Chemical probes are small molecules that selectively modulate a protein of interest and play a crucial role in understanding protein function and validating new drug targets. Covalent modality represents an important expansion of the repertoire of drug hunters to target poorly ligandable proteins.

Criteria for chemical probes have been established to describe the potency, selectivity, target engagement, and properties of small-molecule probes that are qualified to enable the interrogation and validation of drug targets. However, these definitions have been tailored to reversibly acting modulators but fall short in their applicability to other modalities.

Hartung et al. propose quality criteria for covalent inhibitors are different from those for reversible inhibitors. The authors highlight several examples of suitable probe and pathfinder compounds, including the GPX4 inhibitor ML210, the PARP16 inhibitor DB008, the UCHL1 inhibitor IMP-1710, and the cMyc binder EN4.

Covalent inhibitors have shown promise in targeting proteins that were previously considered undruggable. For example, the KRasG12C inhibitor has shown cellular functional activities at concentrations below 1 μM. The development of covalent inhibitors requires significant efforts to characterize and validate them as a prerequisite for conclusive use in biomedical research and target validation studies.

In my experience it cannot be overstated how important is the SAR of the non-covalent part. It is the positioning through the non-covalent part that facilitates the formation of the covalent bond and drives selectivity.

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Sometimes you get showered with micro-crystals or heavy precipitate. This often means conditions are too supersaturated, causing excessive nucleation.

The goal is to generate as few nuclei as possible to grow larger crystals. To achieve this, you can decrease the protein or precipitant concentration, or use a larger drop  slowing down equilibration.

Another trick is microseeding into conditions which don’t nucleate spontaneously – leading to a focus on the growth phase instead of the nucleation phase.  Additionally, by using a  dilution series of the  seed stock, it is possible to precisely adjust the  number of introduced pre-formed nuclei (seeds) which directly affects the final number and size of the grown crystals. This can allow a few crystals to outgrow the rest. Additives or detergents might help by reducing non-specific nucleation as well.

In short, gentle tinkering to dial back nucleation can convert “too many tiny crystals” into a few sizable ones.

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You’ve got the right construct in the right host – now how you induce expression can make all the difference. A common mistake is blasting cells with a high concentration of IPTG and incubating at 37 °C, only to get mountains of misfolded protein.

In reality, less is often more in expression optimization. Try inducing at a lower temperature (like 18 °C or 20 °C overnight); cooler temps give proteins more time to fold properly, often boosting soluble yield. Adjusting IPTG concentration can help too – sometimes a little (e.g. 0.1 mM instead of 1 mM) is enough to trigger expression without stressing the cells. Also consider the cell density at induction: inducing at mid-log (OD₆₀₀ ~0.6) versus late-log can impact yields and protein quality.

Another powerful approach is auto-induction media, which slowly induce expression as the culture grows to high density – great for obtaining high yields without constant supervision.

Finally, don’t overlook shaking speed and aeration; sample oxygenation can improve protein production in bacteria. Tuning these parameters requires a bit of trial and error, but the payoff is big: higher yields and properly folded protein.

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Fusion tags can be a protein producer’s best friend. Attaching a tag like His₆, GST, or MBP can dramatically simplify purification and even improve solubility for tricky proteins. For instance, MBP or GST tags often help keep a normally aggregation-prone protein folded by acting as a chaperoning partner. ⁣
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The trade-off? ⁣
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Tags add extra length and may interfere with your protein’s function or crystallization if not removed. It’s important to choose a tag that suits your goal: His-tags are small and great for quick IMAC purification; larger tags like MBP increase yield of soluble protein but almost always require a cleavage step before final analyses. ⁣

Also consider tag placement – N-terminal vs C-terminal – as it can affect folding or activity. Use tags strategically: they’re extremely helpful tools, but plan ahead for whether (and how) to remove them later. ⁣
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The DbTACs platform is a novel technology that enables selectively targeted protein degradation. It is designed to precisely degrade proteins of interest. The approach is compatible with various warheads such as small molecules, antibodies, or DNA motifs.

DbTCACs stands for DNA framework-based PROTACs. In comparison to PROTACs, the DbTACs platform uses a DNA framework-engineered chimera instead of a small molecule-based chimera, which provides a more stable and versatile platform for targeted protein degradation. Additionally, the DbTACs platform is able to target “undruggable” proteins and degrade proteins in a time-dependent manner, which are not features of PROTACs.

The platform uses a click chemistry-mediated programmable linker to achieve simultaneous selective multi-target proteolysis. The universality of the platform is highlighted by the following features: (1) precise degradation of POIs; (2) simultaneous selective multi-target proteolysis; (3) compatibility with various warheads; (4) a DNA framework-engineered chimera instead of a small molecule-based chimera; (5) a more stable and versatile platform for targeted protein degradation; (6) the ability to target “undruggable” proteins; (7) the ability to degrade proteins in a time-dependent manner; and (8) the ability to analyze the stability of the DbTACs using 2% agarose gel electrophoresis.

The DbTACs platform is a significant advancement in the field of drug discovery as it provides a new approach for selectively targeted protein degradation. The ability to precisely degradePOIs and achieve simultaneous selective multi-target proteolysis is a valuable tool for drug discovery. The platform is also compatible with various warheads, such as small molecules, antibodies, or DNA motifs, which provides versatility in targeting different kinds of proteins. The DbTACs platform can be used to develop new drugs for various diseases, including cancer, neurodegenerative diseases, and autoimmune disorders.

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