Figuring out an unusual natural product’s activity can be a difficult but rewarding exercise. Deep evolutionary time has provided us with a bizarre range of chemical structures that are presumably not being synthesized by organisms for the sheer fun of it – these things are acting as signaling molecules, antifeedants, poisons for the competition, pheromones and who knows what else. Many biochemical pathways have been discovered and defined by the actions of some natural product (the opiate receptors and the cardiac glycosides are two of the classic examples) although sometimes this has taken a very long time indeed (as in the cases of salicylic acid and quinine).
Here’s a new paper on a macrolide from a sponge in the Gulf of Mexico, lasolonide A. It’s long been known to be active against several tumor cell lines and with a unique profile (and has been synthesized more than once, although it’s interesting that some of those papers have made one enantiomer and some the other). Its exact mode of action has been obscure, although it has been shown to promote hyperphosphorylation in cells (so it might be taking the brakes off several kinases somehow). In general, sensitive cells show loss of adhesion, sudden chromosome condensation, and “blebbing“. These effects are actually reversible (cells can take a lot of messing with), but on longer-term exposure the compound is cytotoxic.
It’s pretty potent – Hap1 cells, for example, show these effects at 20 nM concentration. As the paper notes, several other related compounds have been isolated, and they show a definite SAR – moving double bonds around or messing with the size of the macrocycle make it less potent or completely inactive. So there’s likely a specific target, but what? The team here (Stanford, Albany, and Genentech) did the sort of experiment that one does in these days of modern times: they generated a huge library of random mutations and treated those with a severe dose of the compound, reasoning that any cells that survived were likely to have inactivating mutations in particularly important proteins. This worked just as planned, and pointed to mutations in the gene for a protein called lipid droplet associated hydrolase (LDAH). Deliberately inactivating that gene made several different cell lines far less sensitive to the compound, and introducing active LDAH into that line brought the toxicity right back, so the connection looks solid.
That’s an interesting result. There are an awful lot of such enzymes in the cell, generally working through a serine hydrolase mechanism and often involved in lipid processing. But how does that relate to the toxic mechanism, and why is it so specific to the one hydrolase? It’s not because lasolonide A is an inhibitor for LDAH; the knockout experiments showed that cells can get along just fine without that enzyme activity. Mass spec experiments showed that a side-chain ester in the compound was apparently being cleaved to give another natural product that had already been isolated in the wild, lasolonide F. A serine hydrolase could certainly do something like that, and sure enough, in the LDAH-disrupted cells the amount of lasF decreased sharply, suggesting that it is the active toxic species. It’s a lot more polar than the greasy lasA, of course, and actually isnt very active against cells directly, which tells you that lasA is acting as a prodrug to release the active compound once it gets into the cytoplasm.
LDAH has been studied in the past, and it’s known (thus the name) to associate with the surface of lipid droplets as they emerge from the endoplasmic reticulum. In fact, a particular protein hairpin motif has been identified that anchors it into that interface, and as it happens, mutating that hairpin abolishes lasA toxicity. So the protein apparently has to be sitting at the lipid droplet in order to hydrolyze the natural product: where is the natural product, then? Accumulating in the lipid droplet! Various techniques that increase the lipid droplet content of cells also increased the toxicity of lasA. Subcellular fractionation and mass spec confirmed this – the compound is also in the cytoplasm (although that might be an artifact, to be honest), but definitely partitions into the lipids, whereas lasF (as you’d expect from its polarity) doesn’t show up in there at all.
It would be reasonable to assume that LDAH is not necessarily some wonderfully selective enzyme for lasA, but rather is hydrolyzing it because there’s a ton of it accumulating with it in its lipid droplets. This local concentration effect takes us right back into the world of intracellular condensate droplets. A separate lipid droplet phase is a lot more familiar to organic chemists and cell biologists, as is the idea of compound partitioning into such droplets. But the idea is the same for the protein/RNA droplets that we now know are all over the cell: they’re promoting particular reactions and pathways by increased local concentration.
On another level, this is a big reason for why enzyme active sites work, and the same principle applies to proteins interacting with each other. A lot of cell biology is arranging things so that they’re colocated (or most definitely not colocated!), thus all the compartments, membranes, and organelles. Concentration gradients are what keep us all alive. The currently fashionable targeted protein degradation work is all about hijacking such propinquity for fun and profit – there are clearly a lot of ubiquitinating enzymes that will go to work on whatever’s brought next to them, so now we’re focusing on getting them together. “Control of local concentration” may be an overarching theme of 21st-century drug discovery.
To zoom back in to the topic at hand, this paper raises some interesting questions Can this lipid-droplet mechanism be exploited for prodrug delivery? Is it happening already, and lasA is just allowing us to notice it? Are there other lipid-droplet-associated enzymes that can be taken advantage of? We certainly make enough hydrophobic molecules in this business!