Targeted protein degradation (TPD) is an emerging therapeutic modality in which small molecules are used to recruit targets to the natural protein degradation machinery of the cell. Molecular glue degraders (MGD) are monovalent small molecules that accomplish this by redirecting E3 ubiquitin ligases to target proteins, offering the potential to degrade previously unliganded and ″undruggable″ proteins in cancer, neurodegenerative, and other diseases. While attractive due to their drug-like properties, MGDs are exceptionally hard to discover and have largely been identified serendipitously. The Von Hippel-Lindau (VHL) E3 ligase is the second most widely used effector for TPD, though current VHL-based degraders are primarily large heterobifunctional PROTACs (proteolysis-targeting chimeras) designed using target-based ligands. Here, we have instead pursued target-agnostic discovery of VHL MGDs leveraging proprietary ultra-miniaturized microfluidics devices (Picowells) to facilitate unbiased RNA-seq screening of a biased E3-focused library. This resulted in dGEM3, a novel VHL molecular glue that targets the survival of motor neuron (SMN) complex member GEMIN3 for degradation. Through a combination of cellular, biochemical, and biophysical assays, we have characterized the GEMIN3 degron within its helicase ATP-binding domain, and how the kinetics of ternary complex formation impact degradation. These findings provide insights on the re-programmability of VHL for novel targets using drug-like molecular glues.

Multiple myeloma (MM) is a neoplasm of plasma cells that secrete patient specific monoclonal immunoglobulins. A recognized problem in MM treatment is the early recognition of minimal residual disease (MRD), the major cause of relapse. Current MRD detection methods (multiparameter flow cytometry and next generation sequencing) are based on the analysis of bone marrow plasma cells. Both methods cannot detect extramedullary disease and are unsuitable for serial measurements. We describe the methodology to generate high affinity DNA aptamers that are specific to a patient's monoclonal Fab region. Such aptamers are 2000-fold more sensitive than immunofixation electrophoresis and enabled detection and quantification of MRD in serum when conventional MRD methods assessed complete remission. The aptamer isolation process that requires small volumes of serum is automatable, and Fab specific aptamers are adaptable to multiple diagnostic formats including point-of-care devices.

Protein biopharmaceuticals are highly successful, but their utility is compromised by their propensity to aggregate during manufacture and storage. As aggregation can be triggered by non-native states, whose population is not necessarily related to thermodynamic stability, prediction of poorly-behaving biologics is difficult, and searching for sequences with desired properties is labour-intensive and time-consuming. Here we show that an assay in the periplasm of E. coli linking aggregation directly to antibiotic resistance acts as a sensor for the innate (un-accelerated) aggregation of antibody fragments. Using this assay as a directed evolution screen, we demonstrate the generation of aggregation resistant scFv sequences when reformatted as IgGs. This powerful tool can thus screen and evolve 'manufacturable' biopharmaceuticals early in industrial development. By comparing the mutational profiles of three different immunoglobulin scaffolds, we show the applicability of this method to investigate protein aggregation mechanisms important to both industrial manufacture and amyloid disease.

Deregulation of the PRC2 complex, comprised of the core subunits EZH2, SUZ12, and EED, drives aberrant hypermethylation of H3K27 and tumorigenicity of many cancers. Although inhibitors of EZH2 have shown promising clinical activity, preclinical data suggest that resistance can be acquired through secondary mutations in EZH2 that abrogate drug target engagement. To address these limitations, we have designed several hetero-bifunctional PROTACs (proteolysis-targeting chimera) to efficiently target EED for elimination. Our PROTACs bind to EED (pKD ∼ 9.0) and promote ternary complex formation with the E3 ubiquitin ligase. The PROTACs potently inhibit PRC2 enzyme activity (pIC50 ∼ 8.1) and induce rapid degradation of not only EED but also EZH2 and SUZ12 within the PRC2 complex. Furthermore, the PROTACs selectively inhibit proliferation of PRC2-dependent cancer cells (half maximal growth inhibition [GI50] = 49-58 nM). In summary, our data demonstrate a therapeutic modality to target PRC2-dependent cancer through a PROTAC-mediated degradation mechanism.Copyright © 2019 Elsevier Ltd. All rights reserved.

Chronic myelogenous leukemia (CML) is characterized by the oncogenic fusion protein, BCR-ABL protein kinase, against which clinically useful inhibitors have been developed. An alternative approach to treat CML is to degrade the BCR-ABL protein. Recently, potent degraders against BCR-ABL have been developed by conjugating dasatinib to ligands for E3 ubiquitin ligases. Since the degraders contain the dasatinib moiety, they also inhibit BCR-ABL kinase activity, which complicates our understanding of the impact of BCR-ABL degradation by degraders in CML growth inhibition. To address this issue, we chose DAS-IAP, as a potent BCR-ABL degrader, and developed a structurally related inactive degrader, DAS-meIAP, which inhibits kinase activity but does not degrade the BCR-ABL protein. DAS-IAP showed slightly weaker activity than DAS-meIAP in inhibiting cell growth when CML cells were treated for 48 h. However, DAS-IAP showed sustained growth inhibition even when the drug was removed after short-term treatment, whereas CML cell growth rapidly resumed following removal of DAS-meIAP and dasatinib. Consistently, suppression of BCR-ABL levels and downstream kinase signaling were maintained after DAS-IAP removal, whereas kinase signaling rapidly recovered following removal of DAS-meIAP and dasatinib. These results indicate that BCR-ABL degrader shows more sustained inhibition of CML cell growth than ABL kinase inhibitor.