Well, a large group from Schering-Plough and Merck have published the definitive paper on the real target of S-P's cholesterol absorption inhibitor, ezetimibe (Zetia), which drug I've spoken about here and here. It's published in the preprint section of the Proceedings of the National Academy of Sciences, which is a pretty nice journal to be in, and (for a number of reasons) not one that drug company research finds its way into all that often.
The drug and its equally active metabolite hit a protein in the intestinal wall called NPC1L1. This was the suspect in the previous publication on this topic, but now they've nailed it down. The authors were able to express the protein in cells and get a radioligand binding assay going for it using the isolated membranes, which turned out to be a pain. Depending on how you prepare the membrane and what detergents you use in the assay, the binding could change by a factor of 5 in what were supposed to be samples of the same thing. Only membranes from the cells that had expressed the protein showed any binding, and this result was extended to whole animals by generating a strain of mice with NPC1L1 knocked out, and intestinal membranes from their cells, in turn, no longer bind ezetimibe at all.
The paper hammers things flat in every direction: a series of ezetimibe analogs that bound with a thousand-fold range of potency on the intestinal membranes showed the same spread when tested in the NPC1L1 expressed-protein binding assay. Interestingly, intestinal membranes from different species showed a wide range of binding behavior, with mouse being the weakest and rhesus monkey by far the strongest, and that exactly parallels the drug's behavior when dosed in these animals. Humans are in the middle, by the way. (I should mention that the best chance to get a correlation this nice is with a drug like ezetimibe, which hardly gets out of the gut at all. Different species can vary so much once a compound gets out into circulation that a direct link to a binding assay is often obscured.)
So, this is the drug's target, beyond doubt. The doubt kicks in very quickly, though, when you ask what NPC1L1 does and why ezetimibe's binding to it blocks cholesterol uptake. The paper advances two hypotheses - that cholesterol binds directly to NPC1L1 - that it's the cholesterol transporter itself. Alternatively, it could be regulating the real transporter, and since Schering-Plough had already investigated a lot of the obvious candidates, that might well be a further unknown protein.
Note how long this all took. Nothing good comes quickly in drug discovery. The first compounds in the ezetimibe series were discovered in 1990/91, and efforts to identify the molecular target went on for many years. How long, you have to wonder, would it have taken to find this important pathway without first finding the compound which illuminated it?