Context-specific inhibition of mitochondrial ribosomes by phenicol and oxazolidinone antibiotics.

The antibiotics chloramphenicol (CHL) and oxazolidinones including linezolid (LZD) are known to inhibit mitochondrial translation. This can result in serious, potentially deadly, side effects when used therapeutically. Although the mechanism by which CHL and LZD inhibit bacterial ribosomes has been elucidated in detail, their mechanism of action against mitochondrial ribosomes has yet to be explored. CHL and oxazolidinones bind to the ribosomal peptidyl transfer center (PTC) of the bacterial ribosome and prevent incorporation of incoming amino acids under specific sequence contexts, causing ribosomes to stall only at certain sequences. Through mitoribosome profiling, we show that inhibition of mitochondrial ribosomes is similarly context-specific - CHL and LZD lead to mitoribosome stalling primarily when there is an alanine, serine, or threonine in the penultimate position of the nascent peptide chain. We further validate context-specific stalling through in vitro translation assays. A high resolution cryo-EM structure of LZD bound to the PTC of the human mitoribosome shows extensive similarity to the mode of bacterial inhibition and also suggests potential avenues for altering selectivity. Our findings could help inform the rational development of future, less mitotoxic, antibiotics, which are critically needed in the current era of increasing antimicrobial resistance.

Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance.

Alteration of antibiotic binding sites through modification of ribosomal RNA (rRNA) is a common form of resistance to ribosome-targeting antibiotics. The rRNA-modifying enzyme Cfr methylates an adenosine nucleotide within the peptidyl transferase center, resulting in the C-8 methylation of A2503 (m8A2503). Acquisition of cfr results in resistance to eight classes of ribosome-targeting antibiotics. Despite the prevalence of this resistance mechanism, it is poorly understood whether and how bacteria modulate Cfr methylation to adapt to antibiotic pressure. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. In this study, we performed directed evolution of Cfr under antibiotic selection to generate Cfr variants that confer increased resistance by enhancing methylation of A2503 in cells. Increased rRNA methylation is achieved by improved expression and stability of Cfr through transcriptional and post-transcriptional mechanisms, which may be exploited by pathogens under antibiotic stress as suggested by natural isolates. Using a variant that achieves near-stoichiometric methylation of rRNA, we determined a 2.2 Å cryo-electron microscopy structure of the Cfr-modified ribosome. Our structure reveals the molecular basis for broad resistance to antibiotics and will inform the design of new antibiotics that overcome resistance mediated by Cfr.

Antibiotics treat or prevent infections by killing bacteria or slowing down their growth. A large proportion of these drugs do this by disrupting an essential piece of cellular machinery called the ribosome which the bacteria need to make proteins. However, over the course of the treatment, some bacteria may gain genetic alterations that allow them to resist the effects of the antibiotic. Antibiotic resistance is a major threat to global health, and understanding how it emerges and spreads is an important area of research. Recent studies have discovered populations of resistant bacteria carrying a gene for a protein named chloramphenicol-florfenicol resistance, or Cfr for short. Cfr inserts a small modification in to the ribosome that prevents antibiotics from inhibiting the production of proteins, making them ineffective against the infection. To date, Cfr has been found to cause resistance to eight different classes of antibiotics. Identifying which mutations enhance its activity and protect bacteria is vital for designing strategies that fight antibiotic resistance. To investigate how the gene for Cfr could mutate and make bacteria more resistant, Tsai et al. performed a laboratory technique called directed evolution, a cyclic process which mimics natural selection. Genetic changes were randomly introduced in the gene for the Cfr protein and bacteria carrying these mutations were treated with tiamulin, an antibiotic rendered ineffective by the modification Cfr introduces into the ribosome. Bacteria that survived were then selected and had more mutations inserted. By repeating this process several times, Tsai et al. identified 'super' variants of the Cfr protein that lead to greater resistance. The experiments showed that these variants boosted resistance by increasing the proportion of ribosomes that contained the protective modification. This process was facilitated by mutations that enabled higher levels of Cfr protein to accumulate in the cell. In addition, the current study allowed, for the first time, direct visualization of how the Cfr modification disrupts the effect antibiotics have on the ribosome. These findings will make it easier for clinics to look out for bacteria that carry these 'super' resistant mutations. They could also help researchers design a new generation of antibiotics that can overcome resistance caused by the Cfr protein.

Covalent Ligand Screening Uncovers a RNF4 E3 Ligase Recruiter for Targeted Protein Degradation Applications.

Targeted protein degradation has arisen as a powerful strategy for drug discovery allowing the targeting of undruggable proteins for proteasomal degradation. This approach most often employs heterobifunctional degraders consisting of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and mark proteins of interest for proteasomal degradation. One challenge with this approach, however, is that only a few E3 ligase recruiters currently exist for targeted protein degradation applications, despite the hundreds of known E3 ligases in the human genome. Here, we utilized activity-based protein profiling (ABPP)-based covalent ligand screening approaches to identify cysteine-reactive small-molecules that react with the E3 ubiquitin ligase RNF4 and provide chemical starting points for the design of RNF4-based degraders. The hit covalent ligand from this screen reacted with either of two zinc-coordinating cysteines in the RING domain, C132 and C135, with no effect on RNF4 activity. We further optimized the potency of this hit and incorporated this potential RNF4 recruiter into a bifunctional degrader linked to JQ1, an inhibitor of the BET family of bromodomain proteins. We demonstrate that the resulting compound CCW 28-3 is capable of degrading BRD4 in a proteasome- and RNF4-dependent manner. In this study, we have shown the feasibility of using chemoproteomics-enabled covalent ligand screening platforms to expand the scope of E3 ligase recruiters that can be exploited for targeted protein degradation applications.

NHS-Esters As Versatile Reactivity-Based Probes for Mapping Proteome-Wide Ligandable Hotspots.

Most of the proteome is considered undruggable, oftentimes hindering translational efforts for drug discovery. Identifying previously unknown druggable hotspots in proteins would enable strategies for pharmacologically interrogating these sites with small molecules. Activity-based protein profiling (ABPP) has arisen as a powerful chemoproteomic strategy that uses reactivity-based chemical probes to map reactive, functional, and ligandable hotspots in complex proteomes, which has enabled inhibitor discovery against various therapeutic protein targets. Here, we report an alkyne-functionalized N-hydroxysuccinimide-ester (NHS-ester) as a versatile reactivity-based probe for mapping the reactivity of a wide range of nucleophilic ligandable hotspots, including lysines, serines, threonines, and tyrosines, encompassing active sites, allosteric sites, post-translational modification sites, protein interaction sites, and previously uncharacterized potential binding sites. Surprisingly, we also show that fragment-based NHS-ester ligands can be made to confer selectivity for specific lysine hotspots on specific targets including Dpyd, Aldh2, and Gstt1. We thus put forth NHS-esters as promising reactivity-based probes and chemical scaffolds for covalent ligand discovery.

Covalent Ligand Discovery against Druggable Hotspots Targeted by Anti-cancer Natural Products.

Many natural products that show therapeutic activities are often difficult to synthesize or isolate and have unknown targets, hindering their development as drugs. Identifying druggable hotspots targeted by covalently acting anti-cancer natural products can enable pharmacological interrogation of these sites with more synthetically tractable compounds. Here, we used chemoproteomic platforms to discover that the anti-cancer natural product withaferin A targets C377 on the regulatory subunit PPP2R1A of the tumor-suppressor protein phosphatase 2A (PP2A) complex leading to activation of PP2A activity, inactivation of AKT, and impaired breast cancer cell proliferation. We developed a more synthetically tractable cysteine-reactive covalent ligand, JNS 1-40, that selectively targets C377 of PPP2R1A to impair breast cancer signaling, proliferation, and in vivo tumor growth. Our study highlights the utility of using chemoproteomics to map druggable hotspots targeted by complex natural products and subsequently interrogating these sites with more synthetically tractable covalent ligands for cancer therapy.