Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response.

A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunity-driven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development.

Structure-activity relationship study of small-molecule inhibitor of Atg12-Atg3 protein-protein interaction.

Autophagy is a catabolic process that was described to play a critical role in advanced stages of cancer, wherein it maintains tumor cell homeostasis and growth by supplying nutrients. Autophagy is also described to support alternative cellular trafficking pathways, providing a non-canonical autophagy-dependent inflammatory cytokine secretion mechanism. Therefore, autophagy inhibitors have high potential in the treatment of cancer and acute inflammation. In our study, we identified compound 1 as an inhibitor of the ATG12-ATG3 protein-protein interaction. We focused on the systematic modification of the original hit 1, a casein kinase 2 (CK2) inhibitor, to find potent disruptors of ATG12-ATG3 protein-protein interaction. A systematic modification of the hit structure led us to a wide plethora of compounds that maintain its ATG12-ATG3 inhibitory activity, which could act as a viable starting point to design new compounds with diverse therapeutic applications.

Dual targeting of histone deacetylases and MYC as potential treatment strategy for H3-K27M pediatric gliomas.

Diffuse midline gliomas (DMGs) are aggressive and fatal pediatric tumors of the central nervous system that are highly resistant to treatments. Lysine to methionine substitution of residue 27 on histone H3 (H3-K27M) is a driver mutation in DMGs, reshaping the epigenetic landscape of these cells to promote tumorigenesis. H3-K27M gliomas are characterized by deregulation of histone acetylation and methylation pathways, as well as the oncogenic MYC pathway. In search of effective treatment, we examined the therapeutic potential of dual targeting of histone deacetylases (HDACs) and MYC in these tumors. Treatment of H3-K27M patient-derived cells with Sulfopin, an inhibitor shown to block MYC-driven tumors in vivo, in combination with the HDAC inhibitor Vorinostat, resulted in substantial decrease in cell viability. Moreover, transcriptome and epigenome profiling revealed synergistic effect of this drug combination in downregulation of prominent oncogenic pathways such as mTOR. Finally, in vivo studies of patient-derived orthotopic xenograft models showed significant tumor growth reduction in mice treated with the drug combination. These results highlight the combined treatment with PIN1 and HDAC inhibitors as a promising therapeutic approach for these aggressive tumors.

Diffuse midline gliomas (DMGs) are among the most aggressive and fatal brain cancers in children. They are often associated with changes in histones, the proteins that control gene activity and give chromosomes their structure. Most children with DMGs, for example, share the same anomaly in their histone H3 protein (referred to as the H3-K27M mutation). This change affects how small chemical tags called methyl and acetyl groups can be added onto histone 3, which in turn alters the way the protein can switch genes on and off. As a result, tumours start to develop. One potential therapeutic strategy against DMGs is to use histone deacetylase inhibitors (HDACi), a promising type of drugs which inhibits the enzymes that remove acetyl groups from histones. Patients can develop resistance to HDACi, however, highlighting the need to explore other approaches. One possibility is to treat patients with several types of drugs, each usually targeting a distinct biological process that contributes to the emergence of cancer. This combined approach can have multiple benefits; the drugs potentially amplify each other's effect, for example, and it is also less likely for cells to become resistant to more than one compound at the time. In addition, each drug in the combination can be used in a lower dose to reduce side effects and benefit patients. DMG tumour cells often feature higher activity levels of a protein known as MYC, which can contribute to the growth of the tumour. Algranati, Oren et al. therefore set out to test whether combining an HDACi known as Vorinostat with a drug that blocks MYC activity (Sulfopin) can act as an effective treatment for this cancer. Tumour samples from eight DMG patients were treated with either Sulfopin alone, or Sulfopin in association with Vorinostat. Cells exposed to both drugs were less likely to survive, and additional genetic experiments showed that the combined treatment had resulted in pathways that promote tumour development being blocked. When both Sulfopin and Vorinostat were administered to mice made to grow human DMG tumors, the animals showed a greater reduction in tumor growth. Treatment options for DMG are usually limited, with chemotherapy often being ineffective and surgery impossible. The work by Algranati, Oren et al. suggests that combining HDACi and drugs targeting the MYC pathway is a strategy that should be examined further to determine whether clinical applications are possible.

High-throughput screening identifies broad-spectrum Coronavirus entry inhibitors.

The COVID-19 pandemic highlighted the need for antivirals against emerging coronaviruses (CoV). Inhibiting spike (S) glycoprotein-mediated viral entry is a promising strategy. To identify small molecule inhibitors that block entry downstream of receptor binding, we established a high-throughput screening (HTS) platform based on pseudoviruses. We employed a three-step process to screen nearly 200,000 small molecules. First, we identified hits that inhibit pseudoviruses bearing the SARS-CoV-2 S glycoprotein. Counter-screening against pseudoviruses with the vesicular stomatitis virus glycoprotein (VSV-G), yielded sixty-five SARS-CoV-2 S-specific inhibitors. These were further tested against pseudoviruses bearing the MERS-CoV S glycoprotein, which uses a different receptor. Out of these, five compounds, which included the known broad-spectrum inhibitor Nafamostat, were subjected to further validation and tested against pseudoviruses bearing the S glycoprotein of the Alpha, Delta, and Omicron variants as well as bona fide SARS-CoV-2. This rigorous approach revealed an unreported inhibitor and its derivative as potential broad-spectrum antivirals.

Targeting SRSF2 mutations in leukemia with RKI-1447: A strategy to impair cellular division and nuclear structure.

Spliceosome machinery mutations are common early mutations in myeloid malignancies; however, effective targeted therapies against them are still lacking. In the current study, we used an in vitro high-throughput drug screen among four different isogenic cell lines and identified RKI-1447, a Rho-associated protein kinase inhibitor, as selective cytotoxic effector of SRSF2 mutant cells. RKI-1447 targeted SRSF2 mutated primary human samples in xenografts models. RKI-1447 induced mitotic catastrophe and induced major reorganization of the microtubule system and severe nuclear deformation. Transmission electron microscopy and 3D light microscopy revealed that SRSF2 mutations induce deep nuclear indentation and segmentation that are apparently driven by microtubule-rich cytoplasmic intrusions, which are exacerbated by RKI-1447. The severe nuclear deformation in RKI-1447-treated SRSF2 mutant cells prevents cells from completing mitosis. These findings shed new light on the interplay between microtubules and the nucleus and offers new ways for targeting pre-leukemic SRSF2 mutant cells.

Targeting phenylalanine assemblies as a prospective disease-modifying therapy for phenylketonuria.

Phenylketonuria is characterized by the accumulation of phenylalanine, resulting in severe cognitive and neurological disorders if not treated by a remarkably strict diet. There are two approved drugs today, yet both provide only a partial solution. We have previously demonstrated the formation of amyloid-like toxic assemblies by aggregation of phenylalanine, suggesting a new therapeutic target to be further pursued. Moreover, we showed that compounds that halt the formation of these assemblies also prevent their resulting toxicity. Here, we performed high-throughput screening, searching for compounds with inhibitory effects on phenylalanine aggregation. Morin hydrate, one of the most promising hits revealed during the screen, was chosen to be tested in vivo using a phenylketonuria mouse model. Morin hydrate significantly improved cognitive and motor function with a reduction in the number of phenylalanine brain deposits. Moreover, while phenylalanine levels remained high, we observed a recovery in dopaminergic, adrenergic, and neuronal markers. To conclude, the ability of Morin hydrate to halt phenylalanine aggregation without reducing phenylalanine levels implies the toxic role of the phenylalanine assemblies in phenylketonuria and opens new avenues for disease-modifying treatment.

Open science discovery of potent noncovalent SARS-CoV-2 main protease inhibitors.

We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.

Specific quinone reductase 2 inhibitors reduce metabolic burden and reverse Alzheimer's disease phenotype in mice.

Biological aging can be described as accumulative, prolonged metabolic stress and is the major risk factor for cognitive decline and Alzheimer's disease (AD). Recently, we identified and described a quinone reductase 2 (QR2) pathway in the brain, in which QR2 acts as a removable memory constraint and metabolic buffer within neurons. QR2 becomes overexpressed with age, and it is possibly a novel contributing factor to age-related metabolic stress and cognitive deficit. We found that, in human cells, genetic removal of QR2 produced a shift in the proteome opposing that found in AD brains while simultaneously reducing oxidative stress. We therefore created highly specific QR2 inhibitors (QR2is) to enable evaluation of chronic QR2 inhibition as a means to reduce biological age-related metabolic stress and cognitive decline. QR2is replicated results obtained by genetic removal of QR2, while local QR2i microinjection improved hippocampal and cortical-dependent learning in rats and mice. Continuous consumption of QR2is in drinking water improved cognition and reduced pathology in the brains of AD-model mice (5xFAD), with a noticeable between-sex effect on treatment duration. These results demonstrate the importance of QR2 activity and pathway function in the healthy and neurodegenerative brain and what we believe to be the great therapeutic potential of QR2is as first-in-class drugs.

Identifying a selective inhibitor of autophagy that targets ATG12-ATG3 protein-protein interaction.

Macroautophagy/autophagy is a catabolic process by which cytosolic content is engulfed, degraded and recycled. It has been implicated as a critical pathway in advanced stages of cancer, as it maintains tumor cell homeostasis and continuous growth by nourishing hypoxic or nutrient-starved tumors. Autophagy also supports alternative cellular trafficking pathways, providing a mechanism of non-canonical secretion of inflammatory cytokines. This opens a significant therapeutic opportunity for using autophagy inhibitors in cancer and acute inflammatory responses. Here we developed a high throughput compound screen to identify inhibitors of protein-protein interaction (PPI) in autophagy, based on the protein-fragment complementation assay (PCA). We chose to target the ATG12-ATG3 PPI, as this interaction is indispensable for autophagosome formation, and the analyzed structure of the interaction interface predicts that it may be amenable to inhibition by small molecules. We screened 41,161 compounds yielding 17 compounds that effectively inhibit the ATG12-ATG3 interaction in the PCA platform, and which were subsequently filtered by their ability to inhibit autophagosome formation in viable cells. We describe a lead compound (#189) that inhibited GFP-fused MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) puncta formation in cells with IC50 value corresponding to 9.3 µM. This compound displayed a selective inhibitory effect on the growth of autophagy addicted tumor cells and inhibited secretion of IL1B/IL-1ß (interleukin 1 beta) by macrophage-like cells. Compound 189 has the potential to be developed into a therapeutic drug and its discovery documents the power of targeting PPIs for acquiring specific and selective compound inhibitors of autophagy.Abbreviations: ANOVA: analysis of variance; ATG: autophagy related; CQ: chloroquine; GFP: green fluorescent protein; GLuc: Gaussia Luciferase; HEK: human embryonic kidney; IL1B: interleukin 1 beta; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; PCA: protein-fragment complementation assay; PDAC: pancreatic ductal adenocarcinoma; PMA: phorbol 12-myristate 13-acetate; PPI: protein-protein interaction. VCL: vinculin.

Turning high-throughput structural biology into predictive inhibitor design.

A common challenge in drug design pertains to finding chemical modifications to a ligand that increases its affinity to the target protein. An underutilized advance is the increase in structural biology throughput, which has progressed from an artisanal endeavor to a monthly throughput of hundreds of different ligands against a protein in modern synchrotrons. However, the missing piece is a framework that turns high-throughput crystallography data into predictive models for ligand design. Here, we designed a simple machine learning approach that predicts protein-ligand affinity from experimental structures of diverse ligands against a single protein paired with biochemical measurements. Our key insight is using physics-based energy descriptors to represent protein-ligand complexes and a learning-to-rank approach that infers the relevant differences between binding modes. We ran a high-throughput crystallography campaign against the SARS-CoV-2 main protease (MPro), obtaining parallel measurements of over 200 protein-ligand complexes and their binding activities. This allows us to design one-step library syntheses which improved the potency of two distinct micromolar hits by over 10-fold, arriving at a noncovalent and nonpeptidomimetic inhibitor with 120 nM antiviral efficacy. Crucially, our approach successfully extends ligands to unexplored regions of the binding pocket, executing large and fruitful moves in chemical space with simple chemistry.

Azapeptide activity-based probes for the SARS-CoV-2 main protease enable visualization of inhibition in infected cells.

The COVID-19 pandemic has revealed the vulnerability of the modern, global society. With expected waves of future infections by SARS-CoV-2, treatment options for infected individuals will be crucial in order to decrease mortality and hospitalizations. The SARS-CoV-2 main protease is a validated drug target, for which the first inhibitor has been approved for use in patients. To facilitate future work on this drug target, we designed a solid-phase synthesis route towards azapeptide activity-based probes that are capped with a cysteine-reactive electrophile for covalent modification of the active site of Mpro. This design led to the most potent ABP for Mpro and one of the most potent inhibitors reported thus far. We demonstrate that this ABP can be used to visualize Mpro activity and target engagement by drugs in infected cells.

Sulfamate Acetamides as Self-Immolative Electrophiles for Covalent Ligand-Directed Release Chemistry.

Electrophiles for covalent inhibitors that are suitable for in vivo administration are rare. While acrylamides are prevalent in FDA-approved covalent drugs, chloroacetamides are considered too reactive for such purposes. We report sulfamate-based electrophiles that maintain chloroacetamide-like geometry with tunable reactivity. In the context of the BTK inhibitor ibrutinib, sulfamate analogues showed low reactivity with comparable potency in protein labeling, in vitro, and cellular kinase activity assays and were effective in a mouse model of CLL. In a second example, we converted a chloroacetamide Pin1 inhibitor to a potent and selective sulfamate acetamide with improved buffer stability. Finally, we show that sulfamate acetamides can be used for covalent ligand-directed release (CoLDR) chemistry, both for the generation of "turn-on" probes as well as for traceless ligand-directed site-specific labeling of proteins. Taken together, this chemistry represents a promising addition to the list of electrophiles suitable for in vivo covalent targeting.

Optimization of Covalent MKK7 Inhibitors via Crude Nanomole-Scale Libraries.

High-throughput nanomole-scale synthesis allows for late-stage functionalization (LSF) of compounds in an efficient and economical manner. Here, we demonstrated that copper-catalyzed azide-alkyne cycloaddition could be used for the LSF of covalent kinase inhibitors at the nanoscale, enabling the synthesis of hundreds of compounds that did not require purification for biological assay screening, thus reducing experimental time drastically. We generated crude libraries of inhibitors for the kinase MKK7, derived from two different parental precursors, and analyzed them via the high-throughput In-Cell Western assay. Select inhibitors were resynthesized, validated via conventional biological and biochemical methods such as western blots and liquid chromatography-mass spectrometry (LC-MS) labeling, and successfully co-crystallized. Two of these compounds showed over 20-fold increased inhibitory activity compared to the parental compound. This study demonstrates that high-throughput LSF of covalent inhibitors at the nanomole-scale level can be an auspicious approach in improving the properties of lead chemical matter.

Small molecule inhibitor of Igf2bp1 represses Kras and a pro-oncogenic phenotype in cancer cells.

Igf2bp1 is an oncofetal RNA binding protein whose expression in numerous types of cancers is associated with upregulation of key pro-oncogenic RNAs, poor prognosis, and reduced survival. Importantly, Igf2bp1 synergizes with mutations in Kras to enhance signalling and oncogenic activity, suggesting that molecules inhibiting Igf2bp1 could have therapeutic potential. Here, we isolate a small molecule that interacts with a hydrophobic surface at the boundary of Igf2bp1 KH3 and KH4 domains, and inhibits binding to Kras RNA. In cells, the compound reduces the level of Kras and other Igf2bp1 mRNA targets, lowers Kras protein, and inhibits downstream signalling, wound healing, and growth in soft agar, all in the absence of any toxicity. This work presents an avenue for improving the prognosis of Igf2bp1-expressing tumours in lung, and potentially other, cancer(s).

Site-Specific Labeling of Endogenous Proteins Using CoLDR Chemistry.

Chemical modifications of native proteins can affect their stability, activity, interactions, localization, and more. However, there are few nongenetic methods for the installation of chemical modifications at a specific protein site in cells. Here we report a covalent ligand directed release (CoLDR) site-specific labeling strategy, which enables the installation of a variety of functional tags on a target protein while releasing the directing ligand. Using this approach, we were able to label various proteins such as BTK, K-RasG12C, and SARS-CoV-2 PLpro with different tags. For BTK we have shown selective labeling in cells of both alkyne and fluorophores tags. Protein labeling by traditional affinity methods often inhibits protein activity since the directing ligand permanently occupies the target binding pocket. We have shown that using CoLDR chemistry, modification of BTK by these probes in cells preserves its activity. We demonstrated several applications for this approach including determining the half-life of BTK in its native environment with minimal perturbation, as well as quantification of BTK degradation by a noncovalent proteolysis targeting chimera (PROTAC) by in-gel fluorescence. Using an environment-sensitive "turn-on" fluorescent probe, we were able to monitor ligand binding to the active site of BTK. Finally, we have demonstrated efficient CoLDR-based BTK PROTACs (DC50 < 100 nM), which installed a CRBN binder onto BTK. This approach joins very few available labeling strategies that maintain the target protein activity and thus makes an important addition to the toolbox of chemical biology.

An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 Mpro inhibitor.

Designing covalent inhibitors is increasingly important, although it remains challenging. Here, we present covalentizer, a computational pipeline for identifying irreversible inhibitors based on structures of targets with non-covalent binders. Through covalent docking of tailored focused libraries, we identify candidates that can bind covalently to a nearby cysteine while preserving the interactions of the original molecule. We found âˆ¼11,000 cysteines proximal to a ligand across 8,386 complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In a prospective evaluation, five out of nine predicted covalent kinase inhibitors showed half-maximal inhibitory concentration (IC50) values between 155 nM and 4.5 µM. Application against an existing SARS-CoV Mpro reversible inhibitor led to an acrylamide inhibitor series with low micromolar IC50 values against SARS-CoV-2 Mpro. The docking was validated by 12 co-crystal structures. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.

Non-quaternary oximes detoxify nerve agents and reactivate nerve agent-inhibited human butyrylcholinesterase.

Government-sanctioned use of nerve agents (NA) has escalated dramatically in recent years. Oxime reactivators of organophosphate (OP)-inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) serve as antidotes toward poisoning by OPNAs. The oximes used as therapeutics are quaternary compounds that cannot penetrate the blood-brain barrier (BBB). There remains an urgent need for the development of next generation OPNA therapeutics. We have developed two high-throughput screening (HTS) assays using a fluorogenic NA surrogate, O-ethyl methylphosphonyl O-4-methyl-3-cyano-coumarin (EMP-MeCyC). EMP-MeCyC detoxification and EMP-BChE reactivation screening campaigns of ~155,000 small molecules resulted in the identification of 33 nucleophile candidates, including non-quaternary oximes. Four of the oximes were reactivators of both Sarin- and VX-inhibited BChE and directly detoxified Sarin. One oxime also detoxified VX. The novel reactivators included a non-quaternary pyridine amidoxime, benzamidoxime, benzaldoxime and a piperidyl-ketoxime. The VX-inhibited BChE reactivation reaction rates by these novel molecules were similar to those observed with known bis-quaternary reactivators and faster than mono-quaternary pyridinium oximes. Notably, we discovered the first ketoxime reactivator of OP-ChEs and detoxifier of OPNAs. Preliminary toxicological studies demonstrated that the newly discovered non-quaternary oximes were relatively non-toxic in mice. The discovery of unique non-quaternary oximes opens the door to the design of novel therapeutics and decontamination agents following OPNA exposure.

Efficient maternal to neonatal transfer of antibodies against SARS-CoV-2 and BNT162b2 mRNA COVID-19 vaccine.

BACKGROUNDThe significant risks posed to mothers and fetuses by COVID-19 in pregnancy have sparked a worldwide debate surrounding the pros and cons of antenatal SARS-CoV-2 inoculation, as we lack sufficient evidence regarding vaccine effectiveness in pregnant women and their offspring. We aimed to provide substantial evidence for the effect of the BNT162b2 mRNA vaccine versus native infection on maternal humoral, as well as transplacentally acquired fetal immune response, potentially providing newborn protection.METHODSA multicenter study where parturients presenting for delivery were recruited at 8 medical centers across Israel and assigned to 3 study groups: vaccinated (n = 86); PCR-confirmed SARS-CoV-2 infected during pregnancy (n = 65), and unvaccinated noninfected controls (n = 62). Maternal and fetal blood samples were collected from parturients prior to delivery and from the umbilical cord following delivery, respectively. Sera IgG and IgM titers were measured using the Milliplex MAP SARS-CoV-2 Antigen Panel (for S1, S2, RBD, and N).RESULTSThe BNT162b2 mRNA vaccine elicits strong maternal humoral IgG response (anti-S and RBD) that crosses the placenta barrier and approaches maternal titers in the fetus within 15 days following the first dose. Maternal to neonatal anti-COVID-19 antibodies ratio did not differ when comparing sensitization (vaccine vs. infection). IgG transfer ratio at birth was significantly lower for third-trimester as compared with second trimester infection. Lastly, fetal IgM response was detected in 5 neonates, all in the infected group.CONCLUSIONAntenatal BNT162b2 mRNA vaccination induces a robust maternal humoral response that effectively transfers to the fetus, supporting the role of vaccination during pregnancy.FUNDINGIsrael Science Foundation and the Weizmann Institute Fondazione Henry Krenter.

Tunable Methacrylamides for Covalent Ligand Directed Release Chemistry.

Targeted covalent inhibitors are an important class of drugs and chemical probes. However, relatively few electrophiles meet the criteria for successful covalent inhibitor design. Here we describe α-substituted methacrylamides as a new class of electrophiles suitable for targeted covalent inhibitors. While typically α-substitutions inactivate acrylamides, we show that hetero α-substituted methacrylamides have higher thiol reactivity and undergo a conjugated addition-elimination reaction ultimately releasing the substituent. Their reactivity toward thiols is tunable and correlates with the pKa/pKb of the leaving group. In the context of the BTK inhibitor ibrutinib, these electrophiles showed lower intrinsic thiol reactivity than the unsubstituted ibrutinib acrylamide. This translated to comparable potency in protein labeling, in vitro kinase assays, and functional cellular assays, with improved selectivity. The conjugate addition-elimination reaction upon covalent binding to their target cysteine allows functionalizing α-substituted methacrylamides as turn-on probes. To demonstrate this, we prepared covalent ligand directed release (CoLDR) turn-on fluorescent probes for BTK, EGFR, and K-RasG12C. We further demonstrate a BTK CoLDR chemiluminescent probe that enabled a high-throughput screen for BTK inhibitors. Altogether we show that α-substituted methacrylamides represent a new and versatile addition to the toolbox of targeted covalent inhibitor design.