belactosin-a and cyclopropane

Hormaomycins and belactosins are peptide natural products that contain unusual cyclopropane moieties. Bioinformatics analysis of the corresponding biosynthetic gene clusters showed that two conserved genes, hrmI/belK and hrmJ/belL, were potential candidates for catalyzing cyclopropanation. Using in vivo and in vitro assays, the functions of HrmI/BelK and HrmJ/BelL were established. HrmI and BelK, which are heme oxygenase-like dinuclear iron enzymes, catalyze oxidation of the ϵ-amino group of l-lysine to afford l-6-nitronorleucine. Subsequently, HrmJ and BelL, which are iron- and α-ketoglutarate-dependent oxygenases, effectively convert l-6-nitronorleucine into 3-(trans-2-nitrocyclopropyl)-alanine through C4-C6 bond installation. These observations disclose a novel pathway of cyclopropane ring construction and exemplify the new chemistry involving metalloenzymes in natural product biosynthesis.

Topics: Catalysis; Cyclopropanes; Depsipeptides; Intercellular Signaling Peptides and Proteins; Metalloproteins; Molecular Structure

The natural product belactosin A (1) with a trans-cyclopropane structure is a useful prototype compound for developing potent proteasome (core particle, CP) inhibitors. To date, 1 and its analogues are the only CP ligands that bind to both the nonprimed S1 pocket as well as the primed substrate binding channel; however, these molecules harbor a high IC50 value of more than 1 μM. We have performed structure-activity relationship studies, thereby elucidating unnatural cis-cyclopropane derivatives of 1 that exhibit high potency to primarily block the chymotrypsin-like active site of the human constitutive (cCP) and immunoproteasome (iCP). The most active compound 3e reversibly inhibits cCP and iCP similarly with an IC50 of 5.7 nM. X-ray crystallographic analysis of the yeast proteasome in complex with 3e revealed that the ligand is accommodated predominantly into the primed substrate binding channel and covalently binds to the active site threonine residue via its β-lactone ring-opening.

Topics: Cell Proliferation; Chemistry Techniques, Synthetic; Crystallography, X-Ray; Cyclopropanes; Drug Stability; HCT116 Cells; Humans; Intercellular Signaling Peptides and Proteins; Isomerism; Models, Molecular; Peptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Conformation; Water

The belactosin A analog 2a, having the unnatural cis-cyclopropane structure instead of the trans-cyclopropane structure in belactosin A, is a much more potent proteasome inhibitor than belactosin A. However, its cell growth inhibitory effect is rather lower than that expected from its remarkable proteasome inhibitory effect, probably due to its instability under cellular conditions. We hypothesized that the instability of 2a was due to chemical and enzymatic hydrolysis of the strained β-lactone moiety. Thus, to increase the stability of 2a by chemical modification, its analogs with a sterically more hindered β-lactone moiety and/or cyclopropylic strain-based conformational restriction were designed and synthesized, resulting in the identification of a stabilized analog 6a as a proteasome inhibitor with cell growth inhibitory effects. Our findings suggest that the chemical and biological stability of 2a is significantly affected by the steric hindrance around its β-lactone carbonyl moiety and the conformational flexibility of the molecule.

Topics: Cell Proliferation; Cyclopropanes; Dose-Response Relationship, Drug; Drug Design; HCT116 Cells; Humans; Intercellular Signaling Peptides and Proteins; Molecular Structure; Peptides; Proteasome Endopeptidase Complex; Stereoisomerism; Structure-Activity Relationship

An efficient and convenient methodology for the synthesis of the 3-(trans-2-aminocyclopropyl) alanine and 3-(trans-2-nitrocyclopropyl) alanine moieties found in the core of belactosin A and hormaomycin, respectively, is reported. By using an enantioenriched substituted alpha-nitro diazoester in a diastereoselective intramolecular cyclopropanation reaction, the trans-nitrocyclopropyl alanine moiety can be obtained efficiently in five steps from the initial alpha-nitrocyclopropyl lactone unit, thus achieving the synthesis of the cyclopropane core of the two natural products.

Topics: Alanine; Biological Products; Catalysis; Cyclization; Cyclopropanes; Depsipeptides; Intercellular Signaling Peptides and Proteins; Molecular Structure; Peptides; Stereoisomerism