Tomato Steroidal Glycoalkaloids: Biosynthesis in Solanaceae Plants

            Sarcopenia, or age-related muscle atrophy, is a condition that annually affects ~30 million Americans over the age of 60. The inability to regain muscle strength is a debilitating ailment that plagues elderly people with arthritis or those on bed rest recovering from an illness or injury. Sarcopenia is a challenging condition to ameliorate with pharmacological intervention. Androgenic steroid hormones that promote muscle growth (e.g. testosterone) are inextricably linked with side effects such as prostate growth and severe acne. One aza-steroid compound, MK-0773 (whose structure bears a striking resemblance to the male pattern baldness drug Propecia), was developed by Merck Research Laboratories as an inhibitor of muscle atrophy and is currently in Phase II clinical trials. MK-0773 is a selective androgen receptor modulator or ‘SARM’. Merck describes a SARM as an androgen receptor ligand that can induce anabolism (muscle growth), but with reduced effects on reproductive tissues. This type of bioactivity is largely mediated by tissue selectivity rather than receptor function, efficacy or potency. The Merck team was able to characterize an in vitro ligand screening profile that was predictive of a fully anabolic response without reproductive effects in vivo. Using their pharmacological criteria, which hinged on a profound comprehension of the molecular basis for androgen receptor activation, effective AR ligands such as MK-0773 were developed that exhibited the desired tissue selectivity in humans.
            More recently, researchers at the University of Iowa uncovered a natural product with similar biological properties (to those of MK-0773) using an approach that was fundamentally different from Merck’s more traditional drug discovery methods. The Iowa team led by Christopher M. Adams used an approach that selects molecules on the basis of mRNA expression signatures that they elicit in cells, using a technology called ‘the connectivity map.’ The connectivity map compares gene-expression signatures characteristic of a given disease state with those induced by small molecules. When applied to inhibition of muscle atrophy, tomatidine, a hexacyclic aza-spiro steroid abundant in green tomatoes, emerged as a promising therapeutic development candidate. Tomatidine’s mechanism of action reportedly involves activation of an anabolic kinase (mTORC1), which is distinct from that of MK-0773. Tomatidine is the steroidal aglycone of the complex tomato saponin, a-tomatine (shown above).

            Steroidal glycoalkaloids are secondary plant metabolites found in numerous Solanaceae plant species including tomatoes, eggplants and potatoes. Immature green tomatoes contain up to 500 mg of a-tomatine per kilogram of fresh fruit weight. The saponin is biosynthetically converted to esculeoside A (see below) and degraded to allopregnenolone as the tomato ripens, until it reaches levels of ~5 mg/kg in mature red tomatoes. Tomato glycoalkaloids are thought to protect against the adverse effects of predators such as fungi, bacteria, viruses and insects. Radiolabeling studies conducted in the 1960s indicated that cholesterol is the biosynthetic precursor to most of the glycoalkaloids and sapogenins that occur naturally in tomatoes. Yet, tomatoes and other solanaceous plant species are not generally thought of as significant dietary sources of cholesterol for humans. And with good reason: It turns out that cholesterol does not accumulate in plants, but is immediately and completely converted to other substances. The steroidal glycoalkaloids are biosynthesized from cholesterol via oxidations at C26, C22 and C16, but the exact sequence of the functionalizations remains to be established. The steroidal dehydropiperidine verazine appears to be a biosynthetic branching point, wherein C16-b hydroxylation leads to spirocyclization, eventually culminating in a-tomatine, following C3-O-glycosylation. Alternately, in potato species, C16-a hydroxylation of verazine affords the natural product teinemine. Subsequent S_N2-type nucleophilic substitution of teinemine’s piperidine nitrogen onto C16 with inversion of configuration generates the solanidine glycosides, which are linearly fused cyclic amine structures rather than aza-oxo spirocycles.

            In addition to some early precursor administration studies using radiolabeled substrates, the biosynthetic pathway leading to the steroidal glycoalkaloids was largely arrived at by linking together natural products that have been previously characterized from Solanaceae plants. Yoshinori Fujimoto’s research group at the Tokyo Institute of Technology is currently engaged in studies aimed at further refinement of our understanding of the glycoalkaloid biosynthetic pathway. Fujimoto and co-workers have recently disclosed two reports (one in 2013 and another in 2014) on the mechanism of the C26 amination of cholesterol (depicted above). It was previously reported in the 1970s that amination at C26 occurs upon displacement by a nitrogen-based nucleophile such as arginine. Fujimoto reasoned that a transamination mechanism involving an aldehyde intermediate was a more plausible biosynthetic pathway leading to 26-amino-cholesterol. They prepared a labeled C26 aldehyde containing five deuterium atoms in eleven synthetic operations from 3b-hydroxycholenic acid. When the deuterium-labeled substrate was ‘fed’ to a tomato seedling (Solanum lycopersicum), LC/MS analysis of the biosynthesized tomatine indicated that the labeled aldehyde was indeed incorporated into tomatine. The study supports the notion that a transamination mechanism involving an aldehyde precursor is operative during tomato glycoalkaloid biosynthesis starting from cholesterol. It must also be noted that an alternate deuterium-labeled substrate (the corresponding C26 alcohol) was incorporated into tomatine three times more efficiently than the d₅-aldehyde shown above, which is not entirely consistent with Fujimoto’s transamination hypothesis. The exact structure of the biosynthetically relevant C26 aldehyde derived from cholesterol, which has never been directly observed or characterized, needs to be clarified through further studies.

            In terms of nutrition, ripened red tomatoes are best known for the strong antioxidative effects of lycopenes, which act as highly efficient free radical scavengers and quenchers of reactive oxygen species (ROS). In addition, Japanese researchers recently isolated esculeoside A (structure above) from the ripe fruit of tomatoes (~500 mg/kg ripe tomato). The steroidal alkaloid glycoside inhibited hyperlipidemia and atherosclerosis in apoE-deficient mice by inhibition of acyl coenzyme A: cholesterol acyl-transferase (ACAT) activity, suggesting that esculeoside A may have inhibitory effects on atherosclerosis. Tomatoes, both red and green, should be considered a near-ideal superfood, given that we now understand, at the molecular level, their contribution of beneficial antioxidants, carbohydrates, fiber, vitamins and bioactive steroidal glycoalkaloids to the human diet.