Bifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, forming a target/PROTAC/ligase ternary complex. Despite the importance of this key intermediate species, no detailed validation of a method to directly determine binding parameters for ternary complex kinetics has been reported, and it remains to be addressed whether tuning the kinetics of PROTAC ternary complexes may be an effective strategy to improve the efficiency of targeted protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation in vitro using purified proteins. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of bromodomain and extraterminal domain proteins Brd2, Brd3, and Brd4 to the von Hippel–Lindau E3 ligase (VHL). We reveal marked differences in ternary complex off-rates for different PROTACs that exhibit either positive or negative cooperativity for ternary complex formation relative to binary binding. The positively cooperative degrader MZ1 forms comparatively stable and long-lived ternary complexes with either Brd4BD2 or Brd2BD2 and VHL. Equivalent complexes with Brd3BD2 are destabilized due to a single amino acid difference (Glu/Gly swap) present in the bromodomain. We observe that this difference in ternary complex dissociative half-life correlates to a greater initial rate of intracellular degradation of Brd2 and Brd4 relative to Brd3. These findings establish a novel assay to measure the kinetics of PROTAC ternary complexes and elucidate the important kinetic parameters that drive effective target degradation.

The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We used a structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also causes rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and anti-proliferative effects than compounds that merely inhibit co-repressor interactions. This work establishes the BTB domain as a highly druggable structure, paving the way for the use of other members of this protein family as drug targets. The magnitude of effects elicited by this class of BCL6-degrading compounds exceeds that of our equipotent non-degrading inhibitors, suggesting opportunities for the development of BCL6-based lymphoma therapeutics.

Bifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, forming a target:PROTAC:ligase ternary complex. Despite the importance of this key intermediate species, no detailed validation of a method to directly determine binding parameters for ternary complex kinetics has been reported, and it remains to be addressed whether tuning the kinetics of PROTAC ternary complexes may be an effective strategy to improve the efficiency of targeted protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation in vitro using purified proteins. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of BET proteins Brd2, Brd3 and Brd4 to the E3 ligase VHL. We reveal marked differences in ternary complex off-rates for different PROTACs that exhibit either positive or negative cooperativity for ternary complex formation relative to binary binding. The positively cooperative degrader MZ1 forms comparatively stable and long-lived ternary complexes with either Brd4 BD2 or Brd2 BD2 and VHL. Equivalent complexes with Brd3 BD2 are destabilised due to a single amino acid difference (Glu/Gly swap) present in the bromodomain. We observe that this difference in ternary complex dissociative half-life correlates to a greater initial rate of intracellular degradation of Brd2 and Brd4 relative to Brd3. These findings establish a novel assay to measure the kinetics of PROTAC ternary complexes and elucidate the important kinetic parameters that drive effective target degradation.

Abstract-Prosthetic use and satisfaction in wounded servicemembers and veterans with unilateral upper-limb loss has not been thoroughly explored. Through a national survey, we enrolled 47 participants from the Vietnam conflict and 50 from Operation Iraqi Freedom/Operation Enduring Freedom (OIF/ OEF) with combat-associated major unilateral upper-limb loss. Upper-limb prosthetic devices were used by 70% of the Vietnam group and 76% of the OIF/OEF group. Mechanical/bodypowered upper-limb devices were favored by the Vietnam group, while a combination of myoelectric/hybrid and mechanical/body-powered devices were favored by the OIF/OEF group. Upper-limb devices were completely abandoned in 30% of the Vietnam and 22% of the OIF/OEF groups. Abandonment was more frequent for transhumeral and more proximal levels (42% of Vietnam and 40% of OIF/OEF) than more distal limb-loss levels. Upper-limb prostheses were rejected because of dissatisfaction with the device by significantly fewer (23%) members of the Vietnam group than the OIF/OEF group (45%) (p < 0.001). Most common reasons for rejection included pain, poor comfort, and lack of functionality. A significant paradigm shift has been noted in the OIF/OEF group, who use a greater number and diversity of upper-limb prostheses than the Vietnam group.

Mesenchymal stromal/stem cells (MSC) are promising candidates for cellular therapy of different diseases in humans and in animals. Following the guidelines of the International Society for Cell Therapy, human MSC may be identified by expression of a specific panel of cell surface markers (CD105+, CD73+, CD90+, CD34-, CD14-, or CD11b-, CD79- or CD19-, HLA-DR-). In addition, multiple differentiation potential into at least the osteogenic, adipogenic, and chondrogenic lineage is a main criterion for MSC definition. Human MSC and MSC of a variety of mammals isolated from different tissues meet these criteria. In addition to the abovementioned, they express many more cell surface markers. Yet, these are not uniquely expressed by MSC. The gross phenotypic appearance like marker expression and differentiation potential is similar albeit not identical for MSC from different tissues and species. Similarly, MSC may feature different biological characteristics depending on the tissue source and the isolation and culture procedures. Their versatile biological qualities comprising immunomodulatory, anti-inflammatory, and proregenerative capacities rely largely on the migratory and secretory capabilities of MSC. They are attracted to sites of tissue lesion and secrete factors to promote self-repair of the injured tissue. This is a big perspective for clinical MSC applications in both veterinary and human medicine. Phase I/II clinical trials have been initiated to assess safety and feasibility of MSC therapies in acute and chronic disease settings. Yet, since the mode of MSC action in a specific disease environment is still unknown at large, it is mandatory to unravel the response of MSC from a given source onto a specific disease environment in suitable animal models prior to clinical applications. © 2017 International Society for Advancement of Cytometry.