fam cyclase enzymes in the synthetic operons was employed to control the stereochemistry of the resulting product. Establishing a robust expression system provides a facile route to scalable levels of similar natural and new forms of bioactive hapalindole derivatives and its structural relatives (e.g., fischerindoles, welwitindolinones). Moreover, this versatile expression system represents a promising tool for exploring other functional characteristics of orphan gene products that mediate the remarkable biosynthesis of this important family of natural products.