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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.

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/4746304. COMMENTS ON THE PREPRINT BY Dölle, Adhikari et al. Targeted protein degradation is a very active field at the moment. Many efforts in this area are focusing on transforming known ligands (binders, inhibitors) of proteins with a clear disease relevance into bifunctional (PROTAC-based) degrader molecules. Unlike the traditional antagonist/inhibitor based compounds in preclinical and clinical use that diminish (inhibit) activity of the target, these degrader molecules induce selective degradation of the target. Thus, they remove the target from the proteome. This type of pharmacological activity could be a real benefit when the target in question plays significant scaffolding roles, by engaging multiple binding partners using different regions and binding sites. In such a case, inhibiting individual protein-protein interactions would be highly impractical. However, if the target is degraded, all these PPIs would disappear together with the target! Dölle, Adhikari et al. select one such target - WDR5, a protein that performs different scaffolding roles (i.e. binds different partners) in the context of epigenetic regulation. Because of this, WDR5 has been implicated as a target for drug development and couple of compounds that inhibit WDR5 mediated PPIs have been described. These compounds served here as a starting point for WDR5 selective degrader development. The authors used existing structures of WDR5 bound to the PPI inhibitors to identify surface exposed areas of the molecules that could be modified for degrader molecule development. In brief, each PROTAC (bifunctional degrader) includes a ligand for the target and a ligand that recruits an E3 ubiquitin ligase, connected via a linker. The linker is known to have an impact on the performance of PROTACs and the authors use three chemically distinct types of linkers (PEG based, aliphatic and aromatic). The nature of the E3 ligase is also a major factor that affects degraders' activity, and the authors start by incorporating ligands for cereblon (CRBN), VHL and MDM2. Altogether, they generate number of PROTAC-based degraders featuring different linkers and different ligases. They describe detailed validation steps of their degraders which included: Measuring in vitro (biochemical) affinity between WDR5 and degrader molecules using ITC, showing Kd values in low nM. - They also tested binding via DSF The authors tested that degraders were cell permeable and that they engaged the target using BRET. This is an important step in validation as degrader molecules tend to be larger, leading to concerns that may have difficulty entering cells. They provide evidence that their degraders induce target degradation in cells, including under endogenous conditions. Importantly, they show that negative control compounds (always critical to have on hand) show no activity, and that inhibiting proteasome rescues observed degradation. Additionally, they confirm that mRNA levels of WDR5 did not change, thus further validating that the reason for decrease in protein levels is due to degradation. (They also include additional pieces of evidence that effects on WDR5 protein levels are degradation dependent) Also importantly, the authors show selectivity by quantitative proteomics and demonstrate that WDR5 is the only protein depleted out of more than 5800 identified after 9 hours of treatment (while treatment with individual ligands did not have this effect) Lastly, they show anti-proliferative effects in MV4-11 cells of their best performing degraders (these compounds were VHL-based PROTACs). However, the concentration needed for cellular effects was high (10uM). The authors then showed that this is due to low levels of VHL present. When they overexpressed VHL, the growth inhibitory activity improved. Overall, the work is of high quality and includes appropriate steps for degrader validation. This gives high confidence that WDR5 degraders described in this work are useful as probes for WDR5 biology. For example, what happens to histone methylation once WDR5 is removed? Does removal of WDR5 lead to destabilization (or stabilization) of some of its binding partners (proteomics results suggest that this may not be the case, but would be interesting to dig deeper into this question)? What happens to transcription? What effect does this have on MYC activity (MYC family is known to engage with WDR5)? I am sure the authors and the community have these and many other questions in mind, and I look forward to seeing what new biology they and others can discover with this new generation of tool compounds in hand.

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/4672026. Dear authors, thank you for sharing this preprint with everyone. I recently decided to support research in chemical biology by providing feedback and thoughts on preprints in this field. Your preprint caught my eye, and I hope you will find the comments below useful (I hesitate to call this peer review, but if you would like to share what I wrote with a journal please feel free). Kind regards, Milka Comments to authors: In this preprint the authors combine genetic code expansion (a strategy that allows one to incorporate non-canonical amino acids at a specific position within the protein), with biorthogonal labeling (a type of chemical reaction executed in living systems in a way that is orthogonal to biological/physiological reactions) and optical microscopy to visualize two members of the transmembrane AMPA receptor (AMPAR) regulatory protein (TARP) family, 2 and 8 in live neurons and brain slices. The main problem that the authors set out to address is as follows: - The authors present evidence that antibodies can't recognize endogenous TARP gamma2 and gamma8 in neurons because these proteins are found to be associated with AMPAR's ligand binding domain (LBD) in a manner that masks the epitope. This suggests that strategies like immunostaining using fluorescently labeled antibodies are not appropriate for TARP imaging. Therefore, to solve this problem the authors decided to pursue a completely different strategy that does not rely on antibody use. Overall, this is an important research problem and the approach the authors chose to pursue is appropriate for addressing this problem. Summary of the approach: - The authors incorporated clickable trans-cyclooctene derivatized lysine into TARP gamma2 and gamma8 using established strategy for genetic code expansion. Exposing the modified TARP gamma2 and gamma8 to cell-impermeable tetrazine-dyes resulted in biorthogonal reaction called strain-promoted inverse electron-demand Diels-Alder cycloaddition reaction (SPIEDAC), whereby TARP gamma2 (or gamma8) featuring a modified lysine is covalently labeled with the fluorescent dye. The strategy does not seem to prevent TARP gamma2 and gamma8 from interacting with AMPARs, or affect TARP gamma2 and gamma8 function making this a well-tolerated modification. Additionally, the fluorescent signal is strong enough to allow visualization in primary neurons, as well as organotypic hippocampal slice cultures. Summary of key biological observations: - The key observations made in this study are: o Most of endogenous TARP gamma2 and gamma8 are found to be associated with AMPAR at the surface of hippocampal neurons. o TARP gamma2 was found to be mostly localized to synaptic spines as opposed to extrasynaptic sites, while TARP gamma8 had a more even distribution between the two, while still favoring synaptic spines. o Analysis of visual data allowed for identification of TARP gamma2 synaptic clusters (80 nm in size) that localized within dendritic spines. TARP gamma8 was not observed to form clusters under the conditions used in this work. Concluding remarks: Overall, I found the application of the genetic code expansion in this neurobiology context to be of interest and I can see the potential of this methodology (with further improvements elaborated on by the authors in their Discussion section) for imaging systems that are otherwise difficult to address using optical microscopy.

Targeted protein degradation is fascinating, promising, exciting,intriguing, different, unique... but is it also hyped? As anytechnology that breaks new conceptual grounds and opens previously unforeseen circumstances, targeted protein degradation is subject to the technology hype cycle. In this presentation, I provide the basic introduction to the field, and comment on how we can navigate the hype cycle to advance the field faster.

Many scientists spend years of training in an academic setting, and progress from one academic appointment to another using CVs as they go along to illustrate their educational and scientific achievements. Over time, CVs become long and meandering, not surprising given that CV is short for curriculum vitae aka "a course of life". Unfortunately, most employers outside the academia are not interested in long narratives. More terrifyingly, most companies use HR professionals with little or no scientific background to filter through large numbers of applications and narrow down the candidate pool. To streamline the process, employers want to see a short highlight of achievements, expertise, skills, interests and aptitudes, also known as a Resume. In order to resonate with an employer, Resume has to be clear, succinct, specific, and well-aligned with the responsibilities and requirements of the role that a candidate is being considered for. In this presentation you will learn some basics of conducting an effective job search, how to read job ads in a way that will help you structure your Resume, and learn more about what Resume is and what it should look like.

Small molecules drugs are a long time favorites of those involved in frug discovery and development because these medicines tend to be easier to use andcost less to make. Additionally, theycan be developed to tackle a wide range of diseases and moleculartargets. However, not every target protein in our cells contains a small molecule binding pocket, which means that small molecule drugs have limitations in terms of what indications they can be developed for. Moreover, they often hit more than a single target, which means that they may be toxic or lead to undesirable side-effects. Targeted protein degradation is an emerging interest in drug discovery and development. Small molecule degraders induce degradation of their target, instead of inhibition and exhibit more durable effects at lower concentrations. Additionally, they don't require tight binding and can therefore be made even for targets that lack well-defined binding pockets. All this makes them of interest, and here we use a Strengths-Weaknesses-Opportunities-Threats (SWOT) framework to critically assess the current status of this rapidly evolving field.&nbsp

This presentations was given at the the inaugural Proteome-Targeted Drug Discovery Summit 2019 that brought together people interested in pushing the envelop on small-ish molecule based drug discovery and development. From this perspective degrader molecules have really done a lot over the last 5 years to rekindle our imagination of what modalities we can use to go after the undrugged targets. My view point on this emerging field is one of an outsider looking in, and I therefore asked the audience membersto share what they think, as well as offered some of my suggestions on how to grow the field further. The most important suggestions I shared are: (1) the importance of developing standards and rules - both related how to validate degrader molecules but also how to share data and enforce the standards - and nucleating a community to carry this work forward; (2) the importance of advocating and promoting the use of small molecule degraders as tools for basic research that is highly complementary to existing research tools; and (3) the importance to cast degraders into a broader perspective as just the first and most visible example of a brand new type of small molecules - one that act as recruiters/inducers of neo-activity.&nbsp

Scholarly publishing is in crisis! Scholars are in crisis! Science is in crisis! World is in crisis! With so manycrises comes a great deal of anxiety, fueling fast thinking and reactive behavior. In the world of scientific publishing and science evaluationthis has createda pattern of attack and retreat, warring factions, and myopia resulting in a growing chasm between publishers and content creators. In this Keynote Lecture (presented at the EMUG 2019, a meeting that brings together publishing professionals), I will attempt to break the silos and bridge the chasm through arguing that publishing begins, proceeds and ends with people. My main hypothesis is that placingpeople first (authors, readers, reviewers, editors, publishers) can help move us out of the current impasse the relationship between publishers (journals) and scientists has reached. I will share with the audience some of the realities of the current climate of academic science, and offer to learn form them about the current climate of publishing as a profession. I will put forward one provocative solution to the current problem - decoupling research from authoring, and one more practical and easily implementable solution to the problem: making the relationship between content creators and publishers more personal.&nbsp

Chemical compounds can be powerful tools for biomedical research. But in order to earn the "chemical probe" or "tool compound" designation, a compound must undergo rigorous testing and validation, and satisfy certain chemical and biological criteria. Usingthese compounds (sometimes commonly referred to as "chemicals", "inhibitors", or even "drugs" by those who use them as research tools)that have been tested and validated can help answer functional questions about biology and physiology that genetic manipulations can't access. This may leave users with the impression that any compound that produces a phenotype that they are interested in is the best tool for the job. Unfortunately, this prevalent thinking is dangerous as it often leads to incorrect conclusions about biological phenomena, and subsequently frequently leads to wrong clinical hypotheses. The main point of this presentation is to continue to raise the awareness of the critical need to carefully validate all compounds that are meant to be used as biomedical research tools, as well as nucleate a diverse and inclusive community of chemists, biologists, funders, journals, and vendors that will set and enforce the standards, in hope those will safe guard our research efforts going forward.

This is a flier that will accompany a poster I will present at the 2019 Gordon Research Conference on High-Throughput Chemistry and Chemical Biology. The poster and the flier jointly highlight some key issues surrounding problems of rigor and reproducibility in biomedical sciences due to the inappropriate use of chemical tool compounds. Given that these tool compounds are powerful and essential for advancing functional understanding of biological processes, I propose that it is high time to bring to the table key stakeholders (chemical biologists who are making the tools, biologists who are using them, journals, funders and vendors) and build the way forward. You have an opportunity to way in on this question by scanning the QR code at the bottom of the flier and leaving your thoughts in the comments. Thank you!