MS6105

Sun et al. report 22, an LDH degrading PROTAC (VHL recruiting) based on the pyrazole scaffold discovered by NCATS (10.1021/acs.jmedchem.7b00941). 22 degrades LDHA and LDHB by western blot, while the VHL-binding-disrupted negative control 33 does not. Overall, I do not recommend the use of 22 as an LDH degrader until further data emerges. First, the proteomics results indicated down-regulation of >50 proteins--this may or not may be due to on-target LDH degradation or off-target effects, compromising confidence in 22's specificity. Secondly, the time-course of LDH degradation induced by 22 is very slow, requiring > 24 hours to observe clear LDH loss by western blot (Figure 6C). As described by Vetma et al. (doi.org/10.1021/acschembio.4c00152) this is a hallmark of a PROTAC showing spurious degradation activity through non-specific mechanisms like metabolic inhibition or cytotoxicity. More thorough time-course experiments with 22, including especially a CHX blockade assay demonstrating that 22 enhances the intrinsic degradation rate of LDH, is absolutely essential before qualifying 22 as a chemical probe. Thirdly, there is no hook effect at doses up to 10 uM, and the lack of a demonstrated hook effect is a yellow flag for spurious degradation--higher dosing to clearly demonstrate a hook effect, perhaps with a HiBiT-tagged LDH construct to quantify degradation kinetics, would strengthen the data package. Fourthly, there is no evidence of ternary complex formation--either biochemical or cellular evidence of LDH:VHL proximity-induction by 22 is important to establish. Finally, 22 is absolutely not suitable for in vivo use based on the currently available data. The reported exposure following an IP dose in mice is modest (~ 2 uM) and not reported with PPB or intrinsic IV clearance, making it impossible to ascertain the free exposure; there are also no data demonstrating target-engagement (i.e. LDH degradation) in vivo, meaning 22 cannot be used with confidence in animal models. Overall, this is an interesting first attempt at the development of an LDH degrader, but more data are needed before it can be used with confidence as a chemical probe.