Emerging Strategies in Antimicrobial Drug Discovery: A Review of Target-Based and Phenotypic Screening Approaches

Abstract

Antimicrobial resistance has intensified the need for new chemical matter and new mechanisms of action, yet antibacterial discovery remains unusually vulnerable to attrition between a convincing molecular hypothesis and clinically relevant whole-cell activity. This critical narrative review evaluates target-based and phenotypic screening as complementary discovery architectures rather than competing doctrines, with emphasis on small-molecule antibacterial discovery and transferable lessons for antimicrobial research. Literature published from 1 January 2000 to 28 June 2026 was identified through PubMed, PubMed Central, OpenAlex, DOAJ, Semantic Scholar and Google Scholar, supplemented by citation chaining and verification against primary bibliographic records. The evidence indicates that target-based screening is strongest when target vulnerability, chemical tractability and intracellular exposure are demonstrated early, but genomic essentiality alone is an unreliable proxy for pharmacological susceptibility. Phenotypic screening naturally selects for cellular access and functional consequence, yet its apparent physiological realism can be undermined by artificial culture conditions, rediscovery of known mechanisms, non-specific stress phenotypes and protracted target deconvolution. The most productive emerging approaches therefore collapse the traditional divide: genetically sensitised strains and CRISPR interference connect targets to cellular phenotypes; pathway-directed reporters retain whole-cell permeability filters while enriching for desired biology; bacterial cytological profiling, metabolomics and thermal proteome profiling accelerate mechanism resolution; and direct accumulation measurements make permeability and efflux explicit design variables. Machine-learning approaches expand searchable chemical space, but their value depends on experimentally grounded phenotypic labels and subsequent mechanistic validation. Across these strategies, the central determinant of progress is not whether a campaign begins with a target or a phenotype, but whether it rapidly establishes a coherent chain of evidence linking target vulnerability, compound exposure, target engagement, antibacterial effect, resistance liability and disease-relevant performance. Future discovery programmes should be designed around this convergence from the outset.