A Cytochrome P450‐Mediated Intramolecular Carbon–Carbon Ring Closure in the Biosynthesis of Multidrug‐Resistance‐Reversing Lathyrane Diterpenoids

Abstract The Euphorbiaceae produce a wide variety of bioactive diterpenoids. These include the lathyranes, which have received much interest due to their ability to inhibit the ABC transporters responsible for the loss of efficacy of many chemotherapy drugs. The lathyranes are also intermediates in the biosynthesis of range of other bioactive diterpenoids with potential applications in the treatment of pain, HIV and cancer. We report here a gene cluster from Jatropha curcas that contains the genes required to convert geranylgeranyl pyrophosphate into a number of diterpenoids, including the lathyranes jolkinol C and epi‐jolkinol C. The conversion of casbene to the lathyranes involves an intramolecular carbon–carbon ring closure. This requires the activity of two cytochrome P450s that we propose form a 6‐hydroxy‐5,9‐diketocasbene intermediate, which then undergoes an aldol reaction. The discovery of the P450 genes required to convert casbene to lathyranes will allow the scalable heterologous production of these potential anticancer drugs, which can often only be sourced in limited quantities from their native plant.

The Euphorbiaceae produce aw idev ariety of bioactive diterpenoids.T hese include the lathyranes, which have received much interestd ue to their ability to inhibit the ABC transporters responsible for the loss of efficacy of many chemotherapy drugs. The lathyranes are also intermediates in the biosynthesis of range of other bioactive diterpenoids with potentiala pplications in the treatment of pain, HIV and cancer.W er eport here ag ene clusterf rom Jatrophac urcas that contains the genes required to convert geranylgeranyl pyrophosphate into an umber of diterpenoids, including the lathyranes jolkinol C and epi-jolkinol C. The conversion of casbene to the lathyranes involves an intramolecular carbon-carbon ringc losure. This requires the activity of two cytochrome P450s that we propose form a6 -hydroxy-5,9-diketocasbenei ntermediate, which then undergoes an aldol reaction. The discovery of the P450 genes required to convert casbene to lathyranes will allow the scalable heterologousp roduction of these potential anticancer drugs, which can often only be sourcedi nl imited quantities from their native plant.
The Euphorbiaceae produce ad iverse range of casbene (1)-derived diterpenoids, [1] many of which are providing interesting leads in the development of new pharmaceuticals. These include the lathyranes (Scheme 1), which are inhibitors of ABC transporters that are responsible for the efflux of chemotherapy drugs in multidrug-resistant( MDR) cancers as well as fungal and protozoal pathogens. [2] The lathyranes are also the precursors of many other active diterpenoids including ingenol mebutate,alicenced pharmaceutical used for the treatment of actinic keratosis, prostratin, al ead compound for the treatment of latent HIV infections,a nd resiniferatoxin, an ultrapotent capsaicin analogue that is currently in clinical trial for the treatment of cancer-related intractable pain (Scheme 2). [3] Although the relationship between casbane,t he lathyrane and an umber of other diterpenoid classes was noted severald ecades ago (Scheme 1), [4] the mechanism leadingt ot he ring closure required to convert the 14:3 casbane ring into the 5:11:3 lathyrane ring system has not previously been reported.
Recently,w er eported ad iterpenoid biosynthetic gene cluster in castor (Ricinuscommunis)t hat contained genes encoding diterpene synthasesa nd several cytochrome P450s,i ncluding casbene synthasesa nd casbene-5-oxidases. We also demonstrated the existence of similar clusters in otherE uphorbiaceae including Jatropha curcas,aplant that produces av ariety of diterpenoids including lathyranes, jatropholanes,r hamnofolanes and tiglianes [5] ( Figure S1 in the Supporting Information). Using ar ecently released version of the Jatropha curcas genome, [6] we were able to performf urther in silico analysis of this cluster, and found that it contained an umber of enzyme-encoding genes, including casbene synthases,c ytochrome P450s, alcohol dehydrogenases and "alkenal reductase"-like genes ( Figure 1). The P450 genes were all members of the CYP71Dt ribe, and all Scheme1.The lathyranes as proposed intermediates in the biosynthesis of diterpenoidsw ith tigliane, daphnane, ingenane,r hamnofolane and jatropholane carbon skeletons. but two were part of the CYP726A taxon-specific bloom found so far only in the Euphorbiaceae [7,8] (Figure S2).
Using qPCR, we analysed the expression of the genes present within this cluster ( Figure S3). The majority of the genes for which we were ablet od etect transcripts were most abundantly expressedw ithin the roots.T he exception to this was CYP71D495, which was most abundant in leaves, but still abundant in both stems and roots. Thiso bservation was consistent with the roots of J. curcas being rich in diterpenoids. [5] Phylogenetica nalysiso ft he P450 genes suggested that CYP726A35 was orthologous to CYP726A18a nd CYP726A15 from castor ( Figure S2). The former of these P450s is able to convert casbene into 5-ketocasbene via ah ydroxyl intermedi-ate, whereas the latter catalyses as imilar reaction with neocembrene. [7] When CYP726A35 was transiently coexpressed with casbene synthase in Nicotiana benthamiana leaves, we were able to detect am etabolite (2)w ith am olecular mass of 302.23 ( Figure 2), which we identified by NMR spectroscopy as 6-hydroxy-5-ketocasbene (Scheme3). This diterpenoid has previously been reported to be ap roduct of casbene oxidation by CYP726A14 from castor. [9] As econd P450 from J. curcas (CYP726A20)w as also able to convert casbene into 6-hydroxy-5-ketocasbene. This observation was similar to what we observed in castor,where we identified more than one P450 gene that was able to perform casbene 5-oxidation. [7] In silico analyses of CYP726A35, CYP726A18 and CYP726A15 (neocembrene-5-oxidase) revealed the presenceo faputative plastidial transit peptide ( Figure S4). Fusiono fg reen fluorescentp rotein (GFP) to the Nterminus resulted in the import of transiently expressed GFP into the plastids of N. benthamiana ( Figure S4). CYP726A20 did not contain ap redicted chloroplast transit peptide,a nd,c onsistent with this, fusion of the first 80 amino acids of this protein to GFP did not result in import into plastids. Thus it would appear that in both Jatropha and castor the enzymes catalysing casbene 5-oxidationa re located in both the plastid and the endo-Scheme2.Structures of Euphorbia factor L1 (a lathyrane), ingenol mebutate (an ingenane), prostratin (a tigliane) and resiniferatoxin (a daphnane). The red and blue oxygen atoms highlighted on each of the molecules correspond to the 5a nd 9p ositionso fcasbene, respectively.T he green carboncarbon bond corresponds to the 6and10 positionsofc asbene.  plasmicr eticulum. Both Jatropha enzymes were also able to catalyse6 -hydroxylation. Interestingly,i nc astor, Euphorbia peplus [7] and J. curcas (Figure 1), the plastidial casbene-5-oxidases are adjacentt oacasbene synthase, thus indicatingt he order of these genes may be conserved in the Euphorbiaceae.
Only one other of the cytochrome P450 genes present within the J. curcas gene cluster (CYP71D495) was able to form ap roduct (3)w ith casbene. This was purified and determined to be 9-ketocasbene ( Figure 2, Scheme 3). Interestingly,a ll of the diterpenoids reported in J. curcas ( Figure S1) and the vast majority of those described in the Euphorbiaceae contain either ah ydroxy or keto group at this 9-position. [5,7] Indeed, 9oxidation (in addition to 5-oxidation)a ppearst ob ep resent in all lathyranes, jatropholanes, tiglianes and ingenanes; this suggests that it might be ap rerequisite for 6,10 ring closure.
An umber of enzymes, including polyketide synthases, have previously been reported to perform intramolecular ring closures through aldol reactions. [12] An umber of plant P450s that catalysei ntramolecular rearrangements have alsob een characterised. These include CYP80F1 from Hyoscyamus niger,w hich catalyses an oxidation and rearrangement as well as Papaver somniferum CYP719B1 and Coptis japonica CYP80G2, both of which catalyseC ÀCbond formationsina lkaloid biosynthesis. [13] In the case of the taxaned iterpenoids, P450s catalysing CÀC bond migrations and ring closures have also been reported. [14] However, for the plant terpenoids at least,w eare not aware of any other P450-mediated ring closures involving an aldol reaction.
Our discovery of the steps required to convert casbene into lathyranes could be used to allow scalable production of the lathyranes in heterologous host systems such as yeast or tobacco. Interestingly,j olkinol derivatives have already received interesta sp recursors for the development of semisynthetic lathyranes with increased potency against MDR cancerc ell lines. [15] Our study has also provided insight into the metabolic diversification of the diterpenoidsi nt he Euphorbiaceae, and demonstrated by the fact that the cytochrome P450sa re not only responsible ford ecoration of the terpenes caffold,b ut also for initiating intramolecular ring closures.W ea re continuing to characterise the remaining genes present within the J. curcas diterpenoid cluster and to determine their involvement in the biosynthesis of casbene-derived diterpenoidst hat have been reported from this plant.