Search Results (265)

MK-2048 is a potent second-generation HIV integrase inhibitor that has demonstrated acceptable safety and pharmacokinetics (PKs) in clinical trials of vaginal formulations. The substrate-type interactions between MK-2048 and the transporters/metabolizing enzymes that are highly expressed in the human female reproductive tract (FRT) were evaluated. The interactions between MK-2048 and P-gp/BCRP were investigated using a cellular bidirectional permeability assay, while those between MK-2048 and MRP4 were assessed using a vesicular uptake assay. Reaction phenotyping was performed to characterize the interactions between MK-2048 and CYP1A1 and CYP1B1. Using human cervicovaginal fluids (CVFs), MK-2048’s solubility was determined using a thermodynamic solubility method and its protein binding was determined using a rapid equilibrium dialysis method. Our study shows an efflux of MK-2048 in P-gp/BCRP-overexpressing MDCKII cells, which was reduced by a P-gp/BCRP inhibitor. Uptake of MK-2048 in MRP4/control vesicles was found to be ATP-independent. MK-2048 was metabolized by the CYP1A1 enzyme but not by CYP1B1. These data confirm that MK-2048 is a substrate of P-gp, BCRP, and CYP1A1, but is not a substrate of MRP4 or CYP1B1. MK-2048 displays low solubility and high protein binding in human CVF. This data suggests that MK-2048 may potentially interact with drugs that modulate the activity of P-gp, BCRP, and CYP1A1. Full article

Background/Objectives: Multidrug resistance (MDR) remains a major pathophysiological barrier to effective chemotherapy based on anthracyclines, including daunorubicin (DNR), in the treatment of leukemia. However, conventional population-level measurements of drug uptake do not resolve variability in uptake kinetics among individual leukemia cells, which may influence intracellular drug accumulation and therapeutic response. Methods: In this study, real-time DNR uptake was quantified at the single-cell level using a microfluidic biochip that enabled long-term cellular retention and continuous monitoring. Both wild-type drug-sensitive leukemia cells and a multidrug-resistant mutant overexpressing the P-glycoprotein (P-gp) efflux pump were examined. Results: Kinetic analysis revealed that DNR uptake in drug-sensitive cells was well described by a single dominant uptake process, whereas uptake in MDR cells required a model incorporating two kinetically distinct processes. In both cell populations, pronounced cell-to-cell variation was observed in uptake rates and intracellular drug retention, indicating substantial functional heterogeneity within phenotypically similar cells. This variability persisted following the treatment with an MDR inhibitor and obscured the differences between inhibitor-treated and untreated cells when the uptake was compared across different single cells. To overcome this limitation, a same-single-cell analysis (SASCA) approach was employed, enabling direct comparison of DNR uptake in the same individual cell before and after inhibitor exposure, thereby revealing enhanced intracellular DNR retention and accelerated uptake kinetics following inhibition. Conclusions: Together, these results demonstrate that real-time single-cell kinetic analysis reveals functionally relevant heterogeneity in multidrug-resistant leukemia cells and provides insight into the pathophysiology of MDR that cannot be obtained from population-averaged measurements. Full article

The 2025 approval of the selective NaV1.8 blocker suzetrigine for acute pain marked a pivotal advance in analgesic drug development. Yet the subsequent failure of Vertex’s next-generation NaV1.8 inhibitor VX993 to demonstrate clinical analgesia underscores enduring challenges in translating mechanistic promise into patient benefit. This review examines why promising targets and compounds, spanning NaV and TRP channels, often falter and outlines a path toward more reliable target selection and validation. I first summarize the pain pathway, from nociceptor transduction through spinal processing to cortical perception, emphasizing how inflammation and peripheral sensitization reshape excitability. Historically serendipitous, pain drug discovery now prioritizes molecular precision. Most approved chronic pain therapies act in the CNS and are limited by modest efficacy and adverse effects. Nociceptor-enriched targets (NaV1.7/1.8/1.9; TRP channels) remain attractive, yet redundancy among NaV subtypes and the necessity of blocking targets at the correct anatomical sites complicate translation. Human genetics and multi-omics provide a powerful, unbiased engine for target discovery. Rare high-impact variants offer strong causal hypotheses, while common polygenic contributions illuminate broader susceptibility. Large biobanks increasingly reveal a mismatch between legacy pain targets and genetically supported candidates across neuronal and non-neuronal cells. Human DRG transcriptomics highlight NaV channel redundancy. Human in vitro electrophysiology and PK/PD analyses show suzetrigine achieves ~90–95% NaV1.8 engagement, yet neurons can still fire unless additional channels are blocked. Species differences and drug distribution (including BBB/PNS penetration and P-gp efflux) critically influence efficacy; centrally accessible blockade (e.g., for NaV1.7 or TRPA1) may be necessary to achieve robust analgesia, challenging peripherally restricted strategies. Osteoarthritis illustrates how obesity-driven metabolic inflammation, synovial immune activation, subchondral bone remodeling, and specific nociceptor subtypes converge to drive mechanical pain. Multi-omic integration across diseased human tissues can pinpoint causal processes and cell types, enabling more selective and safer target choices. I propose a practical framework for target validation that integrates: (i) rigorous human genetic support; (ii) cell-type and site-of-action mapping; (iii) human-relevant electrophysiology and PK/PD with verified target engagement; (iv) species-appropriate models; (v) consideration of modality (small molecule, biologic, RNA, targeted protein degradation). Advancing genetically and anatomically aligned targets, tested at the right sites and exposures, offers the best path to genuinely effective, better-tolerated pain therapeutics. Full article

Chronic alcohol exposure disrupts blood–brain barrier (BBB) integrity and promotes neuroinflammation, with P2X7 receptor (P2X7R) signaling playing a critical role. Our prior work in male mice linked P2X7R inhibition to reduced extracellular adenosine triphosphate (eATP) release, modulated extracellular vesicle (EV) cargo, and attenuated neuroinflammation in chronic intermittent ethanol (CIE)-exposed mice. However, sex-specific roles of P2X7R signaling and EV-mediated mechanisms in alcohol-induced neuroinflammation remain unclear. Male and female mice were exposed to ethanol vapor for three weeks and treated with Brilliant Blue G (BBG), a P2X7R inhibitor. Compared to their respective CIE-unexposed controls, brain gene expression of tumor necrosis factor–α (Tnf-α), interleukin-1 beta (Il-1b), interleukin-6 (Il-6), monocyte chemoattractant protein-1 (Mcp-1), and Fas ligand (Fasl) significantly increased in CIE-exposed males, while only Il-1b increased in females. P2X7R inhibition significantly reduced these cytokines. Pericyte immunostaining was decreased by CIE (indicating BBB injury) in male mice only and was restored by P2X7R inhibition with no difference between groups in females. Occludin staining (another BBB marker) did not differ between the treatment groups in male and female animals. Circulating cytokines (Macrophage inflammatory protein-1 alpha (MIP-1α), tumor necrosis factor–α (TNF-α), interleukin-1 beta (IL-1β), and interleukin-27 subunit p28/interleukin-30 (IL-27p28/IL-30) were significantly elevated in CIE-exposed males but not in females, with BBG treatment reducing cytokines in males. Circulating eATP, P2X7Rs, P-glycoprotein (P-gp), EVs, and EV-mtDNA, which we identified in our previous study, were increased in both sexes and partially decreased by P2X7R blockade. Spatial memory was impaired by CIE exposure in males but not females, and this deficit was reversed by BBG treatment. Our findings reveal sex differences in CIE-induced circulating cytokines, neuroinflammation, and memory impairment, with a stronger response in males. However, other markers of cell injury associated with CIE exposure were upregulated in both sexes; P2X7R inhibition effectively mitigated these effects, highlighting the functional relevance of targeting the P2X7R in alcohol-induced injury. Full article

P-glycoprotein (P-gp) is an ATP-driven transporter that effluxes a wide range of xenobiotics from cells, and its overexpression is a primary cause of multidrug resistance (MDR) in cancer. It is well-established that P-gp functions through conformational changes, yet its large-scale structural dynamics at work have been unexplored. Here, we directly visualized single P-gp molecules reconstituted in nanodiscs using high-speed atomic force microscopy (HS-AFM). The HS-AFM movies revealed that P-gp is intrinsically dynamic in its apo state, with its nucleotide-binding domains (NBDs) undergoing large, spontaneous opening and closing motions. However, addition of ATP stabilized a conformation characterized by NBD proximity with a strong tendency toward closure. We then leveraged this dynamic viewpoint to elucidate the relationship between Elacridar’s function and the resulting structural dynamics of P-gp. Elacridar is designed to overcome multidrug resistance (MDR) in cancer and acts as a potent dual inhibitor of both P-gp and the Breast Cancer Resistance Protein (BCRP), effectively blocking the drug efflux function of these transporters. This inhibitor has suggested concentration-dependent function: it is effluxed as a substrate at low concentrations and acts as an inhibitor at high concentrations. Our direct observations revealed that low concentrations induced active dynamics in P-gp, whereas high concentrations severely restricted its motion, leading to a rigid, non-productive state. Our study provides critical insights into how observing molecular motion itself can unravel complex biological mechanisms. Full article

Background/Objectives: Chagas disease, caused by Trypanosoma cruzi, remains a major neglected tropical disease with limited therapeutic options restricted to benznidazole and nifurtimox, both associated with significant toxicity and reduced efficacy during chronic infection. Seeking novel, safe, and sustainable chemotherapeutic candidates, two new saturated cardanol-derived phospholipid analogs—LDT10 and LDT119—were rationally designed based on the molecular scaffold of miltefosine and biosourced from cashew nut shell liquid (CNSL). This study aimed to evaluate the pharmacokinetic properties of these compounds in silico and assess their antiparasitic activity, cytotoxicity, and morphological and ultrastructural effects on all developmental forms of T. cruzi in vitro. Materials and Methods: In silico ADMET predictions (SwissADME, pkCSM) were performed to determine bioavailability, pharmacokinetic behavior, CYP inhibition, mutagenicity, and hepatotoxicity. Antiproliferative activity was evaluated in epimastigotes, trypomastigotes, and intracellular amastigotes using dose–response assays and flow cytometry. Cytotoxicity was assessed in HEPG2 and HFF-1 cells using resazurin-based viability assays. Morphological and ultrastructural alterations were investigated through scanning (SEM) and transmission (TEM) electron microscopy. Reactive oxygen species (ROS) generation was quantified with H₂DCFDA after 4 h and 24 h of exposure. Results: In silico analyses indicated favorable drug-like profiles, high intestinal absorption (>89%), absence of mutagenicity or hepatotoxicity, and non-penetration of the blood–brain barrier. LDT10 was not a P-gp substrate, and LDT119 acted as a P-gp inhibitor, suggesting reduced efflux and higher intracellular retention. Both compounds inhibited epimastigote proliferation with low IC₅₀ values (LDT10: 0.81 µM; LDT119: 1.2 µM at 48 h) and reduced trypomastigote viability (LD₅₀ LDT10: 2.1 ± 2 µM; LDT119: 1.8 ± 0.8 µM). Intracellular amastigotes were highly susceptible (IC₅₀ LDT10: 0.48 µM; LDT119: 0.3 µM at 72 h), with >90% inhibition at higher concentrations. No cytotoxicity was observed in mammalian cells up to 20 µM. SEM revealed membrane wrinkling, pore-like depressions, rounded cell bodies, and multiple flagella, indicating cell division defects. TEM showed Golgi disorganization, autophagic vacuoles, mitochondrial vesiculation, and abnormal kinetoplast replication, while host cells remained structurally preserved. Both compounds induced significant ROS production in trypomastigotes after 24 h in a dose-dependent manner. Conclusions: LDT10 and LDT119 exhibited potent and selective in vitro activity against all developmental stages of T. cruzi, with low micromolar to submicromolar IC₅₀/LD₅₀ values, minimal mammalian cytotoxicity, and extensive morphological and ultrastructural damage consistent with disruption of phospholipid biosynthesis pathways. Combined with favorable in silico pharmacokinetic predictions, these CNSL-derived phospholipid analogs represent promising candidates for future Chagas disease chemotherapy and warrant further in vivo evaluation. Full article

Multidrug resistance (MDR) remains a major obstacle in the treatment of ovarian cancer. MDR is often mediated by the overexpression of ATP-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP). In this study, we evaluated the ability of elacridar, a dual P-gp and BCRP inhibitor, to overcome MDR in W1, an ovarian cancer cell line sensitive to Paclitaxel (PAC) and its PAC-resistant variants. Cells were cultured under both two-dimensional (2D) and three-dimensional (3D) conditions to account for differences in tumor-like microenvironments. The MDR1 gene and P-gp protein expression were determined for the analyzed model; P-gp activity was measured by flow-cytometry and fluorescent observation, with and without elacridar. The MTT tests were carried out to evaluate how elacridar, combined with chemotherapeutics, affects cell viability. Our results demonstrate that elacridar effectively inhibited transporter activity and increased cellular sensitivity to PAC and DOX. The inhibitory effect was observed in both 2D and 3D cultures, although the re-sensitization effect in 3D conditions was less pronounced, reflecting the complexity of tumor-specific resistance mechanisms. These findings highlight elacridar as a promising compound for reversing MDR in ovarian cancer and emphasize the importance of 3D models in preclinical drug evaluation. Further studies in advanced in vitro and in vivo models are required to assess the potential of elacridar better. Full article

Multidrug resistance (MDR) remains a significant obstacle to effective cancer chemotherapy, primarily due to overexpression of P-glycoprotein (P-gp), which reduces intracellular accumulation of cytotoxic drugs. This study evaluated the pharmacological potential of the glycyrrhetinic acid derivative soloxolone N-3-(dimethylamino)propylamide (Sol-DMAP) as a biocompatible P-gp inhibitor with hepatoprotective properties. Using a murine model of P-gp-overexpressing RLS40 lymphosarcoma, we demonstrated that Sol-DMAP significantly enhanced the antitumor efficacy of doxorubicin (DOX) by increasing its intratumoral concentration 4.7-fold without enhancing systemic toxicity. Independently, Sol-DMAP exhibited direct antitumor activity, reducing tumor growth in vivo and inducing apoptosis and G1-phase arrest in RLS40 cells in vitro. In addition, Sol-DMAP mitigated DOX-induced hepatic injury by reducing necrotic and dystrophic changes in liver tissue and restoring heme oxygenase 1 (Hmox1) expression. Further studies in HepG2 cells confirmed that Sol-DMAP activated the NRF2-dependent antioxidant response, upregulating HMOX1, GCLC, GCLM, and NQO1 genes. Molecular docking revealed that Sol-DMAP can disrupt the KEAP1-NRF2 interaction, likely leading to NRF2 activation. Collectively, these findings demonstrate that Sol-DMAP effectively reverses P-gp-mediated MDR while protecting the liver from oxidative stress, highlighting its potential as a multifunctional scaffold for the development of safer and more effective chemotherapeutic adjuvants. Full article

Background/Objective: Acute and chronic pain affect millions of individuals, yet there are currently no molecular imaging tools to directly assess pain-related mechanisms in the central nervous system (CNS). The voltage-gated sodium channel Na_V1.8 plays a pivotal role in neuropathic pain by increasing the excitability of nociceptive neurons following nerve injury or inflammation. In this work, we aimed to develop a novel positron emission tomography (PET) imaging probe for Na_V1.8 to facilitate noninvasive quantification of this target in the CNS and thereby advance our understanding of pain neurobiology. Methods: We selected the compound suzetrigine, a U.S. FDA-approved, highly selective non-opioid Na_V1.8 inhibitor, as the first candidate for a Na_V1.8-targeted PET tracer. The compound was first assessed using in silico docking and CNS multiparameter optimization (MPO) analysis to evaluate target binding and predicted brain penetrability. Radiolabeling was accomplished by O-methylation with [¹¹C]methyl iodide to yield [¹¹C]suzetrigine without structural modification. The tracer was then evaluated using in vitro binding assays, including autoradiography and saturation binding on rat brain tissues, to determine binding parameters (K_D, B_max), and using in vivo PET imaging in rats to assess brain uptake, time–activity curves (TACs), and tracer behavior under baseline and pretreatment conditions. Pretreatment was performed with unlabeled suzetrigine, the P-glycoprotein (P-gp) inhibitor verapamil, and the heterologous Na_V1.8 inhibitor A-803467. Results: In silico docking demonstrated favorable binding of suzetrigine to the Na_V1.8 active site, and the calculated CNS MPO score (>3.5) suggested adequate brain penetration. Radiochemical synthesis of [¹¹C]suzetrigine via O-methylation yielded a high decay-corrected radiochemical yield (19.2 ± 2.7%, n = 3), excellent purity (>98%, n = 3), and moderate molar activity (62.9 ± 51.8 MBq/nmol, n = 3). Autoradiography on rat brain tissue confirmed saturable and selective binding of [¹¹C]suzetrigine to Na_V1.8. Saturation binding assays revealed a B_max = 93 fmol/mg and a K_D = 0.1 nM, supporting the imageability of Na_V1.8 in the brain using this tracer. In vivo PET imaging in rats demonstrated rapid and sufficient brain uptake but revealed unexpected tracer behavior: signal intensity markedly increased following pretreatment with either unlabeled suzetrigine or the P-gp inhibitor verapamil, and showed a slight increase after pretreatment with the heterologous Na_V1.8 inhibitor A-803467. Detailed analysis of PET images, TACs, and normalized area-under-curve (AUC) values indicated that these atypical uptake patterns were primarily attributable to P-gp-mediated effects, although additional factors may also contribute. Conclusions: [¹¹C]Suzetrigine exhibits high affinity, good brain uptake, and selective target engagement in vitro, supporting its potential as a first-in-class Na_V1.8-PET tracer. However, in vivo performance is confounded by P-gp-mediated efflux and possibly other mechanisms that limit accurate quantification of Na_V1.8 in the living brain. These findings underscore the critical role of efflux transporters in CNS radiotracer development and highlight the need for design strategies that mitigate P-gp interaction when targeting ion channels in the brain. Future studies will include imaging under constant P-gp inhibition, arterial blood sampling for radiometabolite analysis and full kinetic modeling, and evaluation in non-human primates to assess translational feasibility. Full article

Carfilzomib (CFZ) is a cornerstone in the treatment of relapsed multiple myeloma (MM). However, its efficacy is limited by resistance mediated by the overexpression of the ABC-transporter P-glycoprotein (P-gp). The signaling pathways driving the emergence of P-gp in MM remain unclear. To investigate this, we generated CFZ-resistant AMO-1/CFZ cells with P-gp overexpression by long-term selection. RNA sequencing of control AMO-1 and AMO-1/CFZ, sorted into two subpopulations, P-gp HIGH and P-gp LOW, implicated the Ras/MEK/ERK pathway as the most likely signaling cascade involved in P-gp upregulation. We therefore evaluated two clinically used MAPK pathway inhibitors, cobimetinib and ulixertinib, for their ability to re-sensitize AMO-1/CFZ cells to CFZ. Co-administration at non-toxic concentrations enhanced sensitivity 5-fold with cobimetinib and 17-fold with ulixertinib. Analysis of the combined MTT assay results, rhodamine efflux experiments, molecular docking, and Western blotting revealed distinct actions. Ulixertinib primarily functions as a potent direct P-gp inhibitor. Conversely, non-toxic concentrations of cobimetinib sensitizes cells by suppressing MAPK signaling, though it also exhibits P-gp inhibition at higher concentrations. At the IC₅₀ concentration, both inhibitors reduced P-gp expression. In conclusion, combining CFZ with MAPK pathway inhibitors like cobimetinib or ulixertinib represents a promising strategy to overcome P-gp-mediated resistance in MM. Full article

Phosphoinositide 3-kinase (PI3K) represents a pivotal therapeutic target implicated in cellular proliferation, metabolic processes, and oncogenic mechanisms. This research delineates a comprehensive in silico methodology for identifying effective, pharmacokinetically favorable PI3K inhibitors. Structure-based molecular docking was executed targeting the ATP-binding pocket of PI3K, revealing that the highest-ranked compound, MOL ID: 11325, demonstrated a significant binding affinity, reflected by a docking score of −8.558 kcal/mol. ADMET and SwissADME profiling confirmed that molecule 11325 is Lipinski-compliant, P-gp non-substrate, has a bioavailability score of 0.55, no PAINS or Brenk alerts, and a favorable synthetic accessibility (2.68), supporting its drug-likeness and development potential. A 100 ns molecular dynamics simulation confirmed the stability of the PI3K–ligand complex, demonstrating minimal deviations in root mean square deviation (RMSD) and root mean square fluctuation (RMSF). The binding free energy, determined through the MMGBSA method, exhibited a favorable value (ΔG_bind ≈ −58.6 kcal/mol), thereby corroborating the ligand’s affinity. The FEL analysis revealed distinct low-energy states, while the PCA indicated minimal structural fluctuations, confirming a stable and specific binding mode. Molecule 11325 was designated as a novel, drug-like, and dynamically stable PI3K inhibitor by this integrated computational approach, indicating that it requires additional preclinical validation for therapeutic development. Full article

Background: Multidrug resistance (MDR), primarily driven by P-glycoprotein (P-gp)-mediated drug efflux, presents a significant challenge in cancer therapy, contributing to chemotherapy failure and poor patient outcomes. Objectives: In this study, we explored the potential of manidipine (MA), a clinically approved calcium channel blocker, to reverse P-gp-mediated MDR through modulation of calcium signaling via nuclear factor of activated T cells 2 (NFAT2). Methods: Paclitaxel (PTX) resistance ABCB1-overexpressing cancer in vitro and in vivo were used for evualting the anti-MDR effects of MA, as well as the underlying mechanism with siRNA of NFAT2. Results: We found that MA at non-toxic concentrations (0.6–5.4 μM) significantly sensitize drug-resistant colorectal (HCT-8/T) and non-small cell lung (A549/T) cells to PTX, reducing its IC₅₀ by up to 1328-fold in vitro models. Mechanistically, MA inhibited P-gp efflux activity without altering its expression, as shown by an increased intracellular accumulation of doxorubicin and Flutax-2 (2.3- and 3.1-fold, respectively) and dose-dependent modulation of ATPase activity (EC₅₀ = 4.16 μM). Notably, MA reduced intracellular calcium levels (52% reduction, p < 0.001) and downregulated NFAT2, an oncogene overexpressed in resistant cells. In vivo, MA (3.5 mg/kg) synergizes with PTX to inhibit tumor growth by 68% (p < 0.001) in A549/T xenograft model, without an observable decrease in weight. Conclusions: In sum, all these results position MA as a novel NFAT2 inhibitor to overcome P-gp-mediated MDR via modulating calcium signaling, which points to further investigation for its clinical applications. Full article

This study investigated the protective effects of Gardenia jasminoides Ellis polysaccharides (GP) on lipopolysaccharide (LPS)-induced immunosuppression and oxidative stress in mice and explored how GP modulates macrophage polarization through the TLR4/NF-κB signaling axis. The results showed that GP notably restored thymus and spleen indices in LPS-treated mice, markedly decreased the serum concentrations of malondialdehyde, and enhanced superoxide dismutase activity and total antioxidant capacity. In RAW 264.7 macrophage cultures, GP displayed immunostimulatory effects by improving phagocytic activity, promoting NO synthesis, and enhancing the secretion of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. These effects were observed in cells not pretreated with TAK-242 or PDTC; however, they were not observed in cells pretreated with these inhibitors. At 300 µg/mL concentration, GP markedly enhanced the transcriptional levels of iNOS and cytokine genes. Protein analysis revealed significant upregulation of TLR4, MyD88, TRAF6, NF-κB RelA/p65, and phosphorylated p65. Fluorescence imaging confirmed the nuclear translocation of p65. Collectively, these findings indicated that GP reversed systemic immunosuppression and oxidative stress, offering foundational insights for developing natural immune regulators. The observed immunomodulatory properties of GP are likely mediated through the TLR4/NF-κB signaling pathway. Full article

We explored the pharmacology of the P-glycoprotein (P-gp) efflux pump and its role in multidrug resistance. We used Protein Data Bank (PDB) database mining and the artificial intelligence (AI) model Boltz-2.1.1, developed for simultaneous structure and affinity prediction, to explore the multimeric nature of recent P-gp inhibitors. We construct a MARTINI coarse-grained (CG) force field description of P-gp embedded in a model of the endothelial blood–brain barrier. We found that recent P-gp inhibitors have been captured in either monomeric, dimeric, or trimeric states. Our CG model demonstrates the ability of P-gp substrates to permeate and transition across the BBB bilayer. We report a multimodal binding model of P-gp inhibition in which later generations of inhibitors are found in dimeric and trimeric states. We report analyses of P-gp substrates that point to an extended binding surface that explains how P-gp can bind over 300 substrates non-selectively. Our coarse-grained model of substrate permeation into membranes expressing P-gp shows benchmarking similarities to prior atomistic models and provide new insights on far longer timescales. Full article

Background/Objectives: P-glycoprotein (P-gp), an ATP-binding cassette (ABC) transporter, plays a key role in multidrug resistance by actively exporting chemotherapeutic agents and xenobiotics from cells. Overexpression of P-gp significantly reduces intracellular drug accumulation and compromises treatment efficacy. Despite extensive research, clinically approved P-gp inhibitors remain elusive due to toxicity, poor specificity, and limited efficacy. This study investigates DMH1, a selective type I BMP receptor inhibitor, as a novel P-gp inhibitor. Methods: DMH1 cytotoxicity was assessed in P-gp-overexpressing (PC3-TxR, K562/Dox) and P-gp-deficient (PC3) cell lines using MTT assays. P-gp inhibition was evaluated using calcein AM retention and daunorubicin (DNR) accumulation assays. Kinetic analysis determined DMH1’s effect on P-gp-mediated transport (Vmax and Km). ATPase activity assays were performed to assess DMH1’s impact on ATP hydrolysis. Preliminary molecular docking (CB-Dock2) was used to predict DMH1’s binding site on the human P-gp structure (PDB ID: 6QEX). Results: DMH1 showed no cytotoxicity in P-gp-overexpressing or deficient cells. It significantly enhanced intracellular accumulation of Calcein AM and DNR, indicating effective inhibition of P-gp function. Kinetic data revealed that DMH1 reduced Vmax without affecting Km, consistent with noncompetitive, allosteric inhibition. DMH1 also inhibited ATPase activity in a dose-dependent manner. Docking analysis suggested DMH1 may bind to an allosteric site in the transmembrane domain, potentially stabilizing the inward-facing conformation. Conclusions: DMH1 is a promising noncompetitive, allosteric P-gp inhibitor that enhances intracellular drug retention without cytotoxicity, supporting its potential as a lead compound to overcome multidrug resistance and improve chemotherapeutic efficacy. Full article