The study presents significant improvements in proteolysis targeting chimeras (PROTACs), a chemical method for protein knockdown. PROTACs are heterobifunctional molecules that recruit specific proteins to an E3 ubiquitin ligase, leading to protein degradation. The new generation of nonpeptidic PROTACs achieves potent and highly selective downregulation of target proteins in cell culture. These compounds act catalytically, inducing ubiquitination of large quantities of proteins, providing efficacy not limited by equilibrium occupancy. Two PROTACs were shown to reduce protein levels by over 90% at nanomolar concentrations. Mouse studies indicate broad tissue distribution and knockdown of the targeted protein in tumor xenografts. These data demonstrate a protein knockdown system combining the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR. Small molecule-mediated inhibition of protein function is the fundamental paradigm underpinning the efficacy of the vast majority of clinically used agents. However, pharmacologically relevant inhibition is often only achieved upon >90% target engagement, necessitating high dosing levels that can lead to off-target effects. Approaches that directly control cellular protein levels have the potential to offer cellular efficacy not easily achievable with small-molecule inhibitors. The best-investigated methods of reducing cellular protein levels are genetic knockdown approaches based on antisense oligonucleotides, RNA interference (RNAi), CRISPR/Cas9 or related strategies. Despite the clear therapeutic potential, difficulties in achieving sufficient drug concentrations at the targeted site of action, safety challenges due to off-target effects, and poor metabolic stability remain as major obstacles for routine, systemic delivery of nucleic acid-based protein knockdown agents for therapeutic applications. Some success has been achieved in developing knockdown strategies not based on nucleic acid technologies, so-called 'chemical knockdown strategies'. Chemical knockdown typically use a bifunctional small molecule that binds to a protein target while simultaneously engaging the cellular protein quality control machinery, thus 'hijacking' the machinery to degrade the protein target. The study presents a significant improvement to PROTAC technology. This new generation of nonpeptidic PROTAC molecules achieves potent and highly selective downregulation of target proteins in cell culture. Through a series of in vitro and cellular studies, the mechanism is shown to depend on a ternary complex that efficiently induces ubiquitination of substrate and allows subsequent proteasomal degradation. The study also shows a departure from traditional occupancy-limited efficacy, where each PROTAC molecule is able to induce the degradation of multiple substrate protein molecules. In a preliminary mouse study, PROTACs are shown to be capable of targeted protein knockdown in various tissues including solid tumors. The study also demonstrates that PROTACs mediate catalytic ubiquitination, with the ability to induce the ubiquitination and degradation of multiple molecules of the target protein. The PROTACs are highly specificThe study presents significant improvements in proteolysis targeting chimeras (PROTACs), a chemical method for protein knockdown. PROTACs are heterobifunctional molecules that recruit specific proteins to an E3 ubiquitin ligase, leading to protein degradation. The new generation of nonpeptidic PROTACs achieves potent and highly selective downregulation of target proteins in cell culture. These compounds act catalytically, inducing ubiquitination of large quantities of proteins, providing efficacy not limited by equilibrium occupancy. Two PROTACs were shown to reduce protein levels by over 90% at nanomolar concentrations. Mouse studies indicate broad tissue distribution and knockdown of the targeted protein in tumor xenografts. These data demonstrate a protein knockdown system combining the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR. Small molecule-mediated inhibition of protein function is the fundamental paradigm underpinning the efficacy of the vast majority of clinically used agents. However, pharmacologically relevant inhibition is often only achieved upon >90% target engagement, necessitating high dosing levels that can lead to off-target effects. Approaches that directly control cellular protein levels have the potential to offer cellular efficacy not easily achievable with small-molecule inhibitors. The best-investigated methods of reducing cellular protein levels are genetic knockdown approaches based on antisense oligonucleotides, RNA interference (RNAi), CRISPR/Cas9 or related strategies. Despite the clear therapeutic potential, difficulties in achieving sufficient drug concentrations at the targeted site of action, safety challenges due to off-target effects, and poor metabolic stability remain as major obstacles for routine, systemic delivery of nucleic acid-based protein knockdown agents for therapeutic applications. Some success has been achieved in developing knockdown strategies not based on nucleic acid technologies, so-called 'chemical knockdown strategies'. Chemical knockdown typically use a bifunctional small molecule that binds to a protein target while simultaneously engaging the cellular protein quality control machinery, thus 'hijacking' the machinery to degrade the protein target. The study presents a significant improvement to PROTAC technology. This new generation of nonpeptidic PROTAC molecules achieves potent and highly selective downregulation of target proteins in cell culture. Through a series of in vitro and cellular studies, the mechanism is shown to depend on a ternary complex that efficiently induces ubiquitination of substrate and allows subsequent proteasomal degradation. The study also shows a departure from traditional occupancy-limited efficacy, where each PROTAC molecule is able to induce the degradation of multiple substrate protein molecules. In a preliminary mouse study, PROTACs are shown to be capable of targeted protein knockdown in various tissues including solid tumors. The study also demonstrates that PROTACs mediate catalytic ubiquitination, with the ability to induce the ubiquitination and degradation of multiple molecules of the target protein. The PROTACs are highly specific