Bivalent PROTACs work drive protein degradation by simultaneously binding a target protein and an E3 ligase and forming a productive ternary complex. We hypothesized that increasing binding valency within a PROTAC could enhanced degradation. Here, we designed trivalent PROTACs consisting of a bivalent BET inhibitor and an E3 ligand, tethered via a branched linker. We identified VHL-based SIM1 as a low picomolar BET degrader, with preference for BRD2. Compared to bivalent PROTACs, SIM1 showed more sustained and higher degradation efficacy, which led to more potent anti-cancer activity. Mechanistically, SIM1 simultaneously engages with high avidity both BET bromodomains in a cis intramolecular fashion and forms a 1:1:1 ternary complex with VHL exhibiting positive cooperativity and high cellular stability with prolonged residence time. Collectively, our data along with favorable in vivo pharmacokinetics demonstrate that augmenting the binding valency of proximity-induced modalities can be an enabling strategy for advancing functional outcomes.

Bivalent PROTACs work drive protein degradation by simultaneously binding a target protein and an E3 ligase and forming a productive ternary complex. We hypothesized that increasing binding valency within a PROTAC could enhanced degradation. Here, we designed trivalent PROTACs consisting of a bivalent BET inhibitor and an E3 ligand, tethered via a branched linker. We identified VHL-based SIM1 as a low picomolar BET degrader, with preference for BRD2. Compared to bivalent PROTACs, SIM1 showed more sustained and higher degradation efficacy, which led to more potent anti-cancer activity. Mechanistically, SIM1 simultaneously engages with high avidity both BET bromodomains in a cis intramolecular fashion and forms a 1:1:1 ternary complex with VHL exhibiting positive cooperativity and high cellular stability with prolonged residence time. Collectively, our data along with favorable in vivo pharmacokinetics demonstrate that augmenting the binding valency of proximity-induced modalities can be an enabling strategy for advancing functional outcomes.

On the basis of the three-dimensional pharmacophore model of opioid κ agonists, we simplified the structure of nalfurafine (selective κ agonist) to find the essential structural moieties for binding the opioid receptors, especially κ receptor type. As a result, we found that the trans-fused decahydroisoquinoline derivatives without a phenol ring bound the opioid receptor in micromolar order and that both the amide side chain and the nitrogen substituted by the cyclopropylmethyl group were indispensable moieties for eliciting the κ selectivity. The simple decahydroisoquinoline without amide side chain also bound the opioid receptor without receptor type selectivity, suggesting that the message-address concept would be applicable to even these simple derivatives. These findings that the simple decahydroisoquinoline derivatives showed the affinities for the opioid receptors, especially some of the compounds showed κ selectivity, are the first example in the opioid field.Key words opioid; κ receptor; decahydroisoquinoline; nalfurafine; three-dimensional pharmacophore model Three types of opioid receptors (μ, δ, κ) are now well established not only by pharmacological studies but also by molecular biological studies.1) Narcotic addiction is believed to be derived from the μ receptor type, and therefore δ and κ types are promising drug targets for analgesics without addiction. To obtain ideal analgesics without addiction and other side effects derived from the μ receptor, we have synthesized various kinds of naltrexone derivatives and have reported selective ligands for κ 2-9) and δ [10][11][12][13][14] receptors. Quite recently, one of our designed κ selective agonists, nalfurafine hydrochloride 2,3,6,8,9) Fig. 1), was launched in Japan as an antipruritic for patients undergoing dialysis. 6,8,9) Although many arylacetamide derivatives such as U-50,488H 15,16) (Fig. 1) and U-69,593 17) were synthesized and developed as κ agonists, all of these derivatives were eliminated from clinical trials as not only analgesics but also as antipruritics because of their serious side effects like psychotomimetic and aversive reactions. 18,19) In contrast, nalfurafine has neither aversive nor addictive effects. 20) Our interest in the differences in the pharmacological effects between nalfurafine and the arylacetamide derivatives led us to conduct a detailed structure activity relationship investigation of nalfurafine derivatives. From these studies, we developed the hypothesis that in the active conformation of nalfurafine (Fig. 2), the C-ring would assume the boat form, thereby elevating the amide side chain to bind the κ receptor. 4,5,21,22) Based on this hypothesis, we designed and synthesized KNT-63 with an oxabicyclo[2.2.2] octane skeleton ( Fig. 1), and confirmed its high affinity for the κ receptor. 5) We also proposed a new three-dimensional pharmacophore model applicable to some κ agonists with various chemotypes. 21,22) Our new pharmacophore model of κ agonists supported the proposed active conformation of nalfurafine and...

<p><b>Bivalent small-molecule degraders, or proteolysis targeting chimeras (PROTACs), work by simultaneously binding a target protein and E3 ubiquitin ligase to produce a ternary complex. To drive target ubiquitination and degradation at low catalytic concentrations, degraders must form appropriately positioned complexes of sufficient stability, aided by intra-complex interactions. We hypothesized these molecular recognition features could be enhanced by increasing binding valency. Here we present trivalent PROTACs as a strategy to boost protein degradation. Our design for a trivalent PROTAC consisted of two BET bromodomain inhibitors and an E3 ligase ligand, each separately tethered via a branched linker. In screening, we identified SIM1, a VHL-based PROTAC, as a highly potent BET degrader, capable of low picomolar degradation for all family members, with preference for BRD2. In functional comparison studies to bivalent PROTACs or inhibitors, SIM1 showed more sustained anti-cancer activity across numerous therapeutically relevant cell lines. Biophysical, biochemical, and cellular mechanistic studies showed SIM1 induces conformational changes upon binding to the BET protein to simultaneously engage with high avidity both its bromodomains in a cis intramolecular fashion. The resulting 1:1:1 complex showed positive cooperativity, high stability and prolonged cellular residence time. We provide proof-of-concept for augmenting the binding valency of proximity-induced modalities as a strategy to leverage both cooperativity and avidity within the ternary complex to advance functional outcomes.</b></p>