Anyone who’s done antibiotic research can tell you about what a slog it is. Just looking at the rate of approval of new ones will tell you that, too – it really is like breaking rocks, except breaking rocks is a lot more straightforward and rewarding most of the time. As I’ve said before, when I look back at all the mammalian cells that I’ve killed with my molecules over the years, and compare that to the experience I had working against gram-negative bacteria, it’s pretty sobering. Killing gram-negative pathogens is hard. And killing them with a compound that (A) hasn’t already been discovered, in one form or another and (B) doesn’t kill everything else it touches is a challenge indeed.
There are two new papers, though, that give a person some hope. And we need some, because resistant bacteria, as everyone has been saying for years now, could really give our industrial civilization fits. Here’s some work by the Hergenrother group at Illinois, though, that sheds light on one of the biggest problems in antibiotic drug discovery: what kinds of structures should we be looking at?
That’s a real puzzle, because antibiotic compounds in general tend to have pretty wooly structures, especially the ones derived from natural products. They tend to be outliers in most any rule-of-thumb property screen, and often break several of them simultaneously. Yet they work, and that’s impressive, since to “work” in this context means to penetrate a lipopolysaccharide outer membrane, survive on the other side of it without being pumped right back out of the bacterial cell entirely, and penetrate that a second inner membrane in quantities sufficient to serve as a drug.
This paper looks through a set of compounds, carefully measuring the degree to which each accumulate in E. coli, and tried to draw some general structural lessons. This sort of thing has been done before, but this effort concentrates on simpler molecules rather than known antibiotics and their derivatives. What they come down to is this:
On the basis of these analyses, the following guiding principles for compound accumulation in E. coli were developed: compounds are most likely to accumulate if they contain a non-sterically encumbered amine, some non-polar functionality, they are rigid and have low globularity.
Simple enough, and you might read that and say “Thanks a lot”, because of the apparent vagueness. But the paper goes on to another key insight: if you already have a compound active against gram-positive bacteria, it likely already has the rigidity and shape needed. What if you just attach an open primary amine to it? They demonstrate this idea on the gyrase inhibitor deoxynybomycin, which is only a gram-positive compound. Hanging a plain methylamine group off it, though, converts it into a compound that accumulates in E. coli and shows antibacterial activity in a strain that approximates wild-type (that is, no efflux pump mutations or other half measures). The authors note that such unhindered primary amines are actually quite rare in screening collections (they’re right), and that biasing new collections towards this functionality and towards the structural features they’ve suggested may well improve screening efforts. The first order of business, though, will be more of those gram-positive conversion jobs, and I think that we will see the fruits of those efforts pretty soon. I hope that this trick works across a number of pathogens (and I hope we find that out soon as well!)
The second paper I referred to is the latest in the series from the Boger group at Scripps, which I last blogged about here. In this latest work (which should, by the time you read this, be here at PNAS) they’ve been modifying vancomycin, the famous “antibiotic of last resort”, which is quite the synthetic challenge (see that link if you haven’t looked at its structure; it’s a beast). They’ve managed to introduce variations that improve the potency of the compound substantially (up to 1000x over native vancomycin!), and in this new work they’ve got a compound that combines greatly improved potency with three separate mechanisms of action. This should make it very hard indeed for bacteria to evolve resistance. That’s already a slow process with vancomycin, due to its odd mechanism of action. It binds to D-Ala-D-Ala residues and interferes with cell wall formation, and the fact that it doesn’t go after an enzyme or active site lets it dodge the evolutionary pressures that can lead to resistance against such direct attacks. Throwing in more mechanisms of action can only slow that down even more, which is a very good thing.
I found it quite interesting that the latest modification is a side chain with an unhindered quaternary (tetramethyl)ammonium group, which seems to have profound (and as yet unexplained) effects on membrane permeability. This makes me wonder several things: are the primary amines seen in the Hergenrother work protonated under the conditions where they’re passing through the bacterial membranes? What would happen if they were also tetramethyl quaternary salts? Can compounds that are active against gram-positive enzyme targets but not against live organisms be rescued with a side-chain quat salt to improve their membrane permeability, in the same way that the Hergenrother paper rescues a gram-negative drug candidate?
You might also wonder if this latest modified vancomycin might start showing some gram-negative activity. Unfortunately, vancomycin in general is not the best candidate mechanism for those species, since its target, cell wall synthesis, is rather different in gram-negative organisms, as mentioned above. Gram-positive organisms have the single thicker cell wall, and disrupting it is a strong approach. The double-layer gram-negative organisms also have different compositions in those layers, making vancomycin’s primary mechanism less effective. But still. . .
This latest vancomycin analog will still (as I mentioned in this press writeup) have to go through the usual whole-animal models of efficacy and toxicity before heading into humans, but preliminary cell-based tox assays are promising. I very much look forward to seeing it progress. The activity it shows, though, now puts the ball right back in the organic synthesis court. The Boger group’s work on these structures is very high level stuff, a major synthetic challenge, and they deserve congratulations for just getting these compounds made in the first place. Finding ways to make them on scale will be another challenge of a similar order.