ABSTRACT
Following antigen-driven expansion in lymph node, transforming growth factor-ß (TGFß) is required for differentiation of skin-recruited CD8+ T cell effectors into epidermal resident memory T (Trm) cells and their epidermal persistence. We found that the source of TGFß -supporting Trm cells was autocrine. In addition, antigen-specific Trm cells that encountered cognate antigen in the skin, and bystander Trm cells that did not, both displayed long-term persistence in the epidermis under steady-state conditions. However, when the active-TGFß was limited or when new T cell clones were recruited into the epidermis, antigen-specific Trm cells were more efficiently retained than bystander Trm cells. Genetically enforced TGFßR signaling allowed bystander Trm cells to persist in the epidermis as efficiently as antigen-specific Trm cells in both contexts. Thus, competition between T cells for active TGFß represents an unappreciated selective pressure that promotes the accumulation and persistence of antigen-specific Trm cells in the epidermal niche.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Epidermis/immunology , Keratinocytes/immunology , T-Lymphocytes, Regulatory/immunology , Transforming Growth Factor beta/metabolism , Animals , Binding, Competitive , Bystander Effect , Cellular Microenvironment , Clone Cells , Immunologic Memory , Mice , Mice, Inbred C57BL , Organ Specificity , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Signal Transduction , T-Cell Antigen Receptor SpecificityABSTRACT
The epidermal growth factor receptor ligand Amphiregulin has a well-documented role in the restoration of tissue homeostasis after injury; however, the mechanism by which Amphiregulin contributes to wound repair remains unknown. Here we show that Amphiregulin functioned by releasing bioactive transforming growth factor beta (TGF-ß) from latent complexes via integrin-αV activation. Using acute injury models in two different tissues, we found that by inducing TGF-ß activation on mesenchymal stromal cells (pericytes), Amphiregulin induced their differentiation into myofibroblasts, thereby selectively contributing to the restoration of vascular barrier function within injured tissue. Furthermore, we identified macrophages as a critical source of Amphiregulin, revealing a direct effector mechanism by which these cells contribute to tissue restoration after acute injury. Combined, these observations expose a so far under-appreciated mechanism of how cells of the immune system selectively control the differentiation of tissue progenitor cells during tissue repair and inflammation.
Subject(s)
Amphiregulin/metabolism , Macrophages/metabolism , Pericytes/metabolism , Transforming Growth Factor beta/metabolism , Animals , Cell Differentiation/physiology , Female , Male , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Myofibroblasts/metabolismABSTRACT
Volumetric muscle loss (VML) causes irrecoverable loss of muscle mass and strength and results in permanent disability. VML injury shows extensive fibrosis, which impedes functional tissue regeneration. Our lab has created a biosponge scaffold composed of extracellular matrix (ECM) proteins (i.e., biosponge) that can enhance muscle regeneration and function following VML. In this work, a potent small molecule inhibitor of alpha v-subunit containing integrins known as IDL-2965 was incorporated into the biosponges for localized suppression of fibrosis post-VML. Our results demonstrate that local delivery of IDL-2965 via the biosponges attenuated the deposition of fibrotic tissue preceded by a downregulation of profibrotic genes in VML-injured muscles. The reduction in fibrotic tissue had no detrimental effects on muscle mass, function, size, or vascularity. Overall, these findings suggest that the codelivery of biosponges and IDL-2965 is a safe and effective strategy for the mitigation of fibrotic tissue deposition in VML-injured muscles.
Subject(s)
Muscle, Skeletal , Muscular Diseases , Humans , Muscle, Skeletal/metabolism , Muscular Diseases/pathology , Wound Healing , Extracellular Matrix Proteins/metabolism , FibrosisABSTRACT
The intestinal protozoan Cryptosporidium is a leading cause of diarrheal disease and mortality in young children. There is currently no fully effective treatment for cryptosporidiosis, which has stimulated interest in anticryptosporidial development over the last â¼10 years, with numerous lead compounds identified, including several tRNA synthetase inhibitors. Here, we report the results of a dairy calf efficacy trial of the methionyl-tRNA (Cryptosporidium parvum MetRS [CpMetRS]) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with Cryptosporidium parvum initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day 5. Parasites shed by each recrudescent calf had different amino acid-altering mutations in the gene encoding CpMetRS (CpMetRS), yielding either an aspartate 243-to-glutamate (D243E) or a threonine 246-to-isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246I CpMetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant-expressing parasites, respectively, had 2093 half-maximal effective concentrations (EC50s) that were 613- and 128-fold that of transgenic parasites with wild-type CpMetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC50 >170-fold. Structural modeling of CpMetRS based on an inhibitor-bound Trypanosoma brucei MetRS crystal structure suggested that the resistance mutations reposition nearby hydrophobic residues, interfering with compound binding while minimally impacting substrate binding. This is the first report of naturally emerging Cryptosporidium drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence.
Subject(s)
Cattle Diseases , Cryptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Animals , Cattle , Cattle Diseases/drug therapy , Child , Child, Preschool , Cryptosporidiosis/drug therapy , Cryptosporidium/genetics , Cryptosporidium parvum/genetics , Drug Resistance/genetics , Feces , HumansABSTRACT
BACKGROUND: Cryptosporidiosis, an enteric protozoon, causes substantial morbidity and mortality associated with diarrhea in children <2 years old in low- to middle-income countries. There is no vaccine and treatments are inadequate. A piperazine-based compound, MMV665917, has in vitro and in vivo efficacy against Cryptosporidium parvum. In this study, we evaluated the efficacy of MMV665917 in gnotobiotic piglets experimentally infected with Cryptosporidium hominis, the species responsible for >75% of diarrhea reported in these children. METHODS: Gnotobiotic piglets were orally challenged with C hominis oocysts, and oral treatment with MMV665917 was commenced 3 days after challenge. Oocyst excretion and diarrhea severity were observed daily, and mucosal colonization and lesions were recorded after necropsy. RESULTS: MMV665917 significantly reduced fecal oocyst excretion, parasite colonization and damage to the intestinal mucosa, and peak diarrheal symptoms, compared with infected untreated controls. A dose of 20 mg/kg twice daily for 7 days was more effective than 10 mg/kg. There were no signs of organ toxicity at either dose, but 20 mg/kg was associated with slightly elevated blood cholesterol and monocytes at euthanasia. CONCLUSIONS: These results demonstrate the effectiveness of this drug against C hominis. Piperazine-derivative MMV665917 may potentially be used to treat human cryptosporidiosis; however, further investigations are required.
Subject(s)
Cryptosporidiosis/drug therapy , Cryptosporidium parvum/drug effects , Diarrhea/drug therapy , Piperazines/pharmacology , Animals , Cryptosporidiosis/parasitology , Diarrhea/parasitology , Disease Models, Animal , Intestinal Mucosa/parasitology , Monocytes/parasitology , Oocysts/drug effects , SwineABSTRACT
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
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
Our previous work identified compound 1 (SLU-2633) as a potent lead compound toward the identification of a novel treatment for cryptosporidiosis, caused by the parasite Cryptosporidium (EC50 = 0.17 µM). While this compound is potent and orally efficacious, the mechanism of action and biological target(s) of this series are currently unknown. In this study, we synthesized 70 compounds to develop phenotypic structure-activity relationships around the aryl "tail" group. In this process, we found that 2-substituted compounds are inactive, confirmed that electron withdrawing groups are preferred over electron donating groups, and that fluorine plays a remarkable role in the potency of these compounds. The most potent compound resulting from this work is SLU-10482 (52, EC50 = 0.07 µΜ), which was found to be orally efficacious with an ED90 < 5 mg/kg BID in a Cryptosporidium-infection mouse model, superior to SLU-2633.
Subject(s)
Cryptosporidiosis , Cryptosporidium , Mice , Animals , Cryptosporidiosis/drug therapy , Fluorine , Structure-Activity RelationshipABSTRACT
Tuberculosis (TB) caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb) is one of the most lethal infectious diseases in the world. Presently, Bacillus Calmette-Guerin, the vaccine approved for use against TB, does not offer complete protection against the disease, which necessitates the development of new therapeutics to treat this infection. Overexpression of transforming growth factor beta (TGF-ß) is associated with pulmonary profibrotic changes. The inactive TGF-ß secreted is activated through its cleavage and release by αv integrins. Integrin-mediated regulation of TGF-ß is considered as a master switch in the profibrotic process and a potential therapeutic target. Thus, in this study, we sought to determine if treatment with a broad range antagonist of integrins, CWHM-12, has the potency to inhibit pulmonary fibrosis and enhance Mtb control in a highly susceptible mouse model of Mtb infection, namely the C3Heb/FeJ (FeJ). CWHM-12 treatment at the early stages of Mtb infection was efficacious in reducing disease severity and inflammation associated with decreased iNOS, MIP-2, and IL-10 production without degradation of collagen. This suggests a potential for CWHM-12 targeting of TGF-ß to be explored as an adjunct therapeutic for early Mtb infection.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Animals , Integrins , Mice , Transforming Growth Factor beta/metabolism , Transforming Growth Factors , Tuberculosis/drug therapy , Tuberculosis/prevention & controlABSTRACT
We previously showed that the anti-fungal drug ciclopirox olamine effectively inhibits replication of herpes simplex virus (HSV)-1 and HSV-2. Given the rise of HSV strains that are resistant to nucleos(t)ide analog treatment, as well as the incomplete efficacy of nucleos(t)ide analogs, new inhibitory compounds must be explored for potential use in the treatment of HSV infection. In the present study, we analyzed 44 compounds derived from the core structure of ciclopirox olamine for inhibitory activity against HSV. Thirteen of these derivative compounds inhibited HSV-2 replication by > 1000- to â¼100,000-fold at 1 µM and displayed EC50 values lower than that of acyclovir, as well as low cytotoxicity, indicating their strong therapeutic potential. Through structural comparison, we also provide evidence for the importance of various structural motifs to the efficacy of ciclopirox and its derivatives, namely hydrophobic groups at R4 and R6 of the ciclopirox core structure. Like ciclopirox, representative analogs exhibit some oral bioavailability but are rapidly cleared in vivo. Together, these results will guide further development of N-hydroxypyridones as HSV therapeutics.
Subject(s)
Herpes Simplex , Herpesvirus 1, Human , Acyclovir/chemistry , Acyclovir/pharmacology , Antifungal Agents/pharmacology , Antifungal Agents/therapeutic use , Antiviral Agents/therapeutic use , Ciclopirox/pharmacology , Ciclopirox/therapeutic use , Herpes Simplex/drug therapy , Herpesvirus 2, Human , Humans , Virus ReplicationABSTRACT
Cryptosporidiosis is caused by infection of the small intestine by Cryptosporidium parasites, resulting in severe diarrhea, dehydration, malabsorption, and potentially death. The only FDA-approved therapeutic is only partially effective in young children and ineffective for immunocompromised patients. Triazolopyridazine MMV665917 is a previously reported anti-Cryptosporidium screening hit with in vivo efficacy but suffers from modest inhibition of the hERG ion channel, which could portend cardiotoxicity. Herein, we describe our initial development of structure-activity relationships of this novel lead series with a particular focus on optimization of the piperazine-urea linker. We have discovered that piperazine-acetamide is a superior linker resulting in identification of SLU-2633, which has an EC50 of 0.17 µM, an improved projected margin versus hERG, prolonged pharmacokinetic exposure in small intestine, and oral efficacy in vivo with minimal systemic exposure. SLU-2633 represents a significant advancement toward the identification of a new effective and safe treatment for cryptosporidiosis.
Subject(s)
Antiprotozoal Agents/pharmacology , Cryptosporidiosis/drug therapy , Cryptosporidium/drug effects , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Antiprotozoal Agents/chemical synthesis , Antiprotozoal Agents/chemistry , Cell Line , Dose-Response Relationship, Drug , Ether-A-Go-Go Potassium Channels/metabolism , Humans , Molecular Structure , Parasitic Sensitivity Tests , Structure-Activity RelationshipABSTRACT
Fibrosis is a final common pathway for many causes of progressive chronic kidney disease (CKD). Arginine-glycine-aspartic acid (RGD)-binding integrins are important mediators of the pro-fibrotic response by activating latent TGF-ß at sites of injury and by providing myofibroblasts information about the composition and stiffness of the extracellular matrix. Therefore, blockade of RGD-binding integrins may have therapeutic potential for CKD. To test this idea, we used small-molecule peptidomimetics that potently inhibit a subset of RGD-binding integrins in a murine model of kidney fibrosis. Acute kidney injury leading to fibrosis was induced by administration of aristolochic acid. Continuous subcutaneous administration of CWHM-12, an RGD integrin antagonist, for 28 days improved kidney function as measured by serum creatinine. CWHM-12 significantly reduced Collagen 1 (Col1a1) mRNA expression and scar collagen deposition in the kidney. Protein and gene expression markers of activated myofibroblasts, a major source of extracellular matrix deposition in kidney fibrosis, were diminished by treatment. RNA sequencing revealed that inhibition of RGD integrins influenced multiple pathways that determine the outcome of the response to injury and of repair processes. A second RGD integrin antagonist, CWHM-680, administered once daily by oral gavage was also effective in ameliorating fibrosis. We conclude that targeting RGD integrins with such small-molecule antagonists is a promising therapeutic approach in fibrotic kidney disease.
Subject(s)
Acute Kidney Injury/drug therapy , Antineoplastic Agents/pharmacology , Integrins/antagonists & inhibitors , Oligopeptides/antagonists & inhibitors , Peptidomimetics/pharmacology , Acute Kidney Injury/metabolism , Acute Kidney Injury/pathology , Animals , Collagen/metabolism , Fibrosis/metabolism , Fibrosis/pathology , Fibrosis/prevention & control , Integrins/metabolism , Male , Mice , Mice, Inbred ICR , Oligopeptides/metabolism , Oligopeptides/pharmacology , Small Molecule Libraries/pharmacologyABSTRACT
The presence and stage of liver fibrosis in patients with nonalcoholic steatohepatitis (NASH) is strongly associated with mortality. Thus, both preventing and reversing fibrosis are critically important approaches to prevent death or the need for liver transplantation from NASH. Recently, fibrosis in several mouse models of organ injury was shown to be prevented and reversed with the potent small molecule, arginine-glycine-aspartic acid tripeptide (RGD)-binding, integrin antagonist (3S)-3-(3-bromo-5-(tert-butyl)phenyl)-3-(2-(3-hydroxy-5-((5-hydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)amino)benzamido)acetamido)propanoic acid (Center for World Health and Medicine [CWHM]-12). We hypothesized that RGD-binding integrins may play an important role in fibrosis progression in NASH. We assessed the efficacy of CWHM-12 in a choline deficient, amino-acid defined, high-fat diet (CDAHFD) mouse model of NASH. Mice were kept on the CDAHFD or a control diet for 10 weeks, and CWHM-12 was delivered by continuous infusion for the final 4 weeks. The parameters of NASH and liver fibrosis were evaluated before and after drug treatment. Hepatic steatosis, liver injury, and inflammation were significantly induced by the CDAHFD at week 6 and did not change by week 10. Hepatic profibrogenic gene expression was induced by the CDAHFD at week 6, further increased at week 10, and decreased by CWHM-12. Fibrosis measured by analysis of liver collagen was reduced by CWHM-12 to levels significantly less than found at 6 weeks, demonstrating the possibility of reversing already established fibrosis despite ongoing injury. Demonstrated mechanisms of the antifibrotic effect of CWHM-12 included loss of activated hepatic stellate cells through apoptosis and suppression of hepatic profibrotic signal transduction by transforming growth factor ß. Conclusion: RGD-binding integrins may be critical in the development of fibrosis in NASH and may represent potential targets for treating patients with NASH to reverse advanced liver fibrosis.
ABSTRACT
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
Proteolytic degradation of the major cartilage macromolecules, aggrecan and type II collagen, is a key pathological event in osteoarthritis (OA). ADAMTS-4 and ADAMTS-5, the primary aggrecanases capable of cartilage aggrecan cleavage, are synthesized as latent enzymes and require prodomain removal for activity. The N-termini of the mature proteases suggest that activation involves a proprotein convertase, but the specific family member responsible for aggrecanase activation in cartilage in situ has not been identified. Here we describe purification of a proprotein convertase activity from human OA cartilage. Through biochemical characterization and the use of siRNA, PACE4 was identified as a proprotein convertase responsible for activation of aggrecanases in osteoarthritic and cytokine-stimulated cartilage. Posttranslational activation of ADAMTS-4 and ADAMTS-5 was observed in the extracellular milieu of cartilage, resulting in aggrecan degradation. These findings suggest that PACE4 represents a novel target for the development of OA therapeutics.
Subject(s)
Cartilage/enzymology , Endopeptidases/chemistry , Enzyme Activation , Proprotein Convertases/metabolism , Serine Endopeptidases/metabolism , ADAM Proteins/metabolism , ADAMTS4 Protein , ADAMTS5 Protein , Aged , Aged, 80 and over , Cartilage/metabolism , Humans , Kinetics , Middle Aged , Models, Biological , Osteoarthritis/metabolism , Procollagen N-Endopeptidase/metabolism , Protein Processing, Post-TranslationalABSTRACT
Cryptosporidiosis causes life-threatening diarrhea in infants, but the best available treatment is only modestly efficacious. Rodents infected with relevant Cryptosporidium species do not develop diarrhea, which complicates drug development. Cryptosporidium parvum infection of dairy calves, however, causes an illness like that seen in infants. Here, the clinical and microbiologic anti-Cryptosporidium efficacy of the piperazine-based compound MMV665917 was demonstrated in neonatal calves. Oral administration of MMV665917 (22 mg/kg once daily) was begun two days after the onset of severe diarrhea and continued for seven days. Treatment resulted in prompt resolution of diarrhea, and reduced total fecal oocyst shedding by ~94%. MMV665917 was useful for treatment, rather than just prophylaxis, since it was safe and effective when administered well after the onset of diarrhea. Furthermore, even though all animals received intensive supportive care, there was a strong trend towards improved secondary health outcomes, including general health, appetite, and dehydration measures amongst treated animals. These data establish MMV665917 as an outstanding lead compound for Cryptosporidium drug development.
Subject(s)
Antiprotozoal Agents/administration & dosage , Antiprotozoal Agents/pharmacology , Cryptosporidiosis/drug therapy , Cryptosporidium/drug effects , Piperazines/administration & dosage , Piperazines/pharmacology , Administration, Oral , Animals , Animals, Newborn , Antinematodal Agents , Cattle , Cryptosporidiosis/parasitology , Diarrhea/drug therapy , Diarrhea/parasitology , Disease Models, Animal , Feces/parasitology , Parasite Load , PiperazineABSTRACT
The angiogenic inducer CYR61 is differentially overexpressed in breast cancer cells exhibiting high levels of Heregulin (HRG), a growth factor closely associated with a metastatic breast cancer phenotype. Here, we examined whether CYR61, independently of HRG, actively regulates breast cancer cell survival and chemosensitivity, and the pathways involved. Forced expression of CYR61 in HRG-negative MCF-7 cells notably upregulated the expression of its own integrin receptor alphavbeta3 (>200 times). Small peptidomimetic alphavbeta3 integrin antagonists dramatically decreased cell viability of CYR61-overexpressing MCF-7 cells, whereas control MCF-7/V remained insensitive. Mechanistically, functional blockade of alphavbeta3 specifically abolished CYR6-induced hyperactivation of ERK1/ERK2 MAPK, whereas the activation status of AKT did not decrease. Moreover, CYR61 overexpression rendered MCF-7 cells significantly resistant (>10-fold) to Taxol-induced cytotoxicity. Remarkably, alphavbeta3 inhibition converted the CYR61-induced Taxol-resistant phenotype into a hypersensitive one. Thus, the augmentation of Taxol-induced apoptotic cell death in the presence of alphavbeta3 antagonists demonstrated a strong synergism as verified by the terminal transferase-mediated dUTP nick-end labeling (TUNEL) assay and by flow cytometric analysis for DNA content. Indeed, functional blockade of alphavbeta3, similarly to the pharmacological MAPK inhibitor U0126, synergistically increased both the proportion of CYR61-overexpressing breast cancer cells in the G2 phase of the cell cycle and the appearance of sub-G1 hypodiploid (apoptotic) cells caused by Taxol. Strikingly, CYR61 overexpression impaired the accumulation of wild-type p53 following Taxol exposure, while inhibition of alphavbeta3 or ERK1/ERK2 MAPK signalings completely restored Taxol-induced upregulation of p53. Moreover, antisense downregulation of CYR61 expression abolished the anchorage-independent growth of breast cancer cells engineered to overexpress HRG, and significantly increased their sensitivity to Taxol. Our data provide evidence that CYR61 is sufficient to promote breast cancer cell proliferation, cell survival, and Taxol resistance through a alphavbeta3-activated ERK1/ERK2 MAPK signaling. The identification of a 'CYR61-alphavbeta3 autocrine loop' in the epithelial compartment of breast carcinoma strongly suggests that targeting alphavbeta3 may simultaneously prevent breast cancer angiogenesis, growth, and chemoresistance.
Subject(s)
Breast Neoplasms/pathology , Cell Survival/physiology , Immediate-Early Proteins/physiology , Integrins/physiology , Intercellular Signaling Peptides and Proteins/physiology , MAP Kinase Signaling System/physiology , Receptors, Vitronectin/physiology , Angiogenesis Inducing Agents , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Cysteine-Rich Protein 61 , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immediate-Early Proteins/genetics , Intercellular Signaling Peptides and Proteins/genetics , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Paclitaxel/pharmacology , Tumor Suppressor Protein p53/geneticsABSTRACT
The involvement of alpha(v)beta3 and alpha(v)beta5 integrins in angiogenesis and the use of integrin antagonists as effective antiangiogenic agents are documented. Radiotherapy is an important therapy option for cancer. It has been shown that ionizing radiation exerts primarily antiangiogenic effects in tumors but has also proangiogenic effects as the reaction of the tumor to protect its own vasculature from radiation damage. Here, we show that combined treatment with S247, an Arg-Gly-Glu peptidomimetic antagonist of alpha(v)beta3 integrin, and external beam radiotherapy are beneficial in local tumor therapy. We found that radiation up-regulates alpha(v)beta3 expression in endothelial cells and consecutively phosphorylates Akt, which may provide a tumor escape mechanism from radiation injury mediated by integrin survival signaling. In the presence of S247, the radiation-induced Akt phosphorylation is strongly inhibited. Our studies on endothelial cell proliferation, migration, tube formation, apoptosis, and clonogenic survival show that the radiosensitivity of endothelial cells is enhanced by the concurrent administration of the integrin antagonist. The in vitro data are successfully translated into human glioma (U87), epidermoid (A431), and prostate cancer (PC3) xenograft models growing s.c. on BALB/c-nu/nu mice. In vivo, the combination of S247 treatment and fractionated radiotherapy (5 x 2.5 Gy) leads to enhanced antiangiogenic and antitumor effects compared with either monotherapies. These results underline the importance of alpha(v)beta3 integrin when tumors protect their microvasculature from radiation-induced damage. The data also indicate that the combination of integrin antagonists and radiotherapy represents a rational approach in local cancer therapy.
Subject(s)
Carcinoma, Squamous Cell/radiotherapy , Glioma/radiotherapy , Integrin alphaVbeta3/antagonists & inhibitors , Neovascularization, Pathologic/prevention & control , Organic Chemicals/therapeutic use , Prostatic Neoplasms/radiotherapy , Animals , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Apoptosis/radiation effects , Carcinoma, Squamous Cell/blood supply , Carcinoma, Squamous Cell/metabolism , Cell Movement/drug effects , Cell Movement/radiation effects , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Combined Modality Therapy , Endothelium, Vascular/metabolism , Endothelium, Vascular/radiation effects , Glioma/blood supply , Glioma/metabolism , Humans , Integrin alphaVbeta3/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Phosphorylation , Prostatic Neoplasms/blood supply , Prostatic Neoplasms/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Tumor Stem Cell Assay , Xenograft Model Antitumor AssaysABSTRACT
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
Integrins expressed on endothelial cells modulate cell migration and survival during angiogenesis. Integrins expressed on carcinoma cells potentiate metastasis by facilitating invasion and movement across blood vessels. We describe the activities of two synthetic low-molecular-weight peptidomimetics of the ligand amino acid sequence arg-gly-asp (RGD) in integrin-based functional assays in vitro. We also evaluate efficacy and potential mechanisms of action in models of both spontaneous and experimental metastasis. Broad-spectrum potency against the family of alpha v subunit-containing integrins was observed, with significantly less potency against alpha5beta1 and alpha(IIb)beta3. Both endothelial and tumor cell migration mediated by alpha(v)beta3 was inhibited, whereas proliferation of endothelial cells but not tumor cells was diminished. Continuous infusion of compound by minipumps or oral administration twice daily significantly reduced metastatic tumor burden in the lungs of mice despite no reduction in growth of 435/HAL primary tumors, and only a slight reduction in tumor cells detected in circulating blood. Delaying treatment in this model until after extensive dissemination of tumor cells to the lungs had occurred, and after primary tumor resection, still produced significant efficacy. Conversely, administration of the agent for only the first 18 h after tumor-cell inoculation into the tail vein also resulted in decreased metastases observed after several weeks. These data suggest these compounds or their relatives have potential to interfere with both early and late steps of metastasis involving tumor and endothelial cell functions. Furthermore, the metastatic process can be effectively inhibited independently of primary tumor growth using integrin antagonists.