ACS Chemical Biology最新文献

MYC is a master regulatory transcription factor whose sustained dysregulation promotes the initiation and maintenance of numerous cancers. While MYC is a regarded as a potenial therapeutic target in cancer, its intrinsically disordered structure has proven to be a formidable barrier toward the development of highly effective small molecule inhibitors. We rationalized that proteolysis targeting chimeras (PROTACs), which might accomplish the targeted degradation of MYC, would achieve more potent cell killing in MYC-driven cancer cells than reversible inhibitors. PROTACs are bifunctional small molecules designed to produce a ternary complex between a target protein and an E3 ligase leading the target’s ubiquitination and degradation by the 26S proteasome. We generated PROTAC MTP3 based on modifications of the previously reported MYC-targeting compound KJ-Pyr-9. We found that MTP3 depletes endogenous full-length MYC proteins and uniquely induces increasing levels of a functional, N-terminally truncated MYC species, tMYC. Furthermore, MTP3 perturbs cellular MYC levels in favor of a tMYC-dominated state whose gene regulatory landscape is not significantly altered compared to that of wild type MYC. Moreover, although it lacks ∼10 kDa of MYC’s N-terminal transactivation domain, tMYC is sufficient to maintain an oncogenic proliferative state. Our results highlight the complexities of proximity-inducing compounds against highly regulated and conformationally dynamic protein targets such as MYC and indicate that PROTACs can induce alternative outcomes beyond target protein degradation.

Epoxyeicosatrienoic acids, or EETs, are signaling molecules formed by the metabolism of arachidonic acid by cytochrome P450 enzymes. They are well-known for their anti-inflammatory effects, their ability to lower blood pressure, and benefits to cardiovascular outcomes. Despite the wealth of data demonstrating their physiological benefits, the putative high-affinity receptor that mediates these effects is yet to be identified. The recent report that the sphingosine-1-phosphate receptor 1 (S1PR1) is a high-affinity receptor for a related epoxy lipid prompted us to ask, "Why has the putative EET receptor not been discovered yet? What information about the discoveries of lipid epoxide receptors can help us identify the putative EET receptor?" In this review, we summarize the evidence supporting that the putative EET receptor exists. We then review the data showing EETs binding to other, low-affinity receptors and the discovery of receptors for similar lipid metabolites that can serve as a model for identifying the putative EET receptor. We hope this review will revitalize the search for this important receptor, which can facilitate the development of anti-inflammatory and cardiovascular therapeutics.

Epoxyeicosatrienoic acids, or EETs, are signaling molecules formed by the metabolism of arachidonic acid by cytochrome P450 enzymes. They are well-known for their anti-inflammatory effects, their ability to lower blood pressure, and benefits to cardiovascular outcomes. Despite the wealth of data demonstrating their physiological benefits, the putative high-affinity receptor that mediates these effects is yet to be identified. The recent report that the sphingosine-1-phosphate receptor 1 (S1PR1) is a high-affinity receptor for a related epoxy lipid prompted us to ask, “Why has the putative EET receptor not been discovered yet? What information about the discoveries of lipid epoxide receptors can help us identify the putative EET receptor?” In this review, we summarize the evidence supporting that the putative EET receptor exists. We then review the data showing EETs binding to other, low-affinity receptors and the discovery of receptors for similar lipid metabolites that can serve as a model for identifying the putative EET receptor. We hope this review will revitalize the search for this important receptor, which can facilitate the development of anti-inflammatory and cardiovascular therapeutics.

Halogenated natural products from marine sources often demonstrate potent activity against microorganisms and cancer cell lines. During the last three decades, the lissoclimide class of chlorinated labdane diterpenoids has been characterized with respect to structure and cytotoxic activity. Recently, our laboratories have developed different strategies to produce a broad range of naturally occurring lissoclimides and designed synthetic analogues. This work led to the discovery of a novel halogen−π dispersion interaction between the C2 chloride of chlorolissoclimide and guanine residues in the tRNA exit (E) site of the ribosome. In this study, we aimed to synthesize lissoclimide analogues bearing different substituents in place of the chloride to investigate the importance of the halogen identity for binding, translation inhibition, and cytotoxicity. With previous access to the protio and chloro compounds (haterumaimide Q and chlorolissoclimide), we synthesized two more halogenated variants, fluorolissoclimide and bromolissoclimide, as well as a methylated analogue, methyllissoclimide, to complete a panel of chemical probes for functional and structural studies. Using an integrative approach, we explored the effects of these analogues on the eukaryotic translational machinery in vivo and in vitro. X-ray cocrystal structures with the eukaryotic ribosome were solved for each probe molecule, and the effects on ribosomal thermal stability and FRET-derived ribosome binding constants were determined. Together, these data provide a detailed understanding of the different modes of binding of lissoclimides and insight into their relative activities, which vary according to the substitutions that interact with the eukaryote-specific ribosomal protein eL42. Ultimately, we learned that the presence of a lissoclimide C2-halogen atom─offering a potentially stabilizing halogen−π interaction─appears to facilitate or to synergize with a hydrogen-bonding interaction between the C7-hydroxyl group and the backbone of the ribosomal protein eL42, leading to stronger translation inhibition. We therefore conclude that the C2-halogen and C7-hydroxyl groups are critical contributors to potency, and this idea is borne out in the observations of reduced biological activities in the absence of either group.

Halogenated natural products from marine sources often demonstrate potent activity against microorganisms and cancer cell lines. During the last three decades, the lissoclimide class of chlorinated labdane diterpenoids has been characterized with respect to structure and cytotoxic activity. Recently, our laboratories have developed different strategies to produce a broad range of naturally occurring lissoclimides and designed synthetic analogues. This work led to the discovery of a novel halogen-π dispersion interaction between the C2 chloride of chlorolissoclimide and guanine residues in the tRNA exit (E) site of the ribosome. In this study, we aimed to synthesize lissoclimide analogues bearing different substituents in place of the chloride to investigate the importance of the halogen identity for binding, translation inhibition, and cytotoxicity. With previous access to the protio and chloro compounds (haterumaimide Q and chlorolissoclimide), we synthesized two more halogenated variants, fluorolissoclimide and bromolissoclimide, as well as a methylated analogue, methyllissoclimide, to complete a panel of chemical probes for functional and structural studies. Using an integrative approach, we explored the effects of these analogues on the eukaryotic translational machinery in vivo and in vitro. X-ray cocrystal structures with the eukaryotic ribosome were solved for each probe molecule, and the effects on ribosomal thermal stability and FRET-derived ribosome binding constants were determined. Together, these data provide a detailed understanding of the different modes of binding of lissoclimides and insight into their relative activities, which vary according to the substitutions that interact with the eukaryote-specific ribosomal protein eL42. Ultimately, we learned that the presence of a lissoclimide C2-halogen atom─offering a potentially stabilizing halogen-π interaction─appears to facilitate or to synergize with a hydrogen-bonding interaction between the C7-hydroxyl group and the backbone of the ribosomal protein eL42, leading to stronger translation inhibition. We therefore conclude that the C2-halogen and C7-hydroxyl groups are critical contributors to potency, and this idea is borne out in the observations of reduced biological activities in the absence of either group.

Spirochetes are especially invasive bacteria that are responsible for several human diseases, including Lyme disease, periodontal disease, syphilis, and leptospirosis. Spirochetes rely on an unusual form of motility based on periplasmic flagella (PFs) to infect hosts and evade the immune system. The flexible hook of these PFs contains a post-translational modification in the form of a lysinoalanine (Lal) cross-link between adjacent subunits of FlgE, which primarily comprise the hook. Lal cross-linking has since been found in key species across the phylum and involves residues that are highly conserved. The requirement of the Lal cross-link for motility of the pathogens Treponema denticola (Td) and Borreliella burgdorferi (Bb) establish Lal as a potential therapeutic target for the development of antimicrobials. Herein, we present the design, development, and application of a NanoLuc-based high-throughput screen that was used to successfully identify two structurally related Lal cross-link inhibitors (hexachlorophene and triclosan) from a library of clinically approved small molecules. A structure-activity relationship study further expanded the inhibitor set to a third compound (dichlorophene), and each inhibitor was demonstrated to biochemically block autocatalytic cross-linking of FlgE from several pathogenic spirochetes with varied mechanisms and degrees of specificity. The most potent inhibitor, hexachlorophene, alters Lal cross-linking in cultured cells of Td and reduces bacterial motility in swimming plate assays. Overall, these results provide a proof-of-concept for the discovery and development of Lal-cross-link inhibitors to combat spirochete-derived illnesses.

Ferritin degradation pathways, particularly NCOA4-mediated ferritinophagy, are crucial for maintaining iron homeostasis. Here, we demonstrate the coexistence of two NCOA4 isoforms, one iron-sulfur cluster-free and one iron-sulfur cluster-bound, in oxygenated cell cultures. Using a combination of spectroscopic and analytical techniques, in vitro characterization of the NCOA4 fragment (383-522), denoted NCOA4-D, revealed a predominance of monomeric species with a relatively stable [2Fe-2S] cluster under normoxic conditions. The results demonstrate distinct interactions between NCOA4-D isoforms and ferritin, underscoring the influence of cellular oxygen and iron concentrations on NCOA4's regulatory functions, pathways, and ferritin's fate. Our findings suggest that different NCOA4-initiated degradation pathways may concurrently occur in cells and highlight the necessity of further exploring the role of the Fe-S cluster in NCOA4 as an iron-sensing mechanism for maintaining cellular iron homeostasis.

Iron is essential for bacterial growth, and Pseudomonas aeruginosa synthesizes the siderophores pyochelin (PCH) and pyoverdine to acquire it. PCH contains a thiazolidine ring that aids in iron chelation but is prone to hydrolysis, leading to the formation of 2-(2-hydroxylphenyl)-thiazole-4-carbaldehyde (IQS). Using mass spectrometry, we demonstrated that PCH undergoes hydrolysis and oxidation in solution, resulting in the formation of aeruginoic acid (AA). This study used proteomic analyses and fluorescent reporters to show that AA, dihydroaeruginoic acid (DHA), and PCH induce the expression of femA, a gene encoding the ferri-mycobactin outer membrane transporter in P. aeruginosa. Notably, the induction by AA and DHA was observed only in strains unable to produce pyoverdine, suggesting their weaker iron-chelating ability compared to that of pyoverdine. ⁵⁵Fe uptake assays demonstrated that both AA-Fe and DHA-Fe complexes are transported via FemA; however, no uptake was observed for PCH-Fe through this transporter. Structural studies revealed that FemA is able to bind AA₂-Fe or DHA₂-Fe complexes. Key interactions are conserved between FemA and these two complexes, with specificity primarily driven by one of the two siderophore molecules. Interestingly, although no iron uptake was noted for PCH through FemA, the transporter also binds PCH-Fe in a similar manner. These findings show that under moderate iron deficiency, when only PCH is produced by P. aeruginosa, degradation products AA and DHA enhance iron uptake by inducing femA expression and facilitating iron transport through FemA. This provides new insights into the pathogen's strategies for iron homeostasis.

Optogenetics and chemogenetics are relatively new biomedical technologies that emerged 20 years ago and have been evolving rapidly since then. This has been made possible by the combined use of genetic engineering, optics, and electrophysiology. With the development of optogenetics and thermogenetics, the molecular tools for cellular control are continuously being optimized, studied, and modified, expanding both their applications and their biomedical uses. The most notable changes have occurred in the basic life sciences, especially in neurobiology and the activation of neurons to control behavior. Currently, these methods of activation have gone far beyond neurobiology and are being used in cardiovascular research, for potential cancer therapy, to control metabolism, etc. In this review, we provide brief information on the types of molecular tools for optogenetic and thermogenetic methods─microbial rhodopsins and proteins of the TRP superfamily─and also consider their applications in the field of activation of non-neuronal tissues and mammalian cells. We also consider the potential of these technologies and the prospects for the use of optogenetics and thermogenetics in biomedical research.

Since the middle of the 20th century, long-distance avian migration has been known to rely partly on geomagnetic field. However, the underlying sensory mechanism is still not fully understood. Cryptochrome-4a (ErCry4a), found in European robin (Erithacus rubecula), a night-migratory songbird, has been suggested to be a magnetic sensory molecule. It is sensitive to external magnetic fields via the so-called radical-pair mechanism. ErCry4a is primarily located in the outer segments of the double-cone photoreceptor cells in the eye, which contain stacked and highly ordered membranes that could facilitate the anisotropic attachment of ErCry4a needed for magnetic compass sensing. Here, we investigate possible interactions of ErCry4a with a model membrane that mimics the lipid composition of outer segments of vertebrate photoreceptor cells using experimental and computational approaches. Experimental results show that the attachment of ErCry4a to the membrane could be controlled by the physical state of lipid molecules (average area per lipid) in the outer leaflet of the lipid bilayer. Furthermore, polarization modulation infrared reflection absorption spectroscopy allowed us to determine the conformation, motional freedom, and average orientation of the α-helices in ErCry4a in a membrane-associated state. Atomistic molecular dynamics studies supported the experimental results. A ∼ 1000 kcal mol^-1 decrease in the interaction energy as a result of ErCry4a membrane binding was determined compared to cases where no protein binding to the membrane occurred. At the molecular level, the binding seems to involve negatively charged carboxylate groups of the phosphoserine lipids and the C-terminal residues of ErCry4a. Our study reveals a potential direct interaction of ErCry4a with the lipid membrane and discusses how this binding could be an essential step for ErCry4a to propagate a magnetic signal further and thus fulfill a role as a magnetoreceptor.