Molecular Pharmacology最新文献

Autophagy is a cellular process responsible for the recycling of misfolded proteins and damaged organelles, contributing to cellular homeostasis and energy production. Tumor cells often exploit this mechanism, particularly through the form of autophagy that is cytoprotective, to survive endogenous and exogenous stress and resist chemotherapeutic agents as well as radiation therapy. Although several autophagy inhibitors have been developed to block the protective form of autophagy, their clinical application is often limited due to a lack of selectivity and significant side effects. In addition to the cytoprotective form, cytotoxic, cytostatic, and nonprotective functions of autophagy have been identified. In this review, we summarize a series of publications, largely from our own laboratory, exploring how various antineoplastic agents trigger different forms of autophagy and assess whether autophagy inhibition or modulation could serve as an effective adjuvant approach to enhance therapeutic responses. Furthermore, we discuss recent advancements in the autophagy field and the potential for improving cancer therapeutic strategies. SIGNIFICANCE STATEMENT: This work provides an overview of our previous work investigating the different forms of autophagy induced by various antineoplastic modalities across different tumor models. The purpose of this effort is to draw tentative conclusions regarding the potential of targeting autophagy as a strategy to enhance the efficacy of these therapeutic agents. Additionally, we offer insights into recent advances in the autophagy field.

The development of isoform-selective histone deacetylase (HDAC) inhibitors offers a promising approach to minimize the adverse effects of nonselective HDAC inhibitors. HDAC6, due to its unique structural and functional properties, regulates critical cellular processes like gene expression, proliferation, senescence, and apoptosis. Inspired by a tryptoline-derived natural product, callophycin A, a series of compounds were synthesized and evaluated for HDAC6 selectivity. In the HDAC enzyme assay, compound 6a stood out as the lead, demonstrating 21-fold higher potency against HDAC6 compared with HDAC1 (HDAC6 IC₅₀ = 83.6 ± 1.1 nM vs HDAC1 IC₅₀ = 1790 ± 1.0 nM). In dose-dependent western blot experiments using H1975 lung cancer cells, a lower concentration of compound 6a (3 μM) induced significantly greater acetylation of α-tubulin compared with histone H3, indicating preferential inhibition of cytoplasmic HDAC6 over the nuclear HDAC1 isoform. Molecular docking of compound 6a at the HDAC6 active site (PDB code: 5EDU) revealed key interactions including π-alkyl contacts via the cap group, π-π stacking through the linker, hydrogen bonding involving the zinc-binding group, and Zn²⁺ chelation by the hydroxamic acid moiety that support its strong and selective binding, consistent with its HDAC6 inhibitory profile. Overall, compound 6a represents a promising prototype for the rational design of selective HDAC6 inhibitors, offering a structural framework for developing safer and more effective therapeutics aimed at HDAC6-driven cancers, thereby advancing targeted drug development in oncology. SIGNIFICANCE STATEMENT: Selective histone deacetylase 6 (HDAC6) inhibitors provide a safer alternative to nonselective HDAC inhibitors, with potential applications in cancer. This study identifies compound 6a as a promising lead with remarkable HDAC6 specificity, offering a foundation for developing targeted and efficient therapeutics.

The ventrolateral periaqueductal gray (vlPAG) plays a critical role in pain modulation. GABAergic neurotransmission within the vlPAG regulates the descending pain pathway. This study investigates the mechanisms through which corticosterone (CORT) modulates GABA release in the vlPAG via putative membrane-associated glucocorticoid receptors (mbGRs). Superfusion of CORT decreases evoked inhibitory postsynaptic currents in a mbGR- and CB1 cannabinoid receptor (CB1R)-dependent manner. Using a depolarization-induced suppression of inhibition protocol to test the effects of CORT on the endocannabinoid system, we find that CORT-mediated signaling enhances 2-arachidonoylglycerol synthesis that is inhibited by the diacylglycerol lipase inhibitor, DO34. CORT prolongs CB1R activation through a Gα_s and protein kinase A-dependent pathway, whereas early depolarization-induced suppression of inhibition-initiated endocannabinoid activation of CB1Rs is independent of protein kinase A. These results highlight the critical role of CORT in the vlPAG in engaging endocannabinoid pathways to inhibit GABA release. The results indicate that CORT activation of putative mbGRs promote activation of the descending pain modulatory pathway through CB1R-mediated inhibition of GABA release in the vlPAG. SIGNIFICANCE STATEMENT: This study provides evidence that corticosterone activates putative membrane glucocorticoid receptors to increase levels of 2-arachidonoylglycerol to activate presynaptic CB1 cannabinoid receptors. These findings reveal mechanisms by which stress modulates the ventrolateral periaqueductal gray and the descending pain circuit.

IT1t and clobenprobit (CB) have been shown to act as anti-inflammatory compounds in dependence of the chemokine receptor C-X-C receptor type 4 (CXCR4) in model systems. Here, the direct interaction between CB and CXCR4 is demonstrated via in silico modeling and bioluminescence resonance energy transfer binding assays at wild-type and mutant versions of CXCR4. The binding site is compared with those of IT1t and AMD3100, two well known CXCR4 ligands. In contrast to AMD3100, IT1t also displays an anti-inflammatory signaling effect. Ligands observed to have this anti-inflammatory effect seem to bind into the minor pocket of CXCR4 impacting the binding of the endogenous ligand CXCL12 only at high concentrations. Based on this observation further compounds thought to bind the minor pocket of CXCR4 were designed and screened for their anti-inflammatory potency. The best of these compounds, NP1411, was tested in its ability to inhibit CXCL12 mediated G protein activation as well as CXCL12 and CB binding. SIGNIFICANCE STATEMENT: This study presents a comprehensive investigation into the binding site of anti-inflammatory compounds at the C-X-C receptor type 4 receptor using in silico and in vitro ligand binding approaches. This opens the opportunity for the development of further therapeutic agents with higher potency and/or efficacy as presented in an initial test at the end of the publication.

Antibodies have played a pivotal role in G protein-coupled receptor (GPCR) research and drug development. Nanobodies, or variable domain heavy chain-only antibodies, have emerged as a next-generation antibody with unique advantages in targeting GPCRs. The first generation of intracellular nanobodies have been instrumental in stabilizing GPCR structures for crystallography and in enabling in vitro GPCR imaging. More recently, extracellular-targeted nanobodies have demonstrated diverse pharmacological profiles, with the ability to modulate GPCR activity, localization, and downstream signaling. With these newly uncovered functional properties, nanobodies can be viewed not only as structural tools but also as modulators of receptor pharmacology. We highlight recent innovations in extracellular GPCR-targeting nanobodies and assess several approaches to accelerate their development as versatile research tools and therapeutics. SIGNIFICANCE STATEMENT: Nanobodies have emerged as a next-generation antibody platform with distinct advantages for targeting G protein-coupled receptors. This review highlights recent advances in extracellular G protein-coupled receptor-targeting nanobodies and explores innovative strategies to accelerate their development as powerful research tools and therapeutic agents.

The retinoid X receptor (RXR) belongs to the nuclear receptor superfamily, which regulates various physiological processes. RXR agonists, classified as rexinoids, exhibit selectivity for RXR over the retinoic acid receptor and have therapeutic potential against cancer, metabolic disorders, and Alzheimer disease (AD). Here, we characterized the biological properties of 6-hydroxy-3'-propyl-[1,1'-biphenyl]-3-propanoic acid (6OHA), a compound synthesized in our laboratory based on the structure of magnaldehyde B, and found that it exhibited potent RXRα agonist activity comparable with that of the clinically used RXR agonist bexarotene, but lower agonist activity toward retinoic acid receptor α and RXRγ. RNA-sequencing-based transcriptome analysis of microglial cells revealed that 6OHA and Bex induced similar gene expression patterns; however, 6OHA was more associated strongly with chemotaxis and response to stimuli. Pharmacokinetic studies showed a higher C_max, faster T_max, and more rapid clearance in both the serum and brain for 6OHA than for Bex. Although 6OHA exhibited a higher area under the concentration-time curve from 0 to 6 hours in serum, its area under the concentration-time curve from 0 to 6 hours in the brain was lower than that of Bex. Together with gene expression data, these findings suggest that 6OHA is a more effective RXR agonist in peripheral tissues while maintaining comparable efficacy in the brain. Furthermore, unlike Bex, 6OHA did not increase serum triglycerides or decrease serum thyrotropin and free thyroxine levels, likely reflecting its distinct pharmacologic profile from that of Bex. Collectively, these results suggest that 6OHA is a promising RXR agonist with minimal adverse effects and potential application in treating cancer, AD, and metabolic disorders. SIGNIFICANCE STATEMENT: Despite the potential of retinoid X receptor (RXR) agonists, their application has been limited by adverse effects. The novel RXR agonist 6OHA exhibits potent RXRα agonist activity while showing lower activities for retinoid acid receptor α and RXRγ than the RXR agonist bexarotene. Moreover, 6OHA also shows favorable pharmacokinetics and a gene expression profile distinct from Bex. These properties may account for the minimal adverse effects of 6OHA and support its potential as a therapeutic RXR agonist.

Transient receptor potential vanilloid 2 (TRPV2) is relevant for diseases like cancer, cardiac dysfunction, and infection, warranting drug development targeting TRPV2. However, this has been complicated by the lack of good modulators targeting TRPV2 and questions about species selectivity, so more detailed molecular insights into channel function and pharmacology are required. Two recent studies identified distinct binding sites on rat (r) and mouse (m) TRPV2 for activation by 2-aminoethoxydiphenyl borate (2-APB). Here we aimed to determine whether the mechanisms for 2-APB sensitivity of TRPV2 indeed differ among these closely related orthologs. Patch clamp electrophysiology revealed that mTRPV2 and human TRPV2 display similar sensitivities to 2-APB when compared with a considerably higher sensitivity of rTRPV2. For both mTRPV2 and rTRPV2, we observed that the exchange of putative 2-APB binding residues within the vanilloid binding pocket alters overall channel sensitivity to 3 TRPV2 pharmacological activators that bind at different sites: 2-APB, cannabidiol, and probenecid. By contrast, the exchange of putative 2-APB binding residues at the S5 binding pocket in both channels resulted in strongly reduced 2-APB sensitivies without reducing sensitivity to cannabidiol and probenecid. rTRPV2 mutants lacking key residues of both binding sites were almost completely 2-APB insensitive. These functional data suggest that the mechanisms accounting for 2-APB sensitivity are similar across mammalian TRPV2 orthologs. Except for serving as a binding site for 2-APB, the vanilloid binding pocket plays a key role in the overall function of TRPV2. These findings are relevant for the emerging framework toward an improved understanding of TRPV2. SIGNIFICANCE STATEMENT: This study resolves the conflict regarding how 2-aminoethoxydiphenyl borate binds to transient receptor potential vanilloid 2 (TRPV2), showing a shared mechanism despite sensitivity differences. These findings enhance TRPV2 modulation insights and highlight species considerations in drug design, aiding the development of selective TRPV2-targeted therapies.

The cycle of GTP binding and hydrolysis controls heterotrimeric G proteins, and mutations reducing GTPase activity result in constitutive G protein signaling. In Gαq (gene: GNAQ) such mutations cause uveal melanoma and Sturge-Weber syndrome. Finding pharmacological agents that inhibit Gαq will be beneficial for research with therapeutic potential. Previously discovered bacterial depsipeptides (FR900359 and YM-254890) bind directly to Gαq and stabilize its inactive complex with GDP, but suffer from limitations of distribution and bioavailability. We used the established Gαq-YM-254890 complex structure to dock small-molecule drugs into the depsipeptide binding site of Gαq. Our in silico screen of 5000 Food Drug and Administration-approved, experimental, and withdrawn drugs predicted that thiazolidinediones are potential ligands of Gαq. Analysis of G protein coupled receptor-stimulated G protein GTPγS binding demonstrated that troglitazone (441 Da) inhibited Gq nucleotide exchange with the IC₅₀ of ∼31.7 μM. The thiazolidinedione analogs, rosiglitazone and pioglitazone, had no effect. High concentrations of troglitazone modestly inhibited Gi₁ and Gs, but not G13. In G protein thermal stability assays, troglitazone and FR900359 stabilized purified Gαq-GDP, indicating direct binding. Consistent with its negative effect on Gq signaling, in MIN6 mouse insulinoma cells, troglitazone inhibited Ca²⁺ mobilization, extracellular regulated protein kinase phosphorylation, and insulin secretion stimulated by the Gq-coupled M3 muscarinic cholinergic receptor. Troglitazone and FR900359 inhibited proliferation of MEL92.1 uveal melanoma cells driven by a GNAQ-Q209L driver mutation, but not of SK-MEL-28 cells driven by BRAF-V600E. Together, our study shows that troglitazone may be a promising new lead for the development of a <500 Da small-molecule therapeutic Gαq inhibitor. SIGNIFICANCE STATEMENT: Troglitazone, unlike other thiazolidinediones, directly binds and inhibits activity of heterotrimeric G protein Gq, with a weaker effect on Gi. Troglitazone may find usage as a repurposed drug scaffold to build novel small-molecule Gαq inhibitors with better bioavailability than depsipeptide Gαq inhibitors.

Malignant melanoma is the most aggressive and deadly skin cancer. Conventional treatment drugs, such as vemurafenib, are prone to resistance, resulting in very low patient survival. This study probed into the antitumor potential of coadministration of atraric acid (AA), a natural compound derived from lichens with multiple biological activities, and vemurafenib in melanoma. Our findings revealed that AA enhances vemurafenib's ability to reduce viability and induce apoptosis in B16-F10 melanoma cells. In vivo studies, including histological analysis, showed that the combination of AA and vemurafenib effectively inhibited melanoma growth and metastasis with minimal side effects. Inhibition of tumor growth by vemurafenib in the presence of AA increased from 20.11% to 23.93% (low dose AA) and 52.06% (high dose AA). Transcriptomic analysis, quantitative reverse transcription polymerase chain reaction, and western blot indicated that AA enhances the antimelanoma effect of vemurafenib was mediated through the modulation of hematopoietic progenitor kinase 1 (HGK), MAP kinase kinase 1 (MEK1), and extracellular signal-regulated kinase (ERK) expression. Molecular docking studies suggested that AA might reduce the expression of MEK1 and ERK by suppressing the phosphorylation of HGK, thereby enhancing vemurafenib inhibition of melanoma growth and metastasis. In conclusion, our study presents AA as a promising candidate that may help enhance the antimelanoma activity of vemurafenib, offering a new avenue for clinical cancer treatment. SIGNIFICANCE STATEMENT: The combination of atraric acid and vemurafenib effectively inhibited melanoma growth and metastasis with minimal side effects. The synergistic effect of atraric acid and vemurafenib is achieved by suppressing the phosphorylation of HGK to reduce the expression of MEK1 and ERK. Atraric acid is a promising candidate in combating chemoresistance in melanoma therapy.

The fourth Transatlantic Early-Career Investigator GPCR Symposium-held on September 5-6, 2024-was a virtual conference intended to provide an accessible, low-cost opportunity for early-stage researchers in the field of G protein-coupled receptor biology to share their work and engage with colleagues. This commentary discusses the symposium's structure and successes, as well as suggesting areas of improvement for future iterations. SIGNIFICANCE STATEMENT: The fourth Transatlantic Early-Career Investigator GPCR Symposium provided an accessible platform for early-career investigators to present their research and engage with peers and senior scientists. By leveraging a virtual format, the symposium addressed the financial and logistical barriers of traditional conferences, fostering scientific discourse and future collaboration. The event's structure facilitated networking opportunities and highlighted the importance of integrating diverse technologies in GPCR research. This symposium's success underscores the need for continued innovation in creating inclusive and impactful scientific forums.