Citation: Lobinska G, Tretyachenko V, Dahan O, Nowak MA, Pilpel Y (2024) The evolutionary safety of mutagenic drugs should be assessed before drug approval. PLoS Biol 22(3): e3002570. https://doi.org/10.1371/journal.pbio.3002570

Published: March 15, 2024

Copyright: © 2024 Lobinska et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the Kimmel Foundation (YP) and the Minerva Center on Live Emulation of Evolution in the Lab (YP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Mutations in pathogens are concerning. They can cause increased virulence or transmissibility, or resistance to therapy or vaccination. A drug that augments the mutation rate of a pathogen is a questionable proposition as its use could benefit the virus by enabling it to evolve faster, yet a growing number of pharmaceutical treatments increase the mutation rate of the viral or microbial pathogens they target [1,2]. If the increase in mutation rate is sufficient, the pathogen can acquire a lethal dosage of mutations, triggering an error catastrophe that prevents adaptation [3] or reduces the basic reproductive ratio to below one [4]. The concept of an error threshold is related to Muller’s ratchet or mutational meltdown [5,6]. Hence, the mechanism of action of mutagenic drugs is based on a population process. An example is the antiviral molnupiravir, an approved treatment for SARS-CoV-2 infection [7]. For other drugs, increased mutation rates are a byproduct of their intended effect, which relies on interfering with the replication process [8,9].

Below, we propose a scheme of 4 steps for evaluating the evolutionary safety of drugs. For each step, we provide suggestions for experiments, analyses, and tests to be performed (Box 1). Mutagenic drugs should be considered, but we think they need to be evaluated carefully for evolutionary safety, along with more common safety criteria, before they can be approved for use. We would define a mutagenic drug as being evolutionarily safe if it reduced the total amount of viable virus mutants produced during infection. Conversely, a treatment would be evolutionarily unsafe if it increased that quantity. A mutagenic treatment that is evolutionarily safe would be expected to reduce the rate of viral evolution in the population, although this concept has to be understood as a probabilistic statement. Of course, there could always be rare, unforeseen mutational events that lead to unexpected and dangerous variants. Any human intervention in evolutionary processes bears such a residual risk.

The first step will be to determine the genomic structure of the pathogen, derive a quantitative understanding of infection dynamics, and measure the natural mutation rate of the pathogen, including the recombination rate. All these quantities can affect the evaluation of evolutionary safety [10]. Of particular interest is the number of positions in the pathogen’s genome that are lethal to the pathogen when mutated, as the higher that number, the higher the likelihood for evolutionary safety [11].

The second step will consist of the study of the mutagenic potential of the drug. This step should apply both to drugs that are mutagenic by design and to drugs that exert mutagenesis as a by-product of their action. If there is mutagenic potential, then the magnitude of the effect must be evaluated in cell-free systems, treated bacteria, and viruses (as they infect cells in cultures). The mutation rate of the virus under treatment is dependent on drug dosage. Effective dosage in the body can be altered by poor treatment adherence, incomplete absorption, and fluctuations in drug concentration between doses. Therefore, the mutagenic potential should be established for a dynamic range of drug concentrations. Small increases in mutation rate are likely to reduce the evolutionary safety of a drug [10]. The treatment could be tested first in cell cultures: in a laboratory environment, a larger fraction of the pathogen’s genetic diversity could be observed than in vivo. The observed viral diversity in vivo depends on the sampling methods and on the pathogens’ distribution within the body. Different concentrations and regimes of the treatment could be tested, controlling for pharmacokinetic aspects in different parts of the body.

In the third step, the assessment of evolutionary safety would proceed into preclinical and clinical trials. Evolution experiments that were performed in the second step, involving taking the pathogen through serial passaging in cell culture, could be repeated in appropriate animal models that are infected with the pathogen and treated with the drug. Pharmacokinetics of the mutagenic treatment within the body could result in different mutation rates than those measured in vitro. Therefore, the viral mutation rate in treated animals or human patients has to be measured, taking into account factors such as poor treatment adherence or excluded sites in the body where the pathogen can replicate but the drug cannot fully reach. Evolutionary safety can be demonstrated if treatment results in a lower cumulative mutant pathogen load. Evolutionary safety could depend on the patient’s immune response and other parameters [10]. It is conceivable that a drug could be deemed evolutionarily safe in some patients groups but unsafe in others.

The fourth step would occur after the drug had been approved and population-wide prescription of the drug ensued. Characteristic mutational patterns could then be linked to mutagenic treatments. For example, the mutational signature of molnupiravir was detected in sequences of SARS-CoV-2 from countries where it was widely used [8], but the study did not reveal if there was an increase in viral diversity as a consequence of treatment with the drug. The appearance of mutational signatures caused by mutagenic treatment does not in itself exclude the possibility that a drug is evolutionarily safe. However, it should be the case that evolutionarily safe treatments reduce the number of new infections and the total amount of virus mutants present in a population. A goal of evolutionary safety assessment will also be to reveal drug administration regimens and pharmaceutical development directions that maximize evolutionary safety. Lack of treatment adherence may affect evolutionary safety; hence, mutagenic drugs might be restricted to hospital use.

Lethal mutagenesis is a relatively new and potentially important strategy for the treatment of infections. The recent SARS-CoV-2 pandemic has underscored the importance of the production of new antiviral drugs. However, evolutionary safety is not confined to the treatment of SARS-CoV-2, it can be applied to other viruses and multidrug-resistant bacterial strains. For example, phage therapy [9], although promising, raises an evolutionary safety concern as it may lead to unpredictable evolutionary outcomes such as increased target resistance towards both phage and antibiotics [12]. A consistent procedure to assess the evolutionary safety of such mutagenesis treatments would allow us to use them without an otherwise significant risk to humanity. Evolutionary safety also has moral and ethical implications. A potential ethical dilemma could occur if, for example, a higher dose of a drug presents more toxicity or adverse effects but is necessary for increased evolutionary safety.