Chemical biology strategies for directly perturbing protein homeostasis including the degradation tag (dTAG) system provide temporal advantages over genetic approaches and improved selectivity over small molecule inhibitors. We describe dTAGV-1, an exclusively selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12F36V-tagged proteins. dTAGV-1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to degrade recalcitrant oncogenes, supports combination degrader studies and facilitates investigations of protein function in cells and mice.

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35Chemical biology strategies for directly perturbing protein homeostasis including the 36 degradation tag (dTAG) system provide temporal advantages over genetic approaches and 37 improved selectivity over small molecule inhibitors. We describe dTAG V -1, an exclusively 38 selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12 F36V -tagged proteins. 39 dTAG V -1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to 40 degrade recalcitrant oncogenes, supports combination degrader studies and facilitates 41 investigations of protein function in cells and mice. 42 43 We next evaluated the utility of dTAG V -1 for combinatorial degrader studies. dTAG V -1 94 was effectively combined with THAL-SNS-032, a previously reported CRBN-recruiting CDK9 95 degrader 21 (Fig. 1e). Pronounced degradation of LACZ-FKBP12 F36V and CDK9 was observed to 96 levels comparable to those with treatment of each degrader alone, avoiding potential substrate 97 competition effects. 22 To confirm the utility of dTAG V -1 for target validation, we evaluated 98 KRAS G12V degradation, an oncogenic driver of PDAC, in PATU-8902 FKBP12 F36V -KRAS G12V ;; 99 KRAS -/cells. 17 dTAG V -1 treatment led to rapid KRAS G12V degradation, which was rescued by 100 use of dTAG V -1-NEG, pre-treatment with proteasome-inhibitor (carfilzomib) or Nedd8 activating 101 enzyme inhibitor (MLN4924), and VHL knockout, consistent with the mechanism of action of a 102 VHL-recruiting degrader ( Fig. 1f-g and Supplementary Fig. 2a-b). We also observed the 103 expected collapse in cellular signaling and diminished cell proliferation in 2D-monolayer and 3D-104 spheroid cultures upon FKBP12 F36V -KRAS G12V degradation with dTAG V -1 treatment, to levels 105 comparable to CRBN-recruiting dTAG molecules ( Fig. 1h and Supplementary Fig. 2c-e). 106To confirm the in vivo applicability of dTAG V -1, we characterized the pharmacokinetic 107 (PK) and pharmacodynamic (PD) profile of dTAG V -1 in mice. dTAG V -1 demonstrated improved 108properties compared to dTAG-13, with a longer half-life (T1/2 = 4.43, 2.41 h respectively) and 109 greater exposure (AUCinf = 18517, 6140 hr*ng mL -1 respectively) by intraperitoneal 110 administration at 10 mg/kg ( Supplementary Table 1). To report on the PD profile of dTAG 111 molecules, we employed MV4;;11 luciferase-FKBP12 F36V (luc-FKBP12 F36V ) cells 6 that allow non-112 invasive monitoring of bioluminescent signal upon dTAG molecule administration in mice. 113Following tail vein injection of MV4;;11 luc-FKBP12 F36V cells and establishment of leukemic 114 burden, we performed daily bioluminescent measurements 4 h after vehicle, 35 mg/kg dTAG-13 115 or 35 mg/kg dTAG V -1 administration. Striking loss of bioluminescent signal was achieved 4 h 116 after the first administration of dTAG V -1 ( Fig. 1i and Supplementary Fig. 3). Consistent loss of 117 bioluminescent signal was observed 4 h after each of the three dTAG-13 or dTAG V -1 118 administrations. Compared to dTAG-13, improved duration of degradation w...