Small Molecule Inhibitors Research Articles

DNMT3A loss drives a HIF-1-dependent synthetic lethality to HDAC6 inhibition in non-small cell lung cancer

DNMT3A encodes a DNA methyltransferase involved in development, cell differentiation, and gene transcription, which is mutated and aberrant-expressed in cancers. Here, we revealed that loss of DNMT3A promotes malignant phenotypes in lung cancer. Based on the epigenetic inhibitor library synthetic lethal screening, we found that small-molecule HDAC6 inhibitors selectively killed DNMT3A-defective NSCLC cells. Knockdown of HDAC6 by siRNAs reduced cell growth and induced apoptosis in DNMT3A-defective NSCLC cells. However, sensitive cells became resistant when DNMT3A was rescued. Furthermore, the selectivity to HDAC6 inhibition was recapitulated in mice, where an HDAC6 inhibitor retarded tumor growth established from DNMT3A-defective but not DNMT3A parental NSCLC cells. Mechanistically, DNMT3A loss resulted in the upregulation of HDAC6 through decreasing its promoter CpG methylation and enhancing transcription factor RUNX1 binding. Notably, our results indicated that HIF-1 pathway was activated in DNMT3A-defective cells whereas inactivated by HDAC6 inhibition. Knockout of HIF-1 contributed to the elimination of synthetic lethality between DNMT3A and HDAC6. Interestingly, HIF-1 pathway inhibitors could mimic the selective efficacy of HDAC6 inhibition in DNMT3A-defective cells. These results demonstrated HDAC6 as an HIF-1-dependent vulnerability of DNMT3A-defective cancers. Together, our findings identify HDAC6 as a potential HIF-1-dependent therapeutic target for the treatment of DNMT3A-defective cancers like NSCLC.

Developments of Fms-like Tyrosine Kinase 3 Inhibitors as Anticancer Agents for AML Treatment.

FMS-like tyrosine kinase 3 (FLT3) is a commonly mutated gene in acute myeloid leukemia. As a receptor tyrosine kinase (RTK), FLT3 plays a role in the proliferation and differentiation of hematopoietic stem cells. As the most frequent molecular alteration in AML, FLT3 has drawn the attention of many researchers, and a lot of small molecule inhibitors targeting FLT3 have been intensively investigated as potential drugs for AML therapy. In this paper, PubMed and SciFinder® were used as a tool; the publications about "FLT3 inhibitor" and "Acute myeloid leukemia" were surveyed from 2014 to the present with an exclusion of those published as patents. In this study, the structural characterization and biological activities of representative FLT3 inhibitors were summarized. The major challenges and future directions for further research are discussed. Recently, numerous FLT3 inhibitors have been discovered and employed in FLT3-mutated AML treatment. In order to overcome the drug resistance caused by FLT3 mutations, screening multitargets FLT3 inhibitors has become the main research direction. In addition, the emergence of irreversible FLT3 inhibitors also provides new ideas for discovering new FLT3 inhibitors.

Paired comparisons of venetoclax concentration in cerebrospinal fluid, bone marrow, and plasma in acute leukemia patients.

Venetoclax, a small molecule inhibitor of BCL-2, has demonstrated efficacy in treating acute leukemias and has been recommended as one of the first-line anti-leukemia therapies. Although venetoclax has been suggested to probably possess the ability to penetrate the central nervous system (CNS), current data to elucidate the characteristics of venetoclax in cerebrospinal fluid (CSF), bone marrow (BM), and plasma are still lacking. This study investigated the real-world characteristics of venetoclax concentrations in CSF, BM, and plasma in acute leukemia patients. Thirteen acute leukemia patients treated with venetoclax were included, with paired samples of CSF, BM, and plasma collected and venetoclax concentrations measured using LC-MS/MS. With the results, the median venetoclax concentrations were 2030 ng/mL in plasma, 16.7 ng/mL in CSF, and 1390 ng/mL in BM. The percentages of CSF/plasma and BM/plasma were 0.74% and 70.37%, respectively. While no direct correlation was observed between CSF and plasma venetoclax levels, there was a trend toward an improved CSF/plasma percentage over time following the last administration of venetoclax. In contrast, a strong correlation was found between BM and plasma levels. This study demonstrated that venetoclax could reach its effective concentration in most patients, suggesting its potential clinical utility in the management of CNS involvement in acute leukemia.

Potent Small Molecules Inhibitors Discovery through Ligand-based Modelling for Effective Treatment of Parkinson’s Disease

Background: Parkinson’s disease (PD) is a chronic neurodegenerative disease affecting mostly aged people. The disease's symptoms develop gradually over time and include tremors, bradykinesia, rigidity, and postural instability. Current treatment options for PD are only symptom-targeted. Prolyl oligopeptidase (POP) is a serine protease enzymes implicated in PD pathogenesis via an increase in the aggregation of α-synuclein protein in the brain. Aim: This study aims to identify potent anti-PD ligands with inhibitory potential against POP Methods: Ligand-based pharmacophore modeling, Glide extra precision (XP) docking, and post-simulation analysis methods were used. Results: The adopted ligand-based (LB) modeling generated pharmacophoric features, including 1 hydrophobic group, 1 positive ionizable group, 2 aromatic rings, and 2 hydrogen bond acceptors. A total of 23 hits with a Gunner-Henry score of 0.7 and an enrichment factor of 30.24 were obtained as validation protocols, making it an ideal model. The LB model retrieved 177 hit compounds from the 69,543 natural screening ligands available in the Interbioscreen database. Interestingly, ligands 1, 2, 3, 4, and 5 orderly demonstrated higher binding affinities with Glide XP docking of -9.0, -8.8, -8.7, -8.7, -8.7 kcal/mol compared to reference drugs, GSK552 and ZPP with -8.2, and -6.8 kcal/mol respectively. Similarly, their MM/GBSA values were recorded as -54.4, -51.3, -58.4, -49.3, - 33.5, & -32.5 kJ/mol respectively. Further, MD analysis indicated that ligands had higher favorable binding and stability to the receptor. Conclusion: Overall, the study paves the way for developing potential anti-PD therapeutics. The ligands are recommended as adjuvant/single candidate as anti-PD candidates upon further experiment.

Randomized, double-blind, phase 1a single-ascending dose and food effect studies assessing safety and pharmacokinetics of EC5026 in healthy volunteers.

Chronic pain represents a significant unmet medical need, affecting one-fifth of the U.S. population. EC5026 is a small molecule inhibitor of the enzyme soluble epoxide hydrolase (sEH) which is being developed as a novel non-opioid, non-NSAID analgesic. EC5026 prolongs the action of epoxy fatty acids, endogenous analgesic lipid mediators that are rapidly metabolized by sEH. We evaluated the safety and pharmacokinetic profile of EC5026 in two phase I trials, a single-ascending dose (SAD) study and a fed-fasted study. The SAD study evaluated EC5026 doses ranging from 0.5 to 24 mg in healthy volunteers. EC5026 was well tolerated. No treatment-emergent adverse events were considered related to EC5026. No apparent treatment- or dose-related trends in laboratory results, vital signs, physical examinations, or electrocardiograms were observed. A linear, near-dose-proportional increase in exposure was observed with progressive doses in the SAD study; plasma exposure was below or near the lower limit of quantification after 0.5-2 mg doses. Mean half-lives ranged from 41.8 to 59.1 h. for doses of 8-24 mg, supporting a once-daily dosing regimen. In the fed-fasted study using 8 mg EC5026 tablets, higher peak concentrations (66%) and total exposures (53%) were observed under the fed condition. Plasma concentrations declined in a monoexponential manner with mean half-lives of 59.5 h. in the fed state and 66.9 h. in the fasted state. Future clinical trials using EC5026 for the treatment of pain are justified based on the favorable outcomes from both clinical trials along with preclinical evidence of analgesic activity.

Drug-resistant menin variants retain high binding affinity and interactions with MLL1

Menin is an essential oncogenic co-factor of MLL1 fusion proteins in acute leukemias and inhibitors of the menin-MLL1 interaction are under evaluation in clinical trials. Recent studies found emerging resistance to menin inhibitor treatment in leukemia patients as a result of somatic mutations in menin. To understand how patient mutations in menin affect the interaction with MLL1, we performed systematic characterization of the binding affinity of these menin mutants (T349M, M327I, G331R and G331D) and the N-terminal fragment of MLL1. We also determined the crystal structures of menin patient mutants and their complexes with MLL1-derived peptides. We found that drug-resistant mutations in menin occur at a site adjacent to the MLL1 binding site, but they do not affect MLL1 binding to menin. On the contrary, our structural analysis shows that all these point mutations in menin generate steric clash with menin inhibitors. We also found that mutation G331D results in a very slow dissociation of MLL1 from menin and this mutant might be particularly difficult to inhibit with small molecule drugs. This work provides structural information to support the development of a new generation of small molecule inhibitors that overcome resistance caused by menin mutations.

Transforming the Discovery of Targeted Protein Degraders: The Translational Power of Predictive PK/PD Modeling.

Targeted protein degraders (TPDs), an emerging therapeutic modality, are attracting considerable interest with the promise to address disease-related proteins that are not druggable with conventional small molecule inhibitors. Despite their novel mechanism of action, the PK/PD relationship of degraders is still approached with a mindset deeply rooted in inhibitor drugs. Here, we establish how predictive mechanistic modeling specifically tailored to TPDs can significantly enhance the value of the available information during lead generation and optimization. By integrating the results from in vitro assays with routinely collected PK data, modeling accurately predicts degradation in vivo. These predictions transform the prioritization of compounds for in vivo studies as well as the selection of optimal dose schedules and most informative measurement time points with the least number of animals. Moreover, the comprehensive modeling framework (1) identifies the PK/PD driver of targeted protein degradation and subsequent downstream pharmacodynamic effects, and (2) uncovers the fundamental difference between degrader and inhibitor PK/PD relationships. The practical utility of our predictive modeling is demonstrated with relevant use cases. This framework will allow researchers to transition from current, mostly serendipity-based approaches to more sound model-informed decision making. Going forward, the presented predictive PK/PD modeling framework lays out a rational path to incorporate inter-species differences in the pharmacology and thus promises to help with getting the dose right in clinical trials.

Stabilization of the open conformation οf insulin-regulated aminopeptidase by a novel substrate-selective small-molecule inhibitor.

Insulin-regulated aminopeptidase (IRAP) is an enzyme with important biological functions and the target of drug-discovery efforts. We combined in silico screening with a medicinal chemistry optimization campaign to discover a nanomolar inhibitor of IRAP based on a pyrazolylpyrimidine scaffold. This compound displays an excellent selectivity profile versus homologous aminopeptidases, and kinetic analysis suggests it utilizes an uncompetitive mechanism of action when inhibiting the cleavage of a typical dipeptidic substrate. Surprisingly, the compound is a poor inhibitor of the processing of the physiological cyclic peptide substrate oxytocin and a 10mer antigenic epitope precursor but displays a biphasic inhibition profile for the trimming of a 9mer antigenic peptide. While the compound reduces IRAP-dependent cross-presentation of an 8mer epitope in a cellular assay, it fails to block in vitro trimming of select epitope precursors. To gain insight into the mechanism and basis of this unusual selectivity for this inhibitor, we solved the crystal structure of its complex with IRAP. The structure indicated direct zinc(II) engagement by the pyrazolylpyrimidine scaffold and revealed that the compound binds to an open conformation of the enzyme in a pose that should block the conformational transition to the enzymatically active closed conformation previously observed for other low-molecular-weight inhibitors. This compound constitutes the first IRAP inhibitor targeting the active site that utilizes a conformation-specific mechanism of action, provides insight into the intricacies of the IRAP catalytic cycle, and highlights a novel approach to regulating IRAP activity by blocking its conformational rearrangements.

Role of the CDKL1-SOX11 signaling axis in acute kidney injury.

The biology of the cyclin-dependent kinase-like (CDKL) kinase family remains enigmatic. Contrary to their nomenclature, CDKLs do not rely on cyclins for activation and are not involved in cell cycle regulation. Instead, they share structural similarities with mitogen-activated protein kinases and glycogen synthase kinase-3, although their specific functions and associated signaling pathways are still unknown. Previous studies have shown that the activation of CDKL5 kinase contributes to the development of acute kidney injury (AKI) by suppressing the protective SOX9-dependent transcriptional program in tubular epithelial cells. In the current study, we measured the functional activity of all five CDKL kinases and discovered that, in addition to CDKL5, CDKL1 is also activated in tubular epithelial cells during AKI. To explore the role of CDKL1, we generated a germline knockout mouse that exhibited no abnormalities under normal conditions. Notably, when these mice were challenged with bilateral ischemia-reperfusion and rhabdomyolysis, they were found to be protected from AKI. Further mechanistic investigations revealed that CDKL1 phosphorylates and destabilizes SOX11, contributing to tubular dysfunction. In summary, this study has unveiled a previously unknown CDKL1-SOX11 axis that drives tubular dysfunction during AKI.NEW & NOTEWORTHY Identifying and targeting pathogenic protein kinases holds potential for drug discovery in treating acute kidney injury. Our study, using novel germline knockout mice, revealed that Cdkl1 kinase deficiency does not affect mouse viability but provides protection against acute kidney injury. This underscores the importance of Cdkl1 kinase in kidney injury and supports the development of targeted small-molecule inhibitors as potential therapeutics.

A Binuclear Metallohydrolase Model for RelA/SpoT-Homolog (RSH) Hydrolases

When challenged by starvation, bacterial organisms synthesize guanosine penta- and tetraphosphate, collectively denoted as (p)ppGpp, as second messengers to reprogram metabolism towards slower growth and enhanced stress tolerance. When starvation is alleviated, the RelA-SpoT homolog (RSH) hydrolases down-regulate (p)ppGpp, cleaving the 3’-diphosphate to produce GTP or GDP. Metazoan RSH hydrolases (MESH) possess phosphatase activity responsible for converting cytoplasmic NADPH to NADH in mammalian cells. Inhibitor development for this family may therefore provide therapies to combat bacterial infection or metabolic dysregulation. Despite the availability of dozens of high-resolution structures, catalytic mechanisms of RSH hydrolases have remained poorly understood. All RSH hydrolases tightly bind a Mn2+ near its active center, which is believed sufficient for hydrolase activity. In contrast to this notion, we demonstrate, using the (p)ppGpp hydrolase SpoT from Acinetobacter baumannii, that a second divalent cation, presumably a Mg2+ under physiological conditions, is required for efficient catalysis. We also show that SpoT preferentially cleaves 3’-diphosphate over 3’-phosphate substrates, likely due to a key coordination between the β-phosphate and the second metal center. MESH replaces this β-phosphate with the side chain of an aspartate residue, thereby functioning as a phosphatase. We propose a binuclear metallohydrolase model where an invariant ED (Glu-Asp) diad, previously believed to activate the water nucleophile, instead coordinates to a Mg2+ center. The refined molecular and evolutionary blueprint of RSH hydrolases will provide a more reliable foundation for the development of small-molecule inhibitors of this important enzyme family.

634P First results from phase I/II study of CTS2190, a novel small-molecule inhibitor of type I PRMTs, in patients with advanced solid tumors

634P First results from phase I/II study of CTS2190, a novel small-molecule inhibitor of type I PRMTs, in patients with advanced solid tumors

Synthesis and immunotherapy efficacy of a PD-L1 small-molecule inhibitor combined with its 131I-iodide labelled isostructural compound

Although antibody-based immune checkpoint blockades have been successfully used in antitumor immunotherapy, the low response rate is currently the main problem. In this work, a small-molecule programmed cell death-ligand (PD-L1) inhibitor, LG-12, was developed and radiolabeled with 131I to obtain the chemically and biologically identical radiopharmaceutical [131I]LG-12, which aimed to improve the antitumor effect by combination of LG-12 and [131I]LG-12. LG-12 showed high inhibitory activity to PD-1/PD-L1 interaction. The results of cell uptake and biodistribution studies indicated that [131I]LG-12 could specifically bind to PD-L1 in B16-F10 tumors. It could induce immunogenic cell death and the release of high mobility group box 1 and calreticulin. The combination of [131I]LG-12 and LG-12 could significantly inhibit tumor growth and resulted in enhanced antitumor immune response. This PD-L1 small-molecule inhibitor based combination strategy has great potential for tumor treatment.

The RAL Small G Proteins Are Clinically Relevant Targets in Triple Negative Breast Cancer.

Breast cancer (BC) is the most frequent cancer and second-leading cause of cancer deaths in women in the United States. While RAS mutations are infrequent in BC, triple-negative (TN) and HER2-positive (HER2+) BC both exhibit increased RAS activity. Here, we tested the RAS effectors RALA and RALB, which are overexpressed in BC, as tractable molecular targets in these subtypes. While analysis of the breast cancer patient sample data suggests that the RALs are associated with poor outcome in both TNBC and HER2+ BC, our in vivo and in vitro experimental findings revealed the RALs to be essential in only the TNBC cell lines. While testing the response of the BC cell lines to the RAL inhibitors RBC8 and BQU57, we observed no correlation between drug efficacy and cell line dependency on RAL expression for survival, suggesting that these compounds kill via off-target effects. Finally, we report the discovery of a new small molecule inhibitor, OSURALi, which exhibits strong RAL binding, effectively inhibits RAL activation, and is significantly more toxic to RAL-dependent TNBC cells than RAL-independent HER2+ and normal cell lines. These results support the RALs as viable molecular targets in TNBC and the further investigation of OSURALi as a therapeutic agent.

Macrophage MKL1 contributes to cardiac fibrosis in a mouse model of myocardial infarction

AimsCardiac fibrosis is characterized by aberrant collagen deposition in the heart. Macrophage polarization or infiltration is the main reason to accelerate the collagen deposition. We attempted to investigate the involvement of MKL1 in macrophages during the development of cardiac fibrosis. Materials and methodsCardiac fibrosis is induced by myocardial infarction (MI). The MKL1f/f mice were crossed to the Lyz2-cre mice to generate macrophage conditional MKL1 knockout mice (MKL1ΔMφ). In addition, macrophage conditional MKL1 overexpression mice (MKL1Mϕ-OE) were constructed by crossing Lyz2-cre mice to MKL1ΔN200-Rosa26 mice. Key findingsMKL1 expression was significantly increased in macrophages of both ischemic cardiomyopathy (ICM) patients and mice induced to develop myocardial infarction. Deletion of MKL1 in macrophages improved the heart function after MI-induced cardiac fibrosis. Consistently, MKL1Mϕ-OE mice displayed more severe cardiac fibrosis and worsened heart function than the control mice after MI. Moreover, administration of a small-molecule MKL1 inhibitor CCG-1423 also decreased the collagen deposition after MI. SignificanceOur data demonstrate that MKL1 in macrophages contributes to cardiac fibrosis pathogenesis and reinforce the notion that targeting MKL1 may yield effective antifibrotic therapeutics in the heart.

Novel inhibitors of bromodomain and extra-terminal domain trigger cell death in breast cancer cell lines

Small molecule inhibitors targeting the bromodomain and extra-terminal domain (BET) family proteins have emerged as a promising class of anti-cancer drugs. Nevertheless, the clinical advancement of these agents has been significantly hampered by challenges related to their potency, oral bioavailability, or toxicity. In this study, virtual screening approaches were employed to discover novel inhibitors of the bromodomain-containing protein 4 (BRD4) by analyzing their comparable chemical structural features to established BRD4 inhibitors. Several of these compounds exhibited inhibitory effects on BRD4 activity ranging from 60 % to 70 % at 100 µM concentrations, while one compound also exhibited an 84 % inhibition of Sirtuin 2 (SIRT2) activity. Furthermore, a subset of structurally diverse compounds from the BRD4 inhibitors was selected to investigate their anti-cancer properties in both 2D and 3D cell cultures. These compounds exhibited varying effects on cell numbers depending on the specific cell line, and some of them induced cell cycle arrest in the G0/G1 phase in breast cancer (MDA-MB-231) cells. Moreover, all the compounds studied reduced the sizes of spheroids, and the most potent compound exhibited a 90 % decrease in growth at a concentration of 10 µM in T47D cells. These compounds hold potential as epigenetic regulators for future studies.

Protease activated receptor-1 regulates mixed lineage kinase-3 to drive triple-negative breast cancer tumorigenesis

Triple-negative breast cancer (TNBC) is difficult to treat breast cancer subtype due to lack or insignificant expressions of targetable estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2). Therefore, finding a targetable protein or signaling pathway in TNBC would impact patient care. Here, we report that a member of the Mixed Lineage Kinase (MLK) family, MLK3, is an effector of G-protein-coupled protease-activated receptors 1 (PAR1) and targeting MLK3 by a small-molecule inhibitor prevented PAR1-mediated TNBC tumorigenesis. In silico and immunohistochemistry analysis of human breast tumors showed overexpression of PAR1 and MLK3 in TNBC tumors. Treating α-thrombin and PAR1 agonist increased MLK3 and JNK activities and induced cell migration in TNBC cells. The PAR1 positive/high (PAR1+/hi) population of TNBC cells showed aggressive tumor phenotype with increased MLK3 signaling. Moreover, combined inhibition of the PAR1 and MLK3 mitigated the TNBC tumor burden in preclinical TNBC models. Our data suggests that activation of the PAR1-MLK3 axis promotes TNBC tumorigenesis. Therefore, combinatorial therapy targeting MLK3 and PAR1 could effectively reduce TNBC tumor burden.

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.

Stat3 Inhibitors TTI-101 and SH5-07 Suppress Bladder Cancer Cell Survival in 3D Tumor Models.

Bladder cancer (BCa) is one of the most lethal genitourinary malignancies owing to its propensity for recurrence and poor survival. The biochemical pathway, signal transducer and activator of transcription 3 (STAT3), has gained significance as a molecular pathway that promotes proliferation, invasion, and chemoresistance. In this study, we explored the targeting of STAT3 with TTI-101 and SH5-07 in BCa and elucidated the mechanisms in three-dimensional (3D) spheroid and organoid models. We optimized the growth of spheroids from human, rat, and mouse BCa cell lines (J82, NBT-II, and MB49 respectively) and organoids from BBN (N-butyl-N-(4-hydroxybutyl)-nitrosamine)-induced rat bladder tumors. Cell viability was assessed using MTT and trypan blue assays. Intracellular ATP production, ROS production, and calcium AM (CA)/EtBr staining were used to measure the spheroid and organoid inhibition and mitochondrial function. Western blot analysis was performed to evaluate the pharmacodynamic markers involved in cell proliferation, apoptosis, cancer stem cells (CSCs), and STAT3 signaling in BCa. We found that targeting STAT3 (using TTI-101 and SH5-07) significantly reduced the proliferation of BCa spheroids and organoids, which was accompanied by decreased expression of pSTAT3, Cyclin D1, and PCNA. Our data also demonstrated that treatment with STAT3 inhibitors induced ROS production and cell death in BCa spheroids and organoids. STAT3 inhibition-induced cell death was associated with the activation of caspase 3/7 and PARP cleavage. Moreover, TTI-101 and SH5-07 target cancer stem cells by downregulating the expression of CD44 and CD133 in 3D models. This study provides the first evidence for the prevention of BCa with small-molecule inhibitors TTI-101 and SH5-07 via suppression of CSCs and STAT3 signaling.

The Fibrotic Phenotype of Human Precision-Cut Lung Slices Is Maintained after Cryopreservation.

Human precision-cut lung slices (hPCLS) prepared from fibrotic lungs recapitulate the pathophysiological hallmarks of fibrosis. These hallmark features can also be induced by treating non-fibrotic hPCLS with a fibrotic cocktail (FC). As a result, the fibrotic and fibrosis-induced hPCLS are rapidly emerging as preferred models for disease modeling and drug discovery. However, current hPCLS models are limited by tissue viability in culture, as they are usually only viable for one week after harvesting. Here, we demonstrate that the fibrotic hPCLS can be cryopreserved, stored for months, and then thawed on demand without loss of hPCLS viability or protein content for 14 days post-thawing. Cryopreservation also preserves the pro-fibrotic potential of non-fibrotic hPCLS. Specifically, when we treated the thawed non-fibrotic hPCLS with an FC, we observed significant pro-fibrotic cytokine secretion and elevated tissue stiffness. These pro-fibrotic changes were inhibited by the small-molecule tyrosine kinase inhibitor, Nintedanib. Taken together, our work indicates that a feasible solution to prolong the pre-clinical utility of fibrotic and fibrosis-induced hPCLS is cryopreservation. We anticipate that cryopreserved hPCLS will serve as an advantageous predictive model for the evaluation of pro-fibrotic pathways during acute and chronic toxicity testing.

Allosteric coupling of substrate binding and proton translocation in MmpL3 transporter from Mycobacterium tuberculosis.

Infections caused by Mycobacterium spp. are very challenging to treat, and multidrug-resistant strains rapidly spread in human populations. Major contributing factors include the unique physiological features of these bacteria, drug efflux, and the low permeability barrier of their outer membrane. Here, we focus on MmpL3 from Mycobacterium tuberculosis, an essential inner membrane transporter of the resistance-nodulation-division superfamily required for the translocation of mycolic acids in the form of trehalose monomycolates (TMM) from the cytoplasm or plasma membrane to the periplasm or outer membrane. The MmpL3-dependent transport of TMM is essential for the growth of M. tuberculosis in vitro, inside macrophages, and in M. tuberculosis-infected mice. MmpL3 is also a validated target for several recently identified anti-mycobacterial agents. In this study, we reconstituted the lipid transport activity of the purified MmpL3 using a two-lipid vesicle system and established the ability of MmpL3 to actively extract phospholipids from the outer leaflet of a lipid bilayer. In contrast, we found that MmpL3 lacks the ability to translocate the same phospholipid substrate across the plasma membrane indicating that it is not an energy-dependent flippase. The lipid extraction activity was modulated by substitutions in critical charged and polar residues of the periplasmic substrate-binding pocket of MmpL3, coupled to the proton transfer activity of MmpL3 and inhibited by a small molecule inhibitor SQ109. Based on the results, we propose a mechanism of allosteric coupling wherein substrate translocation by MmpL3 is coupled to the energy provided by the downhill transfer of protons. The reconstituted activities will facilitate understanding the mechanism of MmpL3-dependent transport of lipids and the discovery of new therapeutic options for Mycobacterium spp. infections.IMPORTANCEMmpL3 from Mycobacterium tuberculosis is an essential transporter involved in the assembly of the mycobacterial outer membrane. It is also an important target in undergoing efforts to discover new anti-tuberculosis drugs effective against multidrug-resistant strains spreading in human populations. The recent breakthrough structural studies uncovered features of MmpL3 that suggested a possible lipid transport mechanism. In this study, we reconstituted and characterized the lipid transport activity of MmpL3 and demonstrated that this activity is blocked by MmpL3 inhibitors and substrate mimics. We further uncovered the mechanism of how the binding of a substrate in the periplasmic domain is communicated to the transmembrane proton relay of MmpL3. The uncovered mechanism and the developed assays provide new opportunities for mechanistic analyses of MmpL3 function and its inhibition.