Development of a BCL-xL and BCL-2 dual degrader with improved anti-leukemic activity,

PROteolysis-TArgeting Chimeras (PROTACs) have emerged as an innovative drug development platform. However, most PROTACs have been generated empirically because many determinants of PROTAC specificity and activity remain elusive. Through computational modelling of the entire NEDD8-VHL Cullin RING E3 ubiquitin ligase (CRLVHL)/PROTAC/BCL-xL/UbcH5B(E2)-Ub/RBX1 complex, we find that this complex can only ubiquitinate the lysines in a defined band region on BCL-xL. Using this approach to guide our development of a series of ABT263-derived and VHL-recruiting PROTACs, we generate a potent BCL-xL and BCL-2 (BCL-xL/2) dual degrader with significantly improved antitumor activity against BCL-xL/2-dependent leukemia cells. Our results provide experimental evidence that the accessibility of lysines on a target protein plays an important role in determining the selectivity and potency of a PROTAC in inducing protein degradation, which may serve as a conceptual framework to guide the future development of PROTACs.

-Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Statistical methods were not used to determine sample sizes. However, for quantification purposes, a sample size of n = 2 or 3 unless otherwise stated (reproduced two or more independent times) was used to assess reproducibility and robustness of each experiment performed. Sample sizes were based on prior study in the field (For example: Alabi S, Jaime- Figueroa  In general, no data were excluded from the analyses.
To ensure reproducibility of experimental findings, all biochemical or cellular assays were repeated independently at least two times. One representative result for each experiment is presented in the main Figures or the Extended Data Figures. We confirm that all attempts of replication were successful.
Cultured cells were passaged evenly (random distribution) into dishes or flasks for treatment with PROTACs and/or indicated compounds. Due to dose-response nature of the experiments performed, randomization of the samples post-treatment would make data interpretation impossible.
Investigators were not blinded to the nature of their samples during data collection and analysis based on prior study in the field (for example: Alabi S, Jaime- Figueroa  All the used antibodies are commercially available. The antibodies used in a specific species or application have been validated by manufacturers to be used in that species/application and this information is provided in their website and/or antibody datasheets. CST Antibody Validation Principles To ensure our antibodies will work in your experiment, we adhere to the Hallmarks of Antibody Validation™, six complementary strategies that can be used to determine the functionality, specificity, and sensitivity of an antibody in any given assay. CST adapted the work by Uhlen, et. al., ("A Proposal for Validation of Antibodies." Nature Methods (2016)) to build the Hallmarks of Antibody Validation, based on our decades of experience as an antibody manufacturer and our dedication to reproducible science.

CST Hallmarks of Antibody Validation
We guarantee that our antibodies are fit for purpose by carefully tailoring the combination of validation strategies applied to each product. This means customizing our validation process according to the biological role of the target, while considering the sensitivity requirements of the downstream assay, the availability of appropriate testing models, and the relevance of each method to target investigation. 1. Binary Model: Antibody signal is measured in model systems with known presence/absence of target signal. Includes wild-type vs. genetic knockout, targeted induction or silencing. 2. Ranged Expression: Antibody signal strength is measured in cell lines or tissues representing a known continuum of target expression levels. Includes siRNA and heterozygous knockout assays. 3. Orthogonal Data: Antibody signal is correlated to target expression in model systems measured using antibody independent assays. Includes mass spectrometry and in situ hybridization. 4. Multiple Antibodies: Antibody signal is compared to the signal observed using antibodies targeting nonoverlapping epitopes of the target. Includes IP, ChIP, and ChIP-seq. 5. Heterologous Expression: Antibody signal is evaluated in cell lines following heterologous expression of native (or mutated) target protein.