Many disease pathologies can be understood through the elucidation of localized biomolecular networks, or microenvironments. To this end, enzymatic proximity labeling platforms are broadly applied ...for mapping the wider spatial relationships in subcellular architectures. However, technologies that can map microenvironments with higher precision have long been sought. Here, we describe a microenvironment-mapping platform that exploits photocatalytic carbene generation to selectively identify protein-protein interactions on cell membranes, an approach we term MicroMap (μMap). By using a photocatalyst-antibody conjugate to spatially localize carbene generation, we demonstrate selective labeling of antibody binding targets and their microenvironment protein neighbors. This technique identified the constituent proteins of the programmed-death ligand 1 (PD-L1) microenvironment in live lymphocytes and selectively labeled within an immunosynaptic junction.
Over half of new therapeutic approaches fail in clinical trials due to a lack of target validation. As such, the development of new methods to improve and accelerate the identification of cellular ...targets, broadly known as target ID, remains a fundamental goal in drug discovery. While advances in sequencing and mass spectrometry technologies have revolutionized drug target ID in recent decades, the corresponding chemical-based approaches have not changed in over 50 y. Consigned to outdated stoichiometric activation modes, modern target ID campaigns are regularly confounded by poor signal-to-noise resulting from limited receptor occupancy and low crosslinking yields, especially when targeting low abundance membrane proteins or multiple protein target engagement. Here, we describe a broadly general platform for photocatalytic small molecule target ID, which is founded upon the catalytic amplification of target-tag crosslinking through the continuous generation of high-energy carbene intermediates via visible light-mediated Dexter energy transfer. By decoupling the reactive warhead tag from the small molecule ligand, catalytic signal amplification results in unprecedented levels of target enrichment, enabling the quantitative target and off target ID of several drugs including (+)-JQ1, paclitaxel (Taxol), dasatinib (Sprycel), as well as two G-protein-coupled receptors-ADORA2A and GPR40.
The characterization of ligand binding modes is a crucial step in the drug discovery process and is especially important in campaigns arising from phenotypic screening, where the protein target and ...binding mode are unknown at the outset. Elucidation of target binding regions is typically achieved by X-ray crystallography or photoaffinity labeling (PAL) approaches; yet, these methods present significant challenges. X-ray crystallography is a mainstay technique that has revolutionized drug discovery, but in many cases structural characterization is challenging or impossible. PAL has also enabled binding site mapping with peptide- and amino-acid-level resolution; however, the stoichiometric activation mode can lead to poor signal and coverage of the resident binding pocket. Additionally, each PAL probe can have its own fragmentation pattern, complicating the analysis by mass spectrometry. Here, we establish a robust and general photocatalytic approach toward the mapping of protein binding sites, which we define as identification of residues proximal to the ligand binding pocket. By utilizing a catalytic mode of activation, we obtain sets of labeled amino acids in the proximity of the target protein binding site. We use this methodology to map, in vitro, the binding sites of six protein targets, including several kinases and molecular glue targets, and furthermore to investigate the binding site of the STAT3 inhibitor MM-206, a ligand with no known crystal structure. Finally, we demonstrate the successful mapping of drug binding sites in live cells. These results establish μMap as a powerful method for the generation of amino-acid- and peptide-level target engagement data.
State-of-the-art methods in photoproximity labeling center on the targeted generation and capture of short-lived reactive intermediates to provide a snapshot of local protein environments. Diazirines ...are the current gold standard for high-resolution proximity labeling, generating short-lived aryl(trifluoromethyl) carbenes. Here, we present a method to access aryl(trifluoromethyl) carbenes from a stable diazo source via tissue-penetrable, deep red to near-infrared light (600–800 nm). The operative mechanism of this activation involves Dexter energy transfer from photoexcited osmium(II) photocatalysts to the diazo, thus revealing an aryl(trifluoromethyl) carbene. The labeling preferences of the diazo probe with amino acids are studied, showing high reactivity toward heteroatom−H bonds. Upon the synthesis of a biotinylated diazo probe, labeling studies are conducted on native proteins as well as proteins conjugated to the Os photocatalyst. Finally, we demonstrate that the conjugation of a protein inhibitor to the photocatalyst also enables selective protein labeling in the presence of spectator proteins and achieves specific labeling of a membrane protein on the surface of mammalian cells via a two-antibody photocatalytic system.
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We previously reported medicinal chemistry efforts that identified MK-5204, an orally efficacious β-1,3-glucan synthesis inhibitor derived from the natural product enfumafungin. ...Further extensive optimization of the C2 triazole substituent identified 4-pyridyl as the preferred replacement for the carboxamide of MK-5204, leading to improvements in antifungal activity in the presence of serum, and increased oral exposure. Reoptimizing the aminoether at C3 in the presence of this newly discovered C2 substituent, confirmed that the (R) t-butyl, methyl aminoether of MK-5204 provided the best balance of these two key parameters, culminating in the discovery of ibrexafungerp, which is currently in phase III clinical trials. Ibrexafungerp displayed significantly improved oral efficacy in murine infection models, making it a superior candidate for clinical development as an oral treatment for Candida and Aspergillus infections.
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Our previously reported efforts to produce an orally active β-1,3-glucan synthesis inhibitor through the semi-synthetic modification of enfumafungin focused on replacing the C2 ...acetoxy moiety with an aminotetrazole and the C3 glycoside with a N,N-dimethylaminoether moiety. This work details further optimization of the C2 heterocyclic substituent, which identified 3-carboxamide-1,2,4-triazole as a replacement for the aminotetrazole with comparable antifungal activity. Alkylation of either the carboxamidetriazole at C2 or the aminoether at C3 failed to significantly improve oral efficacy. However, replacement of the isopropyl alpha amino substituent with a t-butyl, improved oral exposure while maintaining antifungal activity. These two structural modifications produced MK-5204, which demonstrated broad spectrum activity against Candida species and robust oral efficacy in a murine model of disseminated Candidiasis without the N-dealkylation liability observed for the previous lead.
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Two high-throughput screening hits were investigated for SAR against human factor IXa. Both hits feature a benzamide linked to a 6-5-heteroaryl via an alkyl amine. In the case where ...this system is a benzimidazolyl-ethyl amine the binding potency for the hit was improved >500-fold, from 9μM to 0.016μM. For the other hit, which contains a tetrahydropyrido-indazole amine, potency was improved 20-fold, from 2μM to 0.09μM. X-ray crystal structures were obtained for an example of each class which improved understanding of the binding, and will enable further drug discovery efforts.
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Using structure based drug design (SBDD), a novel class of potent coagulation Factor IXa (FIXa) inhibitors was designed and synthesized. High selectivity over FXa inhibition was ...achieved. Selected compounds demonstrated oral bioavailability in rat IV/PO pharmacokinetic (PK) studies. Finally, the pharmacodynamics (PD) of this class of molecules was evaluated in Thrombin Generation Assay (TGA) in Corn Trypsin Inhibitor (CTI) citrated human plasma and demonstrated characteristics of a FIXa inhibitor.
Tetrahydrofluorenones which possess a C9a-fluoroalkyl substituent were efficiently converted to tetrahydrofluorenones which contain a ring bridging C9a−C2. Conditions include a stepwise sequence of ...conversion to an alkyl bromide followed by treatment with base, and a direct cyclization by treatment with lithium chloride in DMF heated to 150 °C.
Several tetrahydrofluorenones with a triazole fused across C7–C8 showed high levels of ERβ-selectivity and were found to be potent ERβ-agonists. As a class they demonstrate improved oral ...bioavailability in the rat over a parent class of 7-hydroxy-tetrahydrofluorenones. The most selective agonist displayed 5.7
nM affinity and 333-fold selectivity for ERβ.
Several tetrahydrofluorenones with a triazole fused across C7–C8 showed high levels of ERβ-selectivity and were found to be potent ERβ-agonists. As a class they demonstrate improved oral bioavailability in the rat over a parent class of 7-hydroxy-tetrahydrofluorenones. The most selective agonist displayed 5.7
nM affinity and 333-fold selectivity for ERβ.
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