This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/4817943. GENERAL COMMENTS ON PREPRINT BY Frost, Rocha and Ciulli In this preprint, Frost, Rocha and Ciulli report results of how cells respond to compounds previously described as inhibitors of von Hippel–Lindau (VHL) protein as measured by quantitative mass spectrometry (MS)-based proteomics. VHL is a well-studied protein that serves as a substrate recruiting/recognition subunit of Cullin RING E3 ligase CRL2VHL. The best characterized substrate of VHL is HIF1a, a hypoxia-inducible transcription factor that is under strict control of VHL and oxygen levels. Under normal oxygen conditions, HIF1a is hydroxylated on a proline residue and this modification enhances binding with VHL, resulting in HIF1a polyubiquitination and proteasomal degradation. However, under hypoxic conditions, which often occur in solid tumors, HIF1a is not hydroxylated and is therefore stabilized and able to up-regulate cancer-promoting gene transcription. However, in some conditions, upregulating HIF1a could be beneficial (if interested google Roxadustat). Therefore, agents that inhibit VHL-HIF1a complex formation under normoxic (normal O2) conditions emerged as of interest in drug discovery. Additionally, VHL is one of the most frequently hijacked E3 ubiquitin ligase in the context of targeted protein degradation and PROTAC (Proteolysis Targeting Chimera) development. Thus, VHL ligands are currently of very high interest. The same lab has previously developed and characterized several VHL-HIF1a complex disruptors, including VH032 and VH298. Here, they take an important step to examine how these compounds affect cellular proteome. They also examine proteomic effects of proline hydroxylation (PHD) inhibitor (IOX2) - PHD is the enzyme that hydroxylates HIF1a under normoxic conditions - and benchmark everything against proteomic effects of hypoxia. The most interesting insight to emerge from these experiments is that treatment with VHL-HIF1a complex disruptors (aka inhibitors of Protein-Protein interactions (iPPIs)) leads to increase in levels of only two proteins: AMY1 (Amylase 1) and VHL. So, setting amylase aside, treatment with VHL "inhibitors" (more formally inhibitors of VHL-HIF1a binding) increases levels of VHL. This is an unexpected and interesting finding, and the authors follow up to show that the effect is time-dependent, that the effects are at the level of protein not mRNA, that negative controls don't have the same effect, and that this increase in VHL level is likely due to compound-induced increase in VHL protein stability. Importantly, the authors dive deeper into what happens to VHL ad HIF1a levels upon treatment with VH298, and how the effects change upon prolonged treatment and by increasing concentration of VH298. The observations can be summarized as: it is complicated! More specifically: Short treatment with VHL298 leads to disruption of VHL-HIF1a complex and increases levels of HIF1a (seen before and here) Prolonged treatment with VHL298 stabilizes VHL and increases VHL levels, and increased VHL level reduce HIF1a levels (seen here) Therefore, pharmacology of VHL ligands, even in the context of their use for PROTAC development may very well be complicated and future efforts would need to take this into account. Overall, these are interesting results, and in my view the main implications of this work are importance of: (1) performing robust quantitative proteomics experiments as a part of validating/characterizing effects of small molecules; (2) not underestimating complexity of small molecule mechanism of action; and (3) accounting/examining potential differences of acute vs. prolonged treatments and acute vs. prolonged effects when characterizing and validating small molecules.