ABSTRACT
The lysyl oxidase family represents a promising target in stromal targeting of solid tumors due to the importance of this family in crosslinking and stabilizing fibrillar collagens and its known role in tumor desmoplasia. Using small-molecule drug-design approaches, we generated and validated PXS-5505, a first-in-class highly selective and potent pan-lysyl oxidase inhibitor. We demonstrate in vitro and in vivo that pan-lysyl oxidase inhibition decreases chemotherapy-induced pancreatic tumor desmoplasia and stiffness, reduces cancer cell invasion and metastasis, improves tumor perfusion and enhances the efficacy of chemotherapy in the autochthonous genetically engineered KPC model, while also demonstrating antifibrotic effects in human patient-derived xenograft models of pancreatic cancer. PXS-5505 is orally bioavailable, safe and effective at inhibiting lysyl oxidase activity in tissues. Our findings present the rationale for progression of a pan-lysyl oxidase inhibitor aimed at eliciting a reduction in stromal matrix to potentiate chemotherapy in pancreatic ductal adenocarcinoma.
Subject(s)
Pancreatic Diseases , Pancreatic Neoplasms , Humans , Gemcitabine , Protein-Lysine 6-Oxidase , Pancreatic Neoplasms/drug therapyABSTRACT
Scarring is a lifelong consequence of skin injury, with scar stiffness and poor appearance presenting physical and psychological barriers to a return to normal life. Lysyl oxidases are a family of enzymes that play a critical role in scar formation and maintenance. Lysyl oxidases stabilize the main component of scar tissue, collagen, and drive scar stiffness and appearance. Here we describe the development and characterisation of an irreversible lysyl oxidase inhibitor, PXS-6302. PXS-6302 is ideally suited for skin treatment, readily penetrating the skin when applied as a cream and abolishing lysyl oxidase activity. In murine models of injury and fibrosis, topical application reduces collagen deposition and cross-linking. Topical application of PXS-6302 after injury also significantly improves scar appearance without reducing tissue strength in porcine injury models. PXS-6302 therefore represents a promising therapeutic to ameliorate scar formation, with potentially broader applications in other fibrotic diseases.
Subject(s)
Cicatrix , Protein-Lysine 6-Oxidase , Animals , Cicatrix/drug therapy , Collagen , Fibrosis , Mice , Skin , SwineABSTRACT
The discovery of a dual MAO-B/SSAO inhibitor PXS-5131 is reported. The compound offers a compact and rigid three-dimensional structure with superior selectivity over MAO-A. Potency and selectivity are linked to both the double bond geometry and stereochemistry of the allylamine moiety, highlighting the importance of optimal set up of these features in the class of amine oxidase inhibitors. PXS-5131 possesses an attractive preclinical pharmacokinetic profile and has anti-inflammatory properties in models of acute inflammation and neuroinflammation.
Subject(s)
Amine Oxidase (Copper-Containing) , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Monoamine Oxidase , Monoamine Oxidase Inhibitors/pharmacologyABSTRACT
Systemic sclerosis (SSc) is characterised by progressive multiple organ fibrosis leading to morbidity and mortality. Lysyl oxidases play a vital role in the cross-linking of collagens and subsequent build-up of fibrosis in the extracellular matrix. As such, their inhibition provides a novel treatment paradigm for SSc. A novel small molecule pan-lysyl oxidase inhibitor, PXS-5505, currently in clinical development for myelofibrosis treatment was evaluated using in vivo rodent models resembling the fibrotic conditions in SSc. Both lysyl oxidase and lysyl oxidase-like 2 (LOXL2) expression were elevated in the skin and lung of SSc patients. The oral application of PXS-5505 inhibited lysyl oxidase activity in the skin and LOXL2 activity in the lung. PXS-5505 exhibited anti-fibrotic effects in the SSc skin mouse model, reducing dermal thickness and α-smooth muscle actin. Similarly, in the bleomycin-induced mouse lung model, PXS-5505 reduced pulmonary fibrosis toward normal levels, mediated by its ability to normalise collagen/elastin crosslink formation. PXS-5505 also reduced fibrotic extent in models of the ischaemia-reperfusion heart, the unilateral ureteral obstruction kidney, and the CCl4-induced fibrotic liver. PXS-5505 consistently demonstrates potent anti-fibrotic efficacy in multiple models of organ fibrosis relevant to the pathogenesis of SSc, suggesting that it may be efficacious as a novel approach for treating SSc.
Subject(s)
Collagen , Enzyme Inhibitors , Protein-Lysine 6-Oxidase , Scleroderma, Systemic , Animals , Collagen/antagonists & inhibitors , Collagen/metabolism , Disease Models, Animal , Fibrosis , Humans , Mice , Protein-Lysine 6-Oxidase/antagonists & inhibitors , Protein-Lysine 6-Oxidase/metabolism , Rodentia/metabolism , Scleroderma, Systemic/drug therapy , Scleroderma, Systemic/enzymologySubject(s)
Amino Acid Oxidoreductases/pharmacology , Antifibrotic Agents/pharmacology , Biosensing Techniques/instrumentation , Biosensing Techniques/standards , Amino Acid Oxidoreductases/analysis , Amino Acid Oxidoreductases/therapeutic use , Antifibrotic Agents/analysis , Antifibrotic Agents/therapeutic use , Biosensing Techniques/methods , Fibrosis/drug therapy , Fibrosis/prevention & control , HumansABSTRACT
Lysyl oxidase-like 2 (LOXL2) is a secreted enzyme that catalyzes the formation of cross-links in extracellular matrix proteins, namely, collagen and elastin, and is indicated in fibrotic diseases. Herein, we report the identification and subsequent optimization of a series of indole-based fluoroallylamine inhibitors of LOXL2. The result of this medicinal chemistry campaign is PXS-5120A (12k), a potent, irreversible inhibitor that is >300-fold selective for LOXL2 over LOX. PXS-5120A also shows potent inhibition of LOXL3, an emerging therapeutic target for lung fibrosis. Key to the development of this compound was the utilization of a compound oxidation assay. PXS-5120A was optimized to show negligible substrate activity in vitro for related amine oxidase family members, leading to metabolic stability. PXS-5120A, in a pro-drug form (PXS-5129A, 12o), displayed anti-fibrotic activity in models of liver and lung fibrosis, thus confirming LOXL2 as an important target in diseases where collagen cross-linking is implicated.
Subject(s)
Amines/chemistry , Amines/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Protein-Lysine 6-Oxidase/antagonists & inhibitors , Animals , Drug Design , Humans , Mice , Triazoles/chemistryABSTRACT
Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm (MPN) that usually portends a poor prognosis with limited therapeutic options available. Currently, only allogeneic stem cell transplantation is curative in those who are candidates, while administration of the JAK1/2 inhibitor ruxolitinib carries a risk of worsening cytopenia. The limited therapeutic options available highlight the need for the development of novel treatments for PMF. Lysyl oxidase (LOX), an enzyme vital for collagen cross-linking and extracellular matrix stiffening, has been found to be upregulated in PMF. Herein, we evaluate two novel LOX inhibitors, PXS-LOX_1 and PXS-LOX_2, in two animal models of PMF (GATA1low and JAK2V617F-mutated mice). Specifically, PXS-LOX_1 or vehicle was given to 15- to 16-week-old GATA1low mice via intraperitoneal injection at a dose of 15 mg/kg four times a week for 9 weeks. PXS-LOX_1 was found to significantly decrease the bone marrow fibrotic burden and megakaryocyte number compared to vehicle in both male and female GATA1low mice. Given these results, PXS-LOX_1 was then tested in 15- to 17-week-old JAK2V617F-mutated mice at a dose of 30 mg/kg four times a week for 8 weeks. Again, we observed a significant decrease in bone marrow fibrotic burden. PXS-LOX_2, a LOX inhibitor with improved oral bioavailability, was next evaluated in 15- to 17-week-old JAK2V617F-mutated mice at a dose of 5 mg/kg p.o. four times a week for 8 weeks. This inhibitor also resulted in a significant decrease in bone marrow fibrosis, albeit with a more pronounced amelioration in female mice. Taking these results together, PXS-LOX_1 and PXS-LOX_2 appear to be promising new candidates for the treatment of fibrosis in PMF.
Subject(s)
Enzyme Inhibitors/pharmacology , Primary Myelofibrosis/drug therapy , Protein-Lysine 6-Oxidase/antagonists & inhibitors , Animals , Biological Availability , Bone Marrow Neoplasms , Disease Models, Animal , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/therapeutic use , Female , Male , Mice , Sex Factors , Treatment OutcomeABSTRACT
Fibrosis is characterized by the excessive deposition of extracellular matrix and crosslinked proteins, in particular collagen and elastin, leading to tissue stiffening and disrupted organ function. Lysyl oxidases are key players during this process, as they initiate collagen crosslinking through the oxidation of the ε-amino group of lysine or hydroxylysine on collagen side-chains, which subsequently dimerize to form immature, or trimerize to form mature, collagen crosslinks. The role of LOXL2 in fibrosis and cancer is well documented, however the specific enzymatic function of LOXL2 and LOXL3 during disease is less clear. Herein, we describe the development of PXS-5153A, a novel mechanism based, fast-acting, dual LOXL2/LOXL3 inhibitor, which was used to interrogate the role of these enzymes in models of collagen crosslinking and fibrosis. PXS-5153A dose-dependently reduced LOXL2-mediated collagen oxidation and collagen crosslinking in vitro. In two liver fibrosis models, carbon tetrachloride or streptozotocin/high fat diet-induced, PXS-5153A reduced disease severity and improved liver function by diminishing collagen content and collagen crosslinks. In myocardial infarction, PXS-5153A improved cardiac output. Taken together these results demonstrate that, due to their crucial role in collagen crosslinking, inhibition of the enzymatic activities of LOXL2/LOXL3 represents an innovative therapeutic approach for the treatment of fibrosis.
Subject(s)
Amino Acid Oxidoreductases/antagonists & inhibitors , Collagen/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Fibrosis/prevention & control , Myocardial Infarction/prevention & control , Non-alcoholic Fatty Liver Disease/prevention & control , Animals , Carbon Tetrachloride/toxicity , Collagen/drug effects , Collagen/metabolism , Cross-Linking Reagents/chemistry , Elastin/antagonists & inhibitors , Elastin/drug effects , Elastin/metabolism , Extracellular Matrix/drug effects , Fibrosis/chemically induced , Fibrosis/enzymology , Fibrosis/pathology , Male , Mice , Mice, Inbred C57BL , Myocardial Infarction/enzymology , Myocardial Infarction/pathology , Non-alcoholic Fatty Liver Disease/enzymology , Non-alcoholic Fatty Liver Disease/etiology , Non-alcoholic Fatty Liver Disease/pathology , Rats , Rats, WistarABSTRACT
The synthesis of the structure, 1, assigned to the anti-inflammatory natural product myrsinoic acid F is reported together with a means for preparing its Z-isomer 21. While neither of these compounds corresponds to the natural product, both of them are anti-inflammatory agents (as determined using a mouse ear edema assay) with congener 1 being notably more potent than the widely prescribed NSAID indometacin.
Subject(s)
Alkenes/chemical synthesis , Benzofurans/chemical synthesis , Animals , Anti-Inflammatory Agents, Non-Steroidal , Biological Products , Molecular StructureABSTRACT
Lysyl Oxidase-like 2 (LOXL2), a member of the lysyl oxidase family of amine oxidases is known to be important in normal tissue development and homeostasis, as well as the onset and progression of solid tumors. Here we tested the anti-tumor properties of two generations of novel small molecule LOXL2 inhibitor in the MDA-MB-231 human model of breast cancer. We confirmed a functional role for LOXL2 activity in the progression of primary breast cancer. Inhibition of LOXL2 activity inhibited the growth of primary tumors and reduced primary tumor angiogenesis. Dual inhibition of LOXL2 and LOX showed a greater effect and also led to a lower overall metastatic burden in the lung and liver. Our data provides the first evidence to support a role for LOXL2 specific small molecule inhibitors as a potential therapy in breast cancer.
Subject(s)
Amino Acid Oxidoreductases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Enzyme Inhibitors/pharmacology , Amino Acid Oxidoreductases/genetics , Aminopropionitrile/pharmacology , Animals , Apoptosis , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cancer-Associated Fibroblasts/drug effects , Cancer-Associated Fibroblasts/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Female , Gene Knockdown Techniques , Gene Silencing , Humans , Mice , Neoplasm Metastasis , Neovascularization, Pathologic , Xenograft Model Antitumor AssaysABSTRACT
AIMS: Treatment strategies for breast cancer continue to evolve. No uniformity exists in the UK for the management of node-positive breast cancer patients. Most centres continue to use conventional histopathology of sampled sentinel lymph nodes (SLNs), which requires delayed axillary clearance in up to 25% of patients. Some use touch imprint cytology or frozen section for intraoperative testing, although both have inherent sensitivity issues. An intraoperative molecular diagnostic approach helps to overcome some of these limitations. The aim of this study was to assess the clinical effectiveness of Metasin, a molecular method for the intraoperative evaluation of SLNs. METHODS AND RESULTS: RNA from 3296 lymph nodes from 1836 patients undergoing SLN assessment was analysed with Metasin. Alternate slices of tissue were examined in parallel by histology. Cases deemed to be discordant were analysed by protein gel electrophoresis. There was concordance between Metasin and histology in 94.1% of cases, with a sensitivity of 92% [95% confidence interval (CI) 88-94%] and a specificity of 97% (95% CI 95-97%). Positive and negative predictive values were 88% and 98%, respectively. Over half of the discordant cases (4.4%) were ascribed to tissue allocation bias (TAB). CONCLUSIONS: Clinical validation of the Metasin assay suggests that it is sufficiently sensitive and specific to make it fit for purpose in the intraoperative setting.
Subject(s)
Biomarkers, Tumor/analysis , Breast Neoplasms/pathology , Lymphatic Metastasis/diagnosis , Real-Time Polymerase Chain Reaction/methods , Sentinel Lymph Node/pathology , Female , Humans , Intraoperative Period , Sentinel Lymph Node Biopsy/methodsABSTRACT
Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by a progressive decline in lung function which can be attributed to excessive scarring, inflammation and airway remodelling. Mannose-6-phosphate (M6P) is a strong inhibitor of fibrosis and its administration has been associated with beneficial effects in tendon repair surgery as well as nerve repair after injury. Given this promising therapeutic approach we developed an improved analogue of M6P, namely PXS64, and explored its anti-fibrotic effects in vitro. Normal human lung fibroblasts (NHLF) and human lung fibroblast 19 cells (HF19) were exposed to active recombinant human TGF-ß1 to induce increases in fibrotic markers. rhTGF-ß1 increased constitutive protein levels of fibronectin and collagen in the NHLF cells, whereas HF19 cells showed increased levels of fibronectin, collagen as well as αSMA (alpha smooth muscle actin). PXS64 demonstrated a robust inhibitory effect on all proteins analysed. IPF patient fibroblasts treated with PXS64 presented an improved phenotype in terms of their morphological appearance, as well as a decrease in fibrotic markers (collagen, CTGF, TGF-ß3, tenascin C, αSMA and THBS1). To explore the cell signalling pathways involved in the anti-fibrotic effects of PXS64, proteomics analysis with iTRAQ labelling was performed and the data demonstrated a specific antagonistic effect on the TGF-ß1 pathway. This study shows that PXS64 effectively inhibits the production of extracellular matrix, as well as myofibroblast differentiation during fibrosis. These results suggest that PXS64 influences tissue remodelling by inhibiting TGF-ß1 signalling in NHLF and HF19 cell lines, as well as in IPF patient fibroblasts. Thus PXS64 is a potential candidate for preclinical application in pulmonary fibrosis.
Subject(s)
Fibroblasts/drug effects , Idiopathic Pulmonary Fibrosis/drug therapy , Lung/pathology , Mannosephosphates/therapeutic use , Mannosides/therapeutic use , Organophosphonates/therapeutic use , Prodrugs/pharmacology , Actins/metabolism , Airway Remodeling/drug effects , Animals , Biological Availability , Biomarkers/metabolism , Cell Line , Collagen/metabolism , Fibroblasts/immunology , Fibronectins/metabolism , Humans , Insulin-Like Growth Factor II/genetics , Mannosephosphates/chemistry , Mannosides/chemistry , Mice , Mice, Knockout , Organophosphonates/chemistry , Prodrugs/chemical synthesis , Proteomics , Signal Transduction , Tenascin/metabolism , Transforming Growth Factor beta1/immunologyABSTRACT
Semicarbazide-sensitive amine oxidase (SSAO), also known as vascular adhesion protein-1 (VAP-1), is a member of the copper-dependent amine oxidase family that is associated with various forms of inflammation and fibrosis. To investigate the therapeutic potential of SSAO/VAP-1 inhibition, potent and selective inhibitors with drug-like properties are required. PXS-4681A [(Z)-4-(2-(aminomethyl)-3-fluoroallyloxy)benzenesulfonamide hydrochloride] is a mechanism-based inhibitor of enzyme function with a pharmacokinetic and pharmacodynamic profile that ensures complete, long-lasting inhibition of the enzyme after a single low dose in vivo. PXS-4681A irreversibly inhibits the enzyme with an apparent Ki of 37 nM and a kinact of 0.26 min(-1) with no observed turnover in vitro. It is highly selective for SSAO/VAP-1 when profiled against related amine oxidases, ion channels, and seven-transmembrane domain receptors, and is superior to previously reported inhibitors. In mouse models of lung inflammation and localized inflammation, dosing of this molecule at 2 mg/kg attenuates neutrophil migration, tumor necrosis factor-α, and interleukin-6 levels. These results demonstrate the drug-like properties of PXS-4681A and its potential use in the treatment of inflammation.
Subject(s)
Allyl Compounds/pharmacology , Amine Oxidase (Copper-Containing)/antagonists & inhibitors , Anti-Inflammatory Agents/pharmacology , Cell Adhesion Molecules/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Small Molecule Libraries/pharmacology , Sulfonamides/pharmacology , Adipose Tissue/drug effects , Adipose Tissue/enzymology , Allyl Compounds/chemistry , Allyl Compounds/pharmacokinetics , Allyl Compounds/therapeutic use , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacokinetics , Anti-Inflammatory Agents/therapeutic use , Dermatitis/drug therapy , Dermatitis/enzymology , Dermatitis/immunology , Disease Models, Animal , Dogs , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/therapeutic use , Humans , In Vitro Techniques , Mice , Microsomes/drug effects , Microsomes/enzymology , Models, Biological , Molecular Structure , Pneumonia/drug therapy , Pneumonia/enzymology , Pneumonia/immunology , Rabbits , Rats , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacokinetics , Small Molecule Libraries/therapeutic use , Species Specificity , Sulfonamides/chemistry , Sulfonamides/pharmacokinetics , Sulfonamides/therapeutic useABSTRACT
Operationally simple Au(I)-catalyzed intramolecular hydroarylation (IMHA) reactions of terminal alkynes that proceed in high yield and under very mild conditions are described. These processes involve low catalyst loadings, mild reaction temperatures, and short reaction times, require no cocatalysts or additives, and allow for the generation of a number of important heterocyclic motifs from readily accessible starting materials.
Subject(s)
Alkynes/chemistry , Benzofurans/chemical synthesis , Benzopyrans/chemical synthesis , Coumarins/chemical synthesis , Gold/chemistry , Quinolines/chemical synthesis , Benzofurans/chemistry , Benzopyrans/chemistry , Catalysis , Coumarins/chemistry , Molecular Structure , Quinolines/chemistry , StereoisomerismABSTRACT
The readily available and enzymatically derived cis-1,2-dihydrocatechol 3 has been converted, over 15 steps, into (+)-amabiline, the non-natural enantiomeric form of a crinine-type alkaloid.
Subject(s)
Amaryllidaceae Alkaloids/chemistry , Amaryllidaceae Alkaloids/chemical synthesis , Molecular Structure , StereoisomerismABSTRACT
An expedient first total synthesis of (-)-spirangien A, a potent cytotoxic and antifungal polyketide of myxobacterial origin, is described. By using a common 1,3-diol intermediate obtained by an efficient aldol-reduction sequence for installation of the C15-C18 and C25-C28 stereotetrads and a reagent-controlled boron aldol coupling followed by spiroacetalization, a highly convergent strategy was developed for construction of the elaborate spiroacetal core. Conversion of this advanced spiroacetal intermediate into (+)-spirangien diene, obtained previously by controlled degradation of spirangien A, was then achieved by installation of the truncated side-chain using an allylboration-Peterson sequence. The total synthesis of (-)-spirangien A was then achieved by the controlled attachment of the unsaturated C1-C12 side-chain, avoiding exposure to light. A Stork-Wittig olefination and double Stille cross-coupling sequence was exploited to install the delicate conjugated pentaene chromophore featuring alternating (Z)- and (E)-olefins, leading initially to the methyl ester of spirangien A, which proved significantly more stable than the corresponding free acid. Subsequent careful hydrolysis afforded (-)-spirangien A, validating the relative and absolute configuration.
Subject(s)
Acetals/chemical synthesis , Antimitotic Agents/chemical synthesis , Fatty Acids, Unsaturated/chemical synthesis , Myxococcales/chemistry , Acetals/chemistry , Antimitotic Agents/chemistry , Fatty Acids, Unsaturated/chemistry , StereoisomerismABSTRACT
The first total synthesis of (-)-spirangien A, a cytotoxic and antifungal polyketide of myxobacterial origin, is reported; this exploits a Stork-Wittig olefination and double Stille cross-coupling sequence to install the sensitive pentaene side chain onto a fully elaborated spiroacetal core, leading initially to the methyl ester of spirangien A.
Subject(s)
Acetals/chemical synthesis , Esters/chemistry , Fatty Acids, Unsaturated/chemical synthesis , Acetals/chemistry , Fatty Acids, Unsaturated/chemistry , Methylation , Molecular StructureABSTRACT
[reaction: see text] A revised configurational assignment for the cytotoxic marine macrolide dolastatin 19 is proposed and validated by total synthesis. Key features of the route include an asymmetric vinylogous aldol reaction to install the isolated C13 stereocenter and (E)-trisubstituted alkene, two sequential 1,4-syn boron-mediated aldol reactions, and a Mukaiyama glycosylation to append the l-rhamnose-derived pyranoside.