Evolutionary druggability: leveraging low-dimensional fitness landscapes towards new metrics for antimicrobial applications

Reviewer #1 (Public Review):

The manuscript by Geurrero and colleagues introduces two new metrics that extend the concept of "druggability"- loosely speaking, the potential suitability of a particular drug, target, or drug-target interaction for pharmacological intervention-to collections of drugs and genetic variants. The study draws on previously measured growth rates across a combinatoriality complete mutational landscape involving 4 variants of the TEM-50 (beta lactamase) enzyme, which confers resistance to commonly used beta-lactam antibiotics. To quantify how growth rate - in this case, a proxy for evolutionary fitness - is distributed across allelic variants and drugs, they introduce two concepts: "variant vulnerability" and "drug applicability".

Variant vulnerability is the mean vulnerability (1-normalized growth rate) of a particular variant to a library of drugs, while drug applicability measures the mean across the collection of genetic variants for a given drug. The authors rank the drugs and variants according to these metrics. They show that the variant vulnerability of a particular mutant is uncorrelated with the vulnerability of its one-step neighbors, and analyze how higher-order combinations of single variants (SNPs) contribute to changes in growth rate in different drug environments.

The work addresses an interesting topic and underscores the need for evolution-based metrics to identify candidate pharmacological interventions for treating infections. The authors are clear about the limitations of their approach - they are not looking for immediate clinical applicability - and provide simple new measures of druggability that incorporate an evolutionary perspective, an important complement to the orthodoxy of aggressive, kill-now design principles. I think the ideas here will interest a wide range of readers, but I think the work could be improved with additional analysis - perhaps from evolutionary simulations on the measured landscapes - that tie the metrics to evolutionary outcomes.

Reviewer #2 (Public Review):

The authors introduce the notions of "variant vulnerability" and "drug applicability" as metrics quantifying the sensitivity of a given target variant across a panel of drugs and the effectiveness of a drug across variants, respectively. Given a data set comprising a measure of drug effect (such as growth rate suppression) for pairs of variants and drugs, the vulnerability of a variant is obtained by averaging this measure across drugs, whereas the applicability of a drug is obtained by averaging the measure across variants.

The authors apply the methodology to a data set that was published by Mira et al. in 2015. The data consist of growth rate measurements for a combinatorially complete set of 16 genetic variants of the antibiotic resistance enzyme beta-lactamase across 10 drugs and drug combinations at 3 different drug concentrations, comprising a total of 30 different environmental conditions. For reasons that did not become clear to me, the present authors select only 7 out of 30 environments for their analysis. In particular, for each chosen drug or drug combination, they choose the data set corresponding to the highest drug concentration. As a consequence, they cannot assess to what extent their metrics depend on drug concentration. This is a major concern since Mira et al. concluded in their study that the differences between growth rate landscapes measured at different concentrations were comparable to the differences between drugs. If the new metrics display a significant dependence on drug concentration, this would considerably limit their usefulness.

As a consequence of the small number of variant-drug combinations that are used, the conclusions that the authors draw from their analysis are mostly tentative with weak statistical support. For example, the authors argue that drug combinations tend to have higher drug applicability than single drugs, because a drug combination ranks highest in their panel of 7. However, the effect profile of the single drug cefprozil is almost indistinguishable from that of the top-ranking combination, and the second drug combination in the data set ranks only 5th out of 7.

To assess the environment-dependent epistasis among the genetic mutations comprising the variants under study, the authors decompose the data of Mira et al. into epistatic interactions of different orders. This part of the analysis is incomplete in two ways. First, in their study, Mira et al. pointed out that a fairly large fraction of the fitness differences between variants that they measured were not statistically significant, which means that the resulting fitness landscapes have large statistical uncertainties. These uncertainties should be reflected in the results of the interaction analysis in Figure 4 of the present manuscript. Second, the interpretation of the coefficients obtained from the epistatic decomposition depends strongly on the formalism that is being used (in the jargon of the field, either a Fourier or a Taylor analysis can be applied to fitness landscape data). The authors need to specify which formalism they have employed and phrase their interpretations accordingly.

Reviewer #3 (Public Review):

The authors introduce two new concepts for antimicrobial resistance borrowed from pharmacology, "variant vulnerability" (how susceptible a particular resistance gene variant is across a class of drugs) and "drug applicability" (how useful a particular drug is against multiple allelic variants). They group both terms under an umbrella term "drugability". They demonstrate these features for an important class of antibiotics, the beta-lactams, and allelic variants of TEM-1 beta-lactamase.

The strength of the result is in its conceptual advance and that the concepts seem to work for beta-lactam resistance. However, I do not necessarily see the advance of lumping both terms under "drugability", as this adds an extra layer of complication in my opinion.

I also think that the utility of the terms could be more comprehensively demonstrated by using examples across different antibiotic classes and/or resistance genes. For instance, another good model with published data might have been trimethoprim resistance, which arises through point mutations in the folA gene (although, clinical resistance tends to be instead conferred by a suite of horizontally acquired dihydrofolate reductase genes, which are not so closely related as the TEM variants explored here).

The impact of the work on the field depends on a more comprehensive demonstration of the applicability of these new concepts to other drugs.