Engineered Biosynthesis of Antiprotealide and Other Unnatural Salinosporamide Proteasome Inhibitors

Abstract

A new shunt in the phenylalanine biosynthetic pathway to the nonproteinogenic amino acid L-3-cyclohex-2'-enylalanine was exploited in the marine bacterium Salinispora tropica by mutagenesis to allow for the genetic engineering of unnatural derivatives of the potent proteasome inhibitor salinosporamide A (2) such as antiprotealide (1).

Mining the Cinnabaramide Biosynthetic Pathway to Generate Novel Proteasome Inhibitors

“…[21][22][23] The C-terminal domains of CinA and CinB form the terminal NRPS module of the assembly line, and the highly homologous domain set in the salinosporamide gene cluster is known to activate the nonproteinogenic amino acid b-hydroxy-2'-cyclohexenylalanine. [12,24] Because of the structural similarity of the two compounds, and because the deduced substrate specificity code of the CinB A domain (DLMNVGGV; determined as described previously [25,26] ) showed a significant degree of similarity to that of the SalB A domain (DLLSNGGV), we propose elongation of the cinnabaramide intermediate by extension with b-hydroxy-2'-cyclohexenylalanine (Scheme 2 B). The resulting PCP-tethered cinnabaramide intermediate is then thought to undergo an intramolecular condensation promoted by the ketosynthase (CinC) to generate the g-lactam ring, which provides the CinB-bound alcohol substrate that gives rise to the offloaded b-lactone product.…”

“…Similar enzymes have also been described in the salinosporamide biosynthesis pathway-it was suggested that these were involved in cyclohexenyl-alanine building block formation-and inactivation of a prephenate dehydratase homologue gene (salX) in the sal cluster led to the elimination of all natural salinosporamides. [12,27] It has been reported that the salinosporamide biosynthetic enzymes responsible for the activation and incorporation of the native sal-specific amino acid exhibit relaxed substrate specificities towards a number of aliphatic amino acids. [28] In that work, Moore and co-workers used a comprehensive approach that combined chemical synthesis with metabolic engineering to generate a series of salinosporamide analogues with altered bioactivities against human cancer cell lines.…”

“…Interestingly, the salinosporamide megasynthetase was shown to tolerate both secondary and tertiary carbons in the g-position of the amino acid precursor, but substitution in the bposition was also not accepted. [12] The authors suggested that the latter substitution might directly affect the function of the cytochrome P450 enzyme (SalD) in the pathway.…”

“…JS360 is known to produce cinnabaramides A-G, each exhibiting a g-lactam-b-lactone bicyclic ring structure attached to a cyclohexenyl unit, together with a hexyl side chain. [9] With the exception of the hexyl side chain, significant structure similarity was found between these compounds and the salinosporamides (Scheme 1), a class of PKS/NRPS antitumor metabolites isolated from the marine actinomycetes Salinispora tropica CNB-440 [10][11][12] and Salinospora…”

Salinosporamid‐Naturstoffe: potente Inhibitoren des 20S‐Proteasoms als vielversprechende Krebs‐Chemotherapeutika

“…[78] Die Löschung von salX hob die Biosynthese aller Salinosporamide auf und ermöglichte die gerichtete mutagenetische Produktion einer Serie nichtnatürlicher Salinosporamid-Derivate (siehe Abschnitt 6.2). [79,80] Die anscheinend fehlenden Enzyme (also diejenigen, die an der Bildung von 44 aus 43 beteiligt sind) sowie die beiden fehlenden Reduktasen wurden noch nicht identifiziert und könnten sich außerhalb des sal-Lokus befinden. Die Hydroxygruppe an der Seitenkette wird schließ-lich durch die P450-Hydroxylase SalD eingeführt, wie durch Inaktivierung des entsprechenden Gens und der daraus resultierenden Produktion von Salinosporamid J (30) nachgewiesen wurde; diese Oxidation findet, wie sich aufgrund anderer, biochemisch charakterisierter P450-Enzyme vermuten lässt, [79] wahrscheinlich statt, während die Aminosäure 39 an die PCP-Domäne von SalB gebunden ist.…”