Ribosomes are essential macromolecular machines responsible for protein synthesis in both humans and bacteria, governing the viability of cells across the superkingdoms. Comprising intricate assemblies of ribosomal RNA and ribosomal proteins, ribosomes form through a coordinated, stepwise maturation process involving numerous assembly factors. Eukaryotic ribosome assembly is inherently more complex due to the increased size of the ribosome and the requirement for transport across the nucleolar membrane, a process in which ATPases play a critical role. In both prokaryotes and eukaryotes, ribosomes comprise a small and a large subunit; the large subunit is designated 50S in bacteria and 60S in eukaryotes. The bacterial ribosome is a well-validated target for antibiotic development. Existing antibiotics primarily act by inhibiting the function of the mature ribosome. However, resistance mechanisms have now evolved against all classes currently in clinical use. In the face of rapid emergence of resistance mechanisms to antibiotics, and a dearth of novel antibiotic classes being produced, there is a great unmet clinical need for novel antibiotic classes and structures. Cancer remains a leading cause of mortality worldwide, accounting for a substantial proportion of global deaths and representing a major public health burden. Cancer cells rely on ribosomal maturation for their proliferation and survival, and a long history of evidence implicates eukaryotic ribosomal dysregulation in tumor progression along with cancer cell proliferation and treatment resistance. Indeed, a number of current cancer therapeutics have retrospectively been found to impair ribosome biogenesis. The human ATPase MIDASIN (MDN1) crucial to 60S ribosomal assembly, is overexpressed in breast cancer and implicated in hormonal cancer therapy resistance. This work leverages recently published atomistic models of key ribosomal assembly intermediates and factors. One characterizes a set of assembly intermediate structures along the bacterial 50S subunit assembly pathway, and the other depicts a crucial interaction between two human 60S assembly factors. These structural insights provide the foundation for computer-aided drug design campaigns that aim to develop a novel antibiotic and a new anticancer therapeutic, respectively, by selectively disrupting species-specific ribosomal assembly pathways. Cryo-Electron Microscopy models of the 50S ribosomal subunit in various stages of its assembly provide unprecedented insight into the molecular mechanisms governing bacterial ribosomal assembly, highlighting opportunities for therapeutic intervention. The bacterial ribosomal protein bL17 has been identified as a promising pharmacological target via both genetic knockout experiments and computational investigations. Previous work has already identified a series of potential bL17 binding compounds. Anticancer strategies targeting human ribosomal assembly have remained underexplored, in part due to the complexity of eukaryotic ribosome biogenesis, which is not yet fully understood. The potential of MDN1 as a pharmacological target to disrupt ribosomal assembly and thereby limit cell viability has been demonstrated in yeast. While MDN1 has been shown to play an essential role in ribosome biogenesis in human cells, its potential as a pharmacological target for anticancer therapy remains unvalidated, and no ligands have been specifically developed against human MDN1. The determination of an X-ray structure of human MDN1 in complex with its ribosomal binding partner, NLE1, represents a significant advance, providing mechanistic insight and a structural framework for rational drug design. This work begins with the biophysical characterization of the binding of an initial hit compound, A14, to bL17, and determination of the in vivo inhibitory effect of A14 on E. coli cell growth. These experimental data informed the computational characterization of the A14 binding mode to bL17 via static and dynamic methods. Ligand-based analysis of A14 enabled its computational derivatization into a subsequent compound series, termed the C series. Of these, compound C07 showed an inhibitory effect on E. coli 50S subunit assembly in vitro. In silico molecular modeling of the putative binding mode of C07 within the predicted binding site of bL17 enabled the generation of a preliminary structure-activity-relationship model characterizing the potential contribution of compound moieties and bL17 protein residues to bL17 affinity and 50S assembly inhibition. This structure-activity-relationship model further guided the computational generation of the D series of compounds. Thus, iterative cycles of ligand-based optimization guided by the results of experimental validation ultimately yielded a series of antibiotic scaffold candidates. In parallel, the X-ray crystallographic model capturing the interaction between the two human ribosomal assembly factors MDN1 and NLE1 provided a template for design of inhibitors targeting this protein-protein interaction. The unliganded surface of the MDN1 MIDAS domain was analyzed via static and dynamic structure-based methods to generate a structure-based 3D pharmacophore for use in virtual screening. The resulting hit compounds were characterized extensively via in vitro and in vivo methods. The results show that compound R06 is capable of inhibiting the MDN1-NLE1 interaction in vitro, leading to 60S assembly defects and cancer cell lethality in vivo. The putative binding mode of R06 was characterized via molecular modeling, to generate a hypothesis on the molecular basis of its demonstrated activity.
