ABSTRACT
ABSTRACT: We have reported the direct repair of the sickle cell mutation in vivo in a disease model using vectorized prime editors after hematopoietic stem cell (HSC) mobilization with granulocyte colony-stimulating factor (G-CSF)/AMD3100. The use of G-CSF for HSC mobilization is a hurdle for the clinical translation of this approach. Here, we tested a G-CSF-free mobilization regimen using WU-106, an inhibitor of integrin α4ß1, plus AMD3100 for in vivo HSC prime editing in sickle cell disease (SCD) mice. Mobilization with WU-106 + AMD3100 in SCD mice was rapid and efficient. In contrast to the G-CSF/AMD3100 approach, mobilization of activated granulocytes and elevation of the key proinflammatory cytokine interleukin-6 in the serum were minimal. The combination of WU-106 + AMD3100 mobilization and IV injection of the prime editing vector together with in vivo selection resulted in â¼23% correction of the SCD mutation in the bone marrow and peripheral blood cells of SCD mice. The treated mice demonstrated phenotypic correction, as reflected by normalized blood parameters and spleen size. Editing frequencies were significantly increased (29%) in secondary recipients, indicating the preferential mobilization/transduction of long-term repopulating HSCs. Using this approach, we found <1% undesired insertions/deletions and no detectable off-target editing at the top-scored potential sites. Our study shows that in vivo transduction to treat SCD can now be done within 2 hours involving only simple IV injections with a good safety profile. The same-day mobilization regimen makes in vivo HSC gene therapy more attractive for resource-poor settings, where SCD does the most damage.
Subject(s)
Anemia, Sickle Cell , Genetic Therapy , Granulocyte Colony-Stimulating Factor , Hematopoietic Stem Cell Mobilization , Animals , Humans , Mice , Anemia, Sickle Cell/therapy , Anemia, Sickle Cell/genetics , Benzylamines , Cyclams/pharmacology , Cyclams/therapeutic use , Disease Models, Animal , Gene Editing , Genetic Therapy/methods , Granulocyte Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cell Mobilization/methods , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/metabolism , Heterocyclic Compounds/pharmacology , Heterocyclic Compounds/therapeutic useABSTRACT
Allogeneic hematopoietic cell transplantation (allo-HCT) is a highly effective, well-established treatment for patients with various hematologic malignancies and non-malignant diseases. The therapeutic benefits of allo-HCT are mediated by alloreactive T cells in donor grafts. However, there is a significant risk of graft-versus-host disease (GvHD), in which the donor T cells recognize recipient cells as foreign and attack healthy organs in addition to malignancies. We previously demonstrated that targeting JAK1/JAK2, mediators of interferon-gamma receptor (IFNGR) and IL-6 receptor signaling, in donor T cells using baricitinib and ruxolitinib results in a significant reduction in GvHD after allo-HCT. Furthermore, we showed that balanced inhibition of JAK1/JAK2 while sparing JAK3 is important for the optimal prevention of GvHD. Thus, we have generated novel JAK1/JAK2 inhibitors, termed WU derivatives, by modifying baricitinib. Our results show that WU derivatives have the potential to mitigate GvHD by upregulating regulatory T cells and immune reconstitution while reducing the frequencies of antigen-presenting cells (APCs) and CD80 expression on these APCs in our preclinical mouse model of allo-HCT. In addition, WU derivatives effectively downregulated CXCR3 and T-bet in primary murine T cells. In summary, we have generated novel JAK inhibitors that could serve as alternatives to baricitinib or ruxolitinib.
Subject(s)
Graft vs Host Disease , Hematopoietic Stem Cell Transplantation , Pyrazoles , Transplantation, Homologous , Animals , Mice , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/metabolism , Azetidines/pharmacology , Disease Models, Animal , Graft vs Host Disease/prevention & control , Graft vs Host Disease/drug therapy , Hematopoietic Stem Cell Transplantation/adverse effects , Janus Kinase 1/antagonists & inhibitors , Janus Kinase 1/metabolism , Janus Kinase 2/metabolism , Janus Kinase 2/antagonists & inhibitors , Janus Kinase Inhibitors/pharmacology , Mice, Inbred C57BL , Purines/pharmacology , Pyrazoles/pharmacology , Sulfonamides/pharmacology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/drug effectsABSTRACT
PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas with limited treatment options and poor survival rates. About half of MPNST cases are associated with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome. Overexpression of TYK2 occurs in the majority of MPNST, implicating TYK2 as a therapeutic target. EXPERIMENTAL DESIGN: The effects of pharmacologic TYK2 inhibition on MPNST cell proliferation and survival were examined using IncuCyte live cell assays in vitro, and downstream actions were analyzed using RNA-sequencing (RNA-seq), qPCR arrays, and validation of protein changes with the WES automated Western system. Inhibition of TYK2 alone and in combination with MEK inhibition was evaluated in vivo using both murine and human MPNST cell lines, as well as MPNST PDX. RESULTS: Pharmacologic inhibition of TYK2 dose-dependently decreased proliferation and induced apoptosis over time. RNA-seq pathway analysis on TYK2 inhibitor-treated MPNST demonstrated decreased expression of cell cycle, mitotic, and glycolysis pathways. TYK2 inhibition resulted in upregulation of the MEK/ERK pathway gene expression, by both RNA-seq and qPCR array, as well as increased pERK1/2 levels by the WES Western system. The compensatory response was tested with dual treatment with TYK2 and MEK inhibitors, which synergistically decreased proliferation and increased apoptosis in vitro. Finally, combination therapy was shown to inhibit growth of MPNST in multiple in vivo models. CONCLUSIONS: These data provide the preclinical rationale for the development of a phase I clinical trial of deucravacitinib and mirdametinib in NF1-assosciated MPNST.
Subject(s)
Nerve Sheath Neoplasms , Neurofibromatosis 1 , Neurofibrosarcoma , Humans , Mice , Animals , Neurofibromatosis 1/drug therapy , Neurofibromatosis 1/genetics , Cell Line , Apoptosis , Mitogen-Activated Protein Kinase Kinases/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , Nerve Sheath Neoplasms/drug therapy , Nerve Sheath Neoplasms/genetics , Nerve Sheath Neoplasms/metabolism , Cell Line, Tumor , TYK2 Kinase/genetics , TYK2 Kinase/metabolism , TYK2 Kinase/pharmacologyABSTRACT
Despite the curative potential of hematopoietic stem cell transplantation (HSCT), conditioning-associated toxicities preclude broader clinical application. Antibody-drug conjugates (ADCs) provide an attractive approach to HSCT conditioning that minimizes toxicity while retaining efficacy. Initial studies of ADC conditioning have largely focused on syngeneic HSCT. However, to treat acute leukemias or induce tolerance for solid organ transplantation, this approach must be expanded to allogeneic HSCT (allo-HSCT). Using murine allo-HSCT models, we show that pharmacologic Janus kinase 1/2 (JAK1/2) inhibition combined with CD45- or cKit-targeted ADCs enables robust multilineage alloengraftment. Strikingly, myeloid lineage donor chimerism exceeding 99% was achievable in fully MHC-mismatched HSCT using this approach. Mechanistic studies using the JAK1/2 inhibitor baricitinib revealed marked impairment of T and NK cell survival, proliferation, and effector function. NK cells were exquisitely sensitive to JAK1/2 inhibition due to interference with IL-15 signaling. Unlike irradiated mice, ADC-conditioned mice did not develop pathogenic graft-versus-host alloreactivity when challenged with mismatched T cells. Finally, the combination of ADCs and baricitinib balanced graft-versus-host disease and graft-versus-leukemia responses in delayed donor lymphocyte infusion models. Our allo-HSCT conditioning strategy exemplifies the promise of immunotherapy to improve the safety of HSCT for treating hematologic diseases.
Subject(s)
Azetidines/pharmacology , Hematopoietic Stem Cell Transplantation , Immunoconjugates/pharmacology , Janus Kinase 1/antagonists & inhibitors , Janus Kinase 2/antagonists & inhibitors , Janus Kinase Inhibitors/pharmacology , Purines/pharmacology , Pyrazoles/pharmacology , Signal Transduction/drug effects , Sulfonamides/pharmacology , Allografts , Animals , Disease Models, Animal , Graft vs Host Disease/genetics , Graft vs Host Disease/immunology , Graft vs Host Disease/prevention & control , Graft vs Leukemia Effect/drug effects , Graft vs Leukemia Effect/genetics , Graft vs Leukemia Effect/immunology , Interleukin-15/genetics , Interleukin-15/immunology , Janus Kinase 1/genetics , Janus Kinase 1/immunology , Janus Kinase 2/genetics , Janus Kinase 2/immunology , Mice , Mice, Inbred BALB C , Mice, Knockout , Signal Transduction/genetics , Signal Transduction/immunologyABSTRACT
The fibrotic encapsulation of implants involves the mechanical activation of myofibroblasts and of pro-fibrotic transforming growth factor beta 1 (TGF-ß1). Here, we show that both softening of the implant surfaces and inhibition of the activation of TGF-ß1 reduce the fibrotic encapsulation of subcutaneous silicone implants in mice. Conventionally stiff silicones (elastic modulus, ~2 MPa) coated with a soft silicone layer (elastic modulus, ~2 kPa) reduced collagen deposition as well as myofibroblast activation without affecting the numbers of macrophages and their polarization states. Instead, fibroblasts around stiff implants exhibited enhanced intracellular stress, increased the recruitment of αv and ß1 integrins, and activated TGF-ß1 signalling. In vitro, the recruitment of αv integrin to focal adhesions and the activation of ß1 integrin and of TGF-ß were higher in myofibroblasts grown on latency-associated peptide (LAP)-coated stiff silicones than on soft silicones. Antagonizing αv integrin binding to LAP through the small-molecule inhibitor CWHM-12 suppressed active TGF-ß signalling, myofibroblast activation and the fibrotic encapsulation of stiff subcutaneous implants in mice.
Subject(s)
Prostheses and Implants , Silicones , Transforming Growth Factor beta , Animals , Fibroblasts , Fibrosis , Foreign-Body Reaction , Mice , Myofibroblasts/pathologyABSTRACT
Allogeneic hematopoietic transplantation (allo-HCT) is a curative therapy for a variety of hematologic malignancies, primarily through immune-mediated clearance of malignant cells. This graft-versus-leukemia (GvL) effect is mediated by alloreactive donor T-cells against recipient malignant cells. Unfortunately, graft versus host disease is a potentially lethal complication of this procedure, also mediated by alloreactive donor T-cells against recipient normal tissues. Graft-versus-host disease (GVHD) remains a key contributor to nonrelapse mortality and long-term morbidity in patients undergoing allo-HCT. Reducing GVHD without interfering with - or ideally while enhancing - GvL, would improve outcomes and increase patient eligibility for allo-HCT. The JAK/STAT signaling pathway acts downstream of over 50 cytokines and is central to a wide variety of inflammatory pathways. These pathways play a role in the development and maintenance of GVHD throughout the disease process and within T-cells, B-cells, macrophages, neutrophils, and natural killer cells. Agents targeting JAK/STAT signaling pathways have shown clinical efficacy and gained US Food and Drug Administration approval for numerous diseases. Here, we review the preclinical and clinical evidence for the role of JAK/STAT signaling in the development and maintenance of GVHD and the utility of blocking agents at preventing and treating GVHD.
Subject(s)
Disease Susceptibility , Graft vs Host Disease/drug therapy , Graft vs Host Disease/etiology , Integrin alpha4beta1/antagonists & inhibitors , Molecular Targeted Therapy , Animals , Biomarkers , Disease Models, Animal , H-2 Antigens/genetics , H-2 Antigens/immunology , Hematopoietic Stem Cell Transplantation , Integrin alpha4beta1/genetics , Integrin alpha4beta1/metabolism , Mice , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Transplantation, HomologousABSTRACT
Mobilized peripheral blood has become the primary source of hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation, with a five-day course of granulocyte colony stimulating factor (G-CSF) as the most common regimen used for HSPC mobilization. The CXCR4 inhibitor, plerixafor, is a more rapid mobilizer, yet not potent enough when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects. We sought to improve hematopoietic stem cell transplantation by developing a new mobilization strategy in mice through combined targeting of the chemokine receptor CXCR2 and the very late antigen 4 (VLA4) integrin. Rapid and synergistic mobilization of HSPCs along with an enhanced recruitment of true HSCs was achieved when a CXCR2 agonist was co-administered in conjunction with a VLA4 inhibitor. Mechanistic studies revealed involvement of CXCR2 expressed on BM stroma in addition to stimulation of the receptor on granulocytes in the regulation of HSPC localization and egress. Given the rapid kinetics and potency of HSPC mobilization provided by the VLA4 inhibitor and CXCR2 agonist combination in mice compared to currently approved HSPC mobilization methods, it represents an exciting potential strategy for clinical development in the future.
Subject(s)
Bone Marrow/metabolism , Hematopoietic Stem Cell Mobilization , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Integrin alpha4beta1 , Receptors, Interleukin-8B , Allografts , Animals , Granulocytes/metabolism , Integrin alpha4beta1/antagonists & inhibitors , Integrin alpha4beta1/genetics , Integrin alpha4beta1/metabolism , Mice , Mice, Inbred BALB C , Mice, Knockout , Receptors, Interleukin-8B/antagonists & inhibitors , Receptors, Interleukin-8B/genetics , Receptors, Interleukin-8B/metabolismABSTRACT
Owing to their exceptional physical, chemical, and mechanical properties, carbon nanotubes (CNTs) have been extensively studied for their effect on cellular behaviors. However, little is known about the process by which cells attach and spread on CNTs and the process for cell attachment and spreading on individual single-walled CNTs has not been studied. Cell adhesion and spreading is essential for cell communication and regulation and the mechanical interaction between cells and the underlying substrate can influence and control cell behavior and function. A limited number of studies have described different adhesion mechanisms, such as cellular process entanglements with multi-walled CNT aggregates or adhesion due to adsorption of serum proteins onto the nanotubes. Here, we hypothesized that cell attachment and spreading to both individual single-walled CNTs and multi-walled CNT aggregates is governed by the same mechanism. Specifically, we suggest that cell attachment and spreading on nanotubes is integrin-dependent and is facilitated by the adsorption of serum and cell-secreted adhesive proteins to the nanotubes.
ABSTRACT
Novel reversed isoniazid (RINH) agents were synthesized by covalently linking isoniazid with various efflux pump inhibitor (EPI) cores and their structural motifs. These RINH agents were then evaluated for anti-mycobacterial activity against sensitive, isoniazid mono-resistant and MDR clinical isolates of M. tuberculosis and a selected number of compounds were also tested ex vivo for intracellular activity as well as in the ethidium bromide (EB) assay for efflux pump inhibition efficacy. The potency of some compounds against various strains of M. tuberculosis (4a-c, 7 and 8; H37Rv-MIC99 ≤1.25⯵M, R5401-MIC99 ≤2.5⯵M, X_61-MIC99 ≤5⯵M) demonstrated the potential of the reversed anti-TB agent strategy towards the development of novel anti-mycobacterial agents to address the rapidly growing issue of resistance. Further, macrophage activity with >90% inhibition by 1a-c and 3b (MIC90 ≤13.42⯵M) and inhibition of EB efflux demonstrated by these compounds are encouraging.
Subject(s)
Antitubercular Agents/chemical synthesis , Drug Design , Isoniazid/chemistry , Animals , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/metabolism , CHO Cells , Cell Line , Cell Survival/drug effects , Cricetinae , Cricetulus , Humans , Ion Pumps/antagonists & inhibitors , Ion Pumps/metabolism , Isoniazid/chemical synthesis , Isoniazid/pharmacology , Microbial Sensitivity Tests , Mycobacterium tuberculosis/drug effects , Structure-Activity RelationshipABSTRACT
Efflux pumps are considered a major potential contributor to the development of various forms of resistance in Mycobacterium tuberculosis leading to the emergence of multidrug-resistant tuberculosis (TB). Verapamil (VER) and tricyclic chemosensitizers such as the phenothiazines are known to possess efflux pump inhibition properties and have demonstrated significant efficacy in various TB disease models. Novel hybrid molecules based on fusion of the VER substructure with various tricyclic, as well as nontricyclic, chemosensitizer cores or their structural motifs are described. These hybrid compounds were evaluated in vitro and ex vivo individually for their intrinsic activity and in combination for their potentiating potential with the frontline anti-TB drugs, rifampin and isoniazid. In addition, efflux pump inhibition was assessed in an ethidium bromide assay. This study led to the identification of novel compounds, termed hybrid efflux pump inhibitors, with intrinsic antimycobacterial activities (MIC90 ≤ 3.17 µg/mL) and intracellular activity in macrophages at a low concentration (≤6.25 µg/mL).
Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Mycobacterium tuberculosis/drug effects , Antitubercular Agents/chemical synthesis , Antitubercular Agents/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Drug Design , Humans , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Microbial Sensitivity Tests , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Tuberculosis/microbiologyABSTRACT
Racecadotril (acetorphan) is a neutral endopeptidase (NEP) inhibitor with known antidiarrheal activity in animals and humans; however, in humans, it suffers from shortcomings that might be improved with newer drugs in this class that have progressed to the clinic for nonenteric disease indications. To identify potentially superior NEP inhibitors with immediate clinical utility for diarrhea treatment, we compared their efficacy and pharmacologic properties in a rat intestinal hypersecretion model. Racecadotril and seven other clinical-stage inhibitors of NEP were obtained or synthesized. Enzyme potency and specificity were compared using purified peptidases. Compounds were orally administered to rats before administration of castor oil to induce diarrhea. Stool weight was recorded over 4 hours. To assess other pharmacologic properties, select compounds were orally administered to normal or castor oil-treated rats, blood and tissue samples collected at multiple time points, and active compound concentrations determined by mass spectroscopy. NEP enzyme activity was measured in tissue homogenates. Three previously untested clinical NEP inhibitors delayed diarrhea onset and reduced total stool output, with little or no effect on intestinal motility assessed by the charcoal meal test. Each was shown to be a potent, highly specific inhibitor of NEP. Each exhibited greater suppression of NEP activity in intestinal and nonintestinal tissues than did racecadotril and sustained this inhibition longer. These results suggest that newer clinical-stage NEP inhibitors originally developed for other indications may be directly repositioned for treatment of acute secretory diarrhea and offer advantages over racecadotril, such as less frequent dosing and potentially improved efficacy.
Subject(s)
Antidiarrheals/therapeutic use , Diarrhea/drug therapy , Endopeptidases/metabolism , Protease Inhibitors/therapeutic use , Thiorphan/analogs & derivatives , Animals , Castor Oil , Charcoal/pharmacology , Diarrhea/chemically induced , Dose-Response Relationship, Drug , Feces , Gastrointestinal Motility/drug effects , Male , Rats , Rats, Wistar , Thiorphan/therapeutic useABSTRACT
BACKGROUND & AIMS: Pancreatic stellate cells (PSCs) regulate the development of chronic pancreatitis (CP) and are activated by the cytokine transforming growth factor ß (TGFB). Integrins of the αv family promote TGFB signaling in mice, probably by interacting with the Arg-Gly-Asp (RGD) sequence of the TGFB latency-associated peptide, which frees TGFB to bind its cellular receptors. However, little is known about the role of integrins in the development of CP. We investigated the effects of small-molecule integrin inhibitors in a mouse model of CP. METHODS: We induced CP in C57BL/6 female mice by repeated cerulein administration. An active RGD peptidomimetic compound (Center for World Health and Medicine [CWHM]-12) was delivered by continuous infusion, starting 3 days before or 5 days after cerulein administration began. Pancreata were collected and parenchymal atrophy, fibrosis, and activation of PSCs were assessed by histologic, gene, and protein expression analyses. We measured CWHM-12 effects on activation of TGFB in co-culture assays in which rat PSC cells (large T immortalized cells [LTC-14]) activate expression of a TGFB-sensitive promoter in reporter cells. RESULTS: Pancreatic tissues of mice expressed messenger RNAs encoding subunits of RGD-binding integrins. Cerulein administration increased expression of these integrins, altered pancreatic cell morphology, and induced fibrosis. The integrin inhibitor CWHM-12 decreased acinar cell atrophy and loss, and substantially reduced fibrosis, activation of PSCs, and expression of genes regulated by TGFB. CWHM-12 also reduced established fibrosis in mice and blocked activation of TGFB in cultured cells. CONCLUSIONS: Based on studies of a mouse model of CP and cultured PSCs, integrins that bind RGD sequences activate PSCs and promote the development of pancreatic fibrogenesis in mice. Small-molecule antagonists of this interaction might be developed for treatment of pancreatic fibrotic diseases.
ABSTRACT
Matrix metalloproteinase-13 (MMP-13) is a zinc-dependent protease responsible for the cleavage of type II collagen, the major structural protein of articular cartilage. Degradation of this cartilage matrix leads to the development of osteoarthritis. We previously have described highly potent and selective carboxylic acid containing MMP-13 inhibitors; however, nephrotoxicity in preclinical toxicology species precluded development. The accumulation of compound in the kidneys mediated by human organic anion transporter 3 (hOAT3) was hypothesized as a contributing factor for the finding. Herein we report our efforts to optimize the MMP-13 potency and pharmacokinetic properties of non-carboxylic acid leads resulting in the identification of compound 43a lacking the previously observed preclinical toxicology at comparable exposures.
Subject(s)
Matrix Metalloproteinase 13/drug effects , Matrix Metalloproteinase Inhibitors/chemical synthesis , Matrix Metalloproteinase Inhibitors/pharmacology , Osteoarthritis/drug therapy , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Tetrazoles/chemical synthesis , Tetrazoles/pharmacology , Animals , Cartilage, Articular/drug effects , Cartilage, Articular/pathology , Collagenases/drug effects , Dogs , Drug Design , Humans , Kidney/metabolism , Macaca fascicularis , Male , Matrix Metalloproteinase Inhibitors/toxicity , Models, Molecular , Organic Anion Transporters, Sodium-Independent/metabolism , Protein Binding , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
Given the rise of parasite resistance to all currently used antimalarial drugs, the identification of novel chemotypes with unique mechanisms of action is of paramount importance. Since Plasmodium expresses a number of aspartic proteases necessary for its survival, we have mined antimalarial datasets for drug-like aspartic protease inhibitors. This effort led to the identification of spiropiperidine hydantoins, bearing similarity to known inhibitors of the human aspartic protease ß-secretase (BACE), as new leads for antimalarial drug discovery. Spiropiperidine hydantoins have a dynamic structure-activity relationship profile with positions identified as being tolerant of a variety of substitution patterns as well as a key piperidine N-benzyl phenol pharmacophore. Lead compounds 4e (CWHM-123) and 12k (CWHM-505) are potent antimalarials with IC50 values against Plasmodium falciparum 3D7 of 0.310 µM and 0.099 µM, respectively, and the former features equivalent potency on the chloroquine-resistant Dd2 strain. Remarkably, these compounds do not inhibit human aspartic proteases BACE, cathepsins D and E, or Plasmodium plasmepsins II and IV despite their similarity to known BACE inhibitors. Although the current leads suffer from poor metabolic stability, they do fit into a drug-like chemical property space and provide a new class of potent antimalarial agents for further study.
Subject(s)
Antimalarials/chemistry , Antimalarials/pharmacology , Hydantoins/chemistry , Hydantoins/pharmacology , Malaria, Falciparum/drug therapy , Plasmodium falciparum/drug effects , Animals , Antimalarials/metabolism , Antimalarials/pharmacokinetics , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolism , Drug Discovery , Humans , Hydantoins/metabolism , Hydantoins/pharmacokinetics , Malaria, Falciparum/parasitology , Mice , Microsomes, Liver/metabolism , Piperidines/chemistry , Piperidines/metabolism , Piperidines/pharmacokinetics , Piperidines/pharmacology , Plasmodium falciparum/enzymology , Plasmodium falciparum/metabolism , Rats , Spiro Compounds/chemistry , Spiro Compounds/metabolism , Spiro Compounds/pharmacokinetics , Spiro Compounds/pharmacologyABSTRACT
New verapamil analogues were synthesized and their inhibitory activities against Mycobacterium tuberculosis H37Rv determined in vitro alone and in combination with rifampicin (RIF). Some analogues showed comparable activity to verapamil and exhibited better synergies with RIF. Molecular docking studies of the binding sites of Rv1258c, a M. tuberculosis efflux protein previously implicated in intrinsic resistance to RIF, suggested a potential rationale for the superior synergistic interactions observed with some analogues.
Subject(s)
ATP-Binding Cassette Transporters/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/chemistry , Mycobacterium tuberculosis/drug effects , Rifampin/pharmacology , Verapamil/analogs & derivatives , Verapamil/pharmacology , ATP-Binding Cassette Transporters/metabolism , Anti-Bacterial Agents/chemical synthesis , Bacterial Proteins/metabolism , Binding Sites/drug effects , Dose-Response Relationship, Drug , Microbial Sensitivity Tests , Molecular Docking Simulation , Molecular Structure , Rifampin/chemistry , Structure-Activity Relationship , Verapamil/chemical synthesis , Verapamil/chemistryABSTRACT
Given the threat of drug resistance, there is an acute need for new classes of antimalarial agents that act via a unique mechanism of action relative to currently used drugs. We have identified a set of druglike compounds within the Tres Cantos Anti-Malarial Set (TCAMS) which likely act via inhibition of a Plasmodium aspartic protease. Structure-activity relationship analysis and optimization of these aminohydantoins demonstrate that these compounds are potent nanomolar inhibitors of the Plasmodium aspartic proteases PM-II and PM-IV and likely one or more other Plasmodium aspartic proteases. Incorporation of a bulky group, such as a cyclohexyl group, on the aminohydantion N-3 position gives enhanced antimalarial potency while reducing inhibition of human aspartic proteases such as BACE. We have identified compound 8p (CWHM-117) as a promising lead for optimization as an antimalarial drug with a low molecular weight, modest lipophilicity, oral bioavailability, and in vivo antimalarial activity in mice.
ABSTRACT
Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency. We used this system to delete the gene encoding α(v) integrin subunit because various α(v)-containing integrins have been suggested as central mediators of fibrosis in multiple organs. Such depletion protected mice from carbon tetrachloride-induced hepatic fibrosis, whereas global loss of ß3, ß5 or ß6 integrins or conditional loss of ß8 integrins in HSCs did not. We also found that Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of the α(v) integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of α(v)-containing integrins by a small molecule (CWHM 12) attenuated both liver and lung fibrosis, including in a therapeutic manner. These data identify a core pathway that regulates fibrosis and suggest that pharmacological targeting of all α(v) integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.
Subject(s)
Integrin alphaV/metabolism , Kidney Diseases/genetics , Kidney/pathology , Liver Cirrhosis/genetics , Pulmonary Fibrosis/genetics , Animals , Cells, Cultured , Drug Evaluation, Preclinical , Female , Fibrosis/genetics , Gene Targeting , Integrin alphaV/genetics , Kidney/metabolism , Kidney Diseases/pathology , Liver Cirrhosis/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myofibroblasts/metabolism , Myofibroblasts/pathology , Pulmonary Fibrosis/pathology , Signal Transduction/physiologyABSTRACT
Foxp3(+) CD4(+) T helper cells called regulatory T (T reg) cells play a key role in controlling reactivity to self-antigens and onset of autoimmunity. T reg cells either arise in thymus and are called natural T reg (nT reg) cells or are generated in the periphery through induction of Foxp3 and are called inducible T reg (iT reg) cells. The relative contributions of iT reg cells and nT reg cells in peripheral tolerance remain unclear as a result of an inability to separate these two subsets of T reg cells. Using a combination of novel TCR transgenic mice with a defined self-antigen specificity and conventional mouse models, we demonstrate that a cell surface molecule, neuropilin-1 (Nrp-1), is expressed at high levels on nT reg cells and can be used to separate nT reg versus iT reg cells in certain physiological settings. In addition, iT reg cells generated through antigen delivery or converted under homeostatic conditions lack Nrp-1 expression. Nrp-1(lo) iT reg cells show similar suppressive activity to nT reg cells in controlling ongoing autoimmune responses under homeostatic conditions. In contrast, their activity might be compromised in certain lymphopenic settings. Collectively, our data show that Nrp-1 provides an excellent marker to distinguish distinct T reg subsets and will be useful in studying the role of nT reg versus iT reg cells in different disease settings.