PAI-1 Deficiency Drives Pulmonary Vascular Smooth Muscle Remodeling and Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PAs). Central to the remodeling process is a switch of pulmonary vascular cells to a proliferative, apoptosis-resistant phenotype. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of urokinase-type and tissue-type plasminogen activators (uPA and tPA), but its role in PAH is unsettled. Here, we report that: (1) PAI-1 is deficient in remodeled small PAs and in early-passage PA smooth muscle and endothelial cells (PASMCs and PAECs) from subjects with PAH compared to controls; (2) PAI-1-/- mice spontaneously develop pulmonary vascular remodeling associated with up-regulation of mTORC1 signaling, pulmonary hypertension (PH), and right ventricle (RV) hypertrophy; and (3) pharmacological inhibition of uPA in human PAH PASMCs suppresses pro-proliferative mTORC1 and SMAD3 signaling, restores PAI-1 levels, reduces proliferation and induces apoptosis in vitro, and prevents the development of SU5416/hypoxia-induced PH and RV hypertrophy in vivo in mice. These data strongly suggest that down-regulation of PAI-1 in small PAs promotes vascular remodeling and PH due to unopposed activation of uPA and consequent up-regulation of mTOR and TGF-b signaling in PASMCs, and call for further studies to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate and/or reverse pulmonary vascular remodeling and PH.

Ion Transport and Inhibitor Binding by Human NHE1: Insights from Molecular Dynamics Simulations and Free Energy Calculations.

The human Na+/H+ exchanger (NHE1) plays a crucial role in maintaining intracellular pH by regulating the electroneutral exchange of a single intracellular H+ for one extracellular Na+ across the plasma membrane. Understanding the molecular mechanisms governing ion transport and the binding of inhibitors is of importance in the development of anticancer therapeutics targeting NHE1. In this context, we performed molecular dynamics (MD) simulations based on the recent cryo-electron microscopy (cryo-EM) structures of outward- and inward-facing conformations of NHE1. These simulations allowed us to explore the dynamics of the protein, examine the ion-translocation pore, and confirm that Asp267 is the ion-binding residue. Our free energy calculations did not show a significant difference between Na+ and K+ binding at the ion-binding site. Consequently, Na+ over K+ selectivity cannot be solely explained by differences in ion binding. Our MD simulations involving NHE1 inhibitors (cariporide and amiloride analogues) maintained stable interactions with Asp267 and Glu346. Our study highlights the importance of the salt bridge between the positively charged acylguanidine moiety and Asp267, which appears to play a role in the competitive inhibitory mechanism for this class of inhibitors. Our computational study provides a detailed mechanistic interpretation of experimental data and serves the basis of future structure-based inhibitor design.

A dual-targeting succinate dehydrogenase and F1Fo-ATP synthase inhibitor rapidly sterilizes replicating and non-replicating Mycobacterium tuberculosis.

Mycobacterial bioenergetics is a validated target space for antitubercular drug development. Here, we identify BB2-50F, a 6-substituted 5-(N,N-hexamethylene)amiloride derivative as a potent, multi-targeting bioenergetic inhibitor of Mycobacterium tuberculosis. We show that BB2-50F rapidly sterilizes both replicating and non-replicating cultures of M. tuberculosis and synergizes with several tuberculosis drugs. Target identification experiments, supported by docking studies, showed that BB2-50F targets the membrane-embedded c-ring of the F1Fo-ATP synthase and the catalytic subunit (substrate-binding site) of succinate dehydrogenase. Biochemical assays and metabolomic profiling showed that BB2-50F inhibits succinate oxidation, decreases the activity of the tricarboxylic acid (TCA) cycle, and results in succinate secretion from M. tuberculosis. Moreover, we show that the lethality of BB2-50F under aerobic conditions involves the accumulation of reactive oxygen species. Overall, this study identifies BB2-50F as an effective inhibitor of M. tuberculosis and highlights that targeting multiple components of the mycobacterial respiratory chain can produce fast-acting antimicrobials.

PAI-1 Deficiency Drives Pulmonary Vascular Smooth Muscle Remodeling and Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive and potentially a rapidly fatal disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PA) leading to increased pulmonary vascular resistance and right heart failure. Central to the remodeling process is a switch of the smooth muscle cells in small PAs (PASMC) to a proliferative, apoptosis-resistant phenotype. There is reason to suspect that the plasminogen activator system may play an important role in the remodeling program in PAH based on its roles in vascular post-injury restenosis, fibrosis, angiogenesis and tumorigenesis. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of the plasminogen activators - urokinase-type and tissue-type (uPA and tPA, respectively). Immunohisto- chemical and immunoblot analyses revealed that PAI-1 was deficient in smooth muscle areas of small remodeled PAs and early-passage PASMC from subjects with PAH compared to non-PAH controls. PAI1-/- male and female mice developed spontaneous pulmonary vascular remodeling and pulmonary hypertension (PH) as evidenced by significant increase in PA medial thickness, systolic right ventricular pressure, and right ventricular hypertrophy. Lastly, the uPA inhibitors upamostat (WX-671) and amiloride analog BB2-30F down-regulated mTORC1 and SMAD3, restored PAI-1 levels, reduced proliferation, and induced apoptosis in human PAH PASMC. We examined the effect of inhibition of uPA catalytic activity by BB2-30F on the development of SU5416/Hypoxia (SuHx)-induced PH in mice. Vehicletreated SuHx-exposed mice had up-regulated mTORC1 in small PAs, developed pulmonary vascular remodeling and PH, as evidenced by significant increase of PA MT, sRVP, RV hypertrophy, and a significant decrease in the pulmonary artery acceleration time/pulmonary ejection time (PAAT/PET) ratio compared to age- and sex-matched normoxia controls, whereas BB2-30F-treated group was protected from all these pathological changes. Taken together, our data strongly suggest that PAI-1 down- regulation in PASMC from human PAH lungs promotes PASMC hyper-proliferation, remodeling, and spontaneous PH due to unopposed uPA activation. Further studies are needed to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate the progression and/or reverse pulmonary vascular remodeling and PH.

Automated Patch Clamp Screening of Amiloride and 5-N,N-Hexamethyleneamiloride Analogs Identifies 6-Iodoamiloride as a Potent Acid-Sensing Ion Channel Inhibitor.

Acid-sensing ion channels (ASICs) are transmembrane sensors of extracellular acidosis and potential drug targets in several disease indications, including neuropathic pain and cancer metastasis. The K+-sparing diuretic amiloride is a moderate nonspecific inhibitor of ASICs and has been widely used as a probe for elucidating ASIC function. In this work, we screened a library of 6-substituted and 5,6-disubstituted amiloride analogs using a custom-developed automated patch clamp protocol and identified 6-iodoamiloride as a potent ASIC1 inhibitor. Follow-up IC50 determinations in tsA-201 cells confirmed higher ASIC1 inhibitory potency for 6-iodoamiloride 94 (hASIC1 94 IC50 = 88 nM, cf. amiloride 11 IC50 = 1.7 µM). A similar improvement in activity was observed in ASIC3-mediated currents from rat dorsal root ganglion neurons (rDRG single-concentration 94 IC50 = 230 nM, cf. 11 IC50 = 2.7 µM). 6-Iodoamiloride represents the amiloride analog of choice for studying the effects of ASIC inhibition on cell physiology.

Antifungal activity of 6-substituted amiloride and hexamethylene amiloride (HMA) analogs.

Fungal infections have become an increasing threat as a result of growing numbers of susceptible hosts and diminishing effectiveness of antifungal drugs due to multi-drug resistance. This reality underscores the need to develop novel drugs with unique mechanisms of action. We recently identified 5-(N,N-hexamethylene)amiloride (HMA), an inhibitor of human Na+/H+ exchanger isoform 1, as a promising scaffold for antifungal drug development. In this work, we carried out susceptibility testing of 45 6-substituted HMA and amiloride analogs against a panel of pathogenic fungi. A series of 6-(2-benzofuran)amiloride and HMA analogs that showed up to a 16-fold increase in activity against Cryptococcus neoformans were identified. Hits from these series showed broad-spectrum activity against both basidiomycete and ascomycete fungal pathogens, including multidrug-resistant clinical isolates.

6-Furopyridine Hexamethylene Amiloride Is a Non-Selective P2X7 Receptor Antagonist.

P2X7 is an extracellular adenosine 5'-triphopshate (ATP)-gated cation channel present on leukocytes, where its activation induces pro-inflammatory cytokine release and ectodomain shedding of cell surface molecules. Human P2X7 can be partially inhibited by amiloride and its derivatives at micromolar concentrations. This study aimed to screen a library of compounds derived from amiloride or its derivative 5-(N,N-hexamethylene) amiloride (HMA) to identify a potential P2X7 antagonist. 6-Furopyridine HMA (6-FPHMA) was identified as a novel P2X7 antagonist and was characterized further. 6-FPHMA impaired ATP-induced dye uptake into human RPMI8226 multiple myeloma cells and human P2X7-HEK293 cells, in a concentration-dependent, non-competitive manner. Likewise, 6-FPHMA blocked ATP-induced Ca2+ fluxes in human P2X7-HEK293 cells in a concentration-dependent, non-competitive manner. 6-FPHMA inhibited ATP-induced dye uptake into human T cells, and interleukin-1ß release within human blood and CD23 shedding from RPMI8226 cells. 6-FPHMA also impaired ATP-induced dye uptake into murine P2X7- and canine P2X7-HEK293 cells. However, 6-FPHMA impaired ATP-induced Ca2+ fluxes in human P2X4-HEK293 cells and non-transfected HEK293 cells, which express native P2Y1, P2Y2 and P2Y4. In conclusion, 6-FPHMA inhibits P2X7 from multiple species. Its poor selectivity excludes its use as a specific P2X7 antagonist, but further study of amiloride derivatives as P2 receptor antagonists is warranted.

Urokinase plasminogen activator as an anti-metastasis target: inhibitor design principles, recent amiloride derivatives, and issues with human/mouse species selectivity.

The urokinase plasminogen activator (uPA) is a widely studied anticancer drug target with multiple classes of inhibitors reported to date. Many of these inhibitors contain amidine or guanidine groups, while others lacking these groups show improved oral bioavailability. Most of the X-ray co-crystal structures of small molecule uPA inhibitors show a key salt bridge with the side chain carboxylate of Asp189 in the S1 pocket of uPA. This review summarises the different classes of uPA inhibitors, their binding interactions and experimentally measured inhibitory potencies and highlights species selectivity issues with attention to recently described 6-substituted amiloride and 5­N,N-(hexamethylene)amiloride (HMA) derivatives.

An amiloride derivative is active against the F1Fo-ATP synthase and cytochrome bd oxidase of Mycobacterium tuberculosis.

Increasing antimicrobial resistance compels the search for next-generation inhibitors with differing or multiple molecular targets. In this regard, energy conservation in Mycobacterium tuberculosis has been clinically validated as a promising new drug target for combatting drug-resistant strains of M. tuberculosis. Here, we show that HM2-16F, a 6-substituted derivative of the FDA-approved drug amiloride, is an anti-tubercular inhibitor with bactericidal properties comparable to the FDA-approved drug bedaquiline (BDQ; Sirturo®) and inhibits the growth of bedaquiline-resistant mutants. We show that HM2-16F weakly inhibits the F1Fo-ATP synthase, depletes ATP, and affects the entry of acetyl-CoA into the Krebs cycle. HM2-16F synergizes with the cytochrome bcc-aa3 oxidase inhibitor Q203 (Telacebec) and co-administration with Q203 sterilizes in vitro cultures in 14 days. Synergy with Q203 occurs via direct inhibition of the cytochrome bd oxidase by HM2-16F. This study shows that amiloride derivatives represent a promising discovery platform for targeting energy generation in drug-resistant tuberculosis.

Disruption of Water Networks is the Cause of Human/Mouse Species Selectivity in Urokinase Plasminogen Activator (uPA) Inhibitors Derived from Hexamethylene Amiloride (HMA).

The urokinase plasminogen activator (uPA) plays a critical role in tumor cell invasion and migration and is a promising antimetastasis target. 6-Substituted analogues of 5-N,N-(hexamethylene)amiloride (HMA) are potent and selective uPA inhibitors that lack the diuretic and antikaliuretic properties of the parent drug amiloride. However, the compounds display pronounced selectivity for human over mouse uPA, thus confounding interpretation of data from human xenograft mouse models of cancer. Here, computational and experimental findings reveal that residue 99 is a key contributor to the observed species selectivity, whereby enthalpically unfavorable expulsion of a water molecule by the 5-N,N-hexamethylene ring occurs when residue 99 is Tyr (as in mouse uPA). Analogue 7 lacking the 5-N,N-hexamethylene ring maintained similar water networks when bound to human and mouse uPA and displayed reduced selectivity, thus supporting this conclusion. The study will guide further optimization of dual-potent human/mouse uPA inhibitors from the amiloride class as antimetastasis drugs.

Screening of 5- and 6-Substituted Amiloride Libraries Identifies Dual-uPA/NHE1 Active and Single Target-Selective Inhibitors.

The K+-sparing diuretic amiloride shows off-target anti-cancer effects in multiple rodent models. These effects arise from the inhibition of two distinct cancer targets: the trypsin-like serine protease urokinase-type plasminogen activator (uPA), a cell-surface mediator of matrix degradation and tumor cell invasiveness, and the sodium-hydrogen exchanger isoform-1 (NHE1), a central regulator of transmembrane pH that supports carcinogenic progression. In this study, we co-screened our library of 5- and 6-substituted amilorides against these two targets, aiming to identify single-target selective and dual-targeting inhibitors for use as complementary pharmacological probes. Closely related analogs substituted at the 6-position with pyrimidines were identified as dual-targeting (pyrimidine 24 uPA IC50 = 175 nM, NHE1 IC50 = 266 nM, uPA selectivity ratio = 1.5) and uPA-selective (methoxypyrimidine 26 uPA IC50 = 86 nM, NHE1 IC50 = 12,290 nM, uPA selectivity ratio = 143) inhibitors, while high NHE1 potency and selectivity was seen with 5-morpholino (29 NHE1 IC50 = 129 nM, uPA IC50 = 10,949 nM; NHE1 selectivity ratio = 85) and 5-(1,4-oxazepine) (30 NHE1 IC50 = 85 nM, uPA IC50 = 5715 nM; NHE1 selectivity ratio = 67) analogs. Together, these amilorides comprise a new toolkit of chemotype-matched, non-cytotoxic probes for dissecting the pharmacological effects of selective uPA and NHE1 inhibition versus dual-uPA/NHE1 inhibition.

Systematic evaluation of structure-property relationships and pharmacokinetics in 6-(hetero)aryl-substituted matched pair analogs of amiloride and 5-(N,N-hexamethylene)amiloride.

The K+-sparing diuretic amiloride elicits anticancer activities in multiple animal models. During our recent medicinal chemistry campaign aiming to identify amiloride analogs with improved properties for potential use in cancer, we discovered novel 6-(hetero)aryl-substituted amiloride and 5-(N,N-hexamethylene)amiloride (HMA) analogs with up to 100-fold higher potencies than the parent compounds against urokinase plasminogen activator (uPA), one of amiloride's putative anticancer targets, and no diuretic or antikaliuretic effects. Here, we report the systematic evaluation of structure-property relationships (lipophilicity, aqueous solubility and in vitro metabolic stability in human and mouse liver microsomes) in twelve matched pair analogs selected from our 6-substituted amiloride and HMA libraries. Mouse plasma stability, plasma protein binding, Caco-2 cell permeability, cardiac ion channel activity and pharmacokinetics in mice (PO and IV) and rats (IV) are described alongside amiloride and HMA comparators for a subset of the four most promising matched-pair analogs. The findings combined with earlier uPA activity/selectivity and other data ultimately drove selection of two analogs (AA1-39 and AA1-41) that showed efficacy in separate mouse cancer metastasis studies.

Characterization of Five Novel Anti-MRSA Compounds Identified Using a Whole-Animal Caenorhabditis elegans/Galleria mellonella Sequential-Screening Approach.

There is a significant need to combat the growing challenge of antibacterial drug resistance. We have previously developed a whole-animal dual-screening platform that first used the nematode Caenorhabditis elegans, to identify low-toxicity antibacterial hits in a high-throughput format. The hits were then evaluated in the wax moth caterpillar Galleria mellonella infection model to confirm efficacy and low toxicity at a whole animal level. This multi-host approach is a powerful tool for revealing compounds that show antibacterial effects and relatively low toxicity at the whole organism level. This paper reports the use of the multi-host approach to identify and validate five new anti-staphylococcal compounds: (1) 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol(PPT), (2) (1S,2S)-2-[2-[[3-(1H-benzimidazol-2-yl)propyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-(1-methylethyl)-2-naphthalenyl cyclopropanecarboxylate dihydrochloride(NNC), (3) 4,5,6,7-tetrabromobenzotriazole (TBB), (4) 3-[2-[2-chloro-4-[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl] benzoic acid(GW4064), and (5) N-(cyclopropylmethoxy)-3,4,5-trifluoro-2-[(4-iodo-2-methylphenyl)amino] benzamide(PD198306). The compounds reduced the severity of methicillin-resistant Staphylococcus aureus (MRSA, strain MW2) infections in both C. elegans and G. mellonella and showed minimal inhibitory concentrations (MICs) in the range of 2-8 µg/mL. Compounds NNC, PPT, and TBB permeabilized MRSA-MW2 cells to SYTOX green, suggesting that they target bacterial membranes. Compound TBB showed synergistic activity with doxycycline and oxacillin against MRSA-MW2, and compounds PPT, NNC, GW4064, and PD198306 synergized with doxycycline, polymyxin-B, gentamicin, and erythromycin, respectively. The study demonstrates the utility of the multi-host approach with follow-up hit characterization for prioritizing anti-MRSA compounds for further evaluation.

Discovery of Cephalosporin-3'-Diazeniumdiolates That Show Dual Antibacterial and Antibiofilm Effects against Pseudomonas aeruginosa Clinical Cystic Fibrosis Isolates and Efficacy in a Murine Respiratory Infection Model.

The formation of biofilms provides a formidable defense for many bacteria against antibiotics and host immune responses. As a consequence, biofilms are thought to be the root cause of most chronic infections, including those occurring on medical indwelling devices, endocarditis, urinary tract infections, diabetic and burn wounds, and bone and joint infections. In cystic fibrosis (CF), chronic Pseudomonas aeruginosa (P. aeruginosa) respiratory infections are the leading cause of morbidity and mortality in adults. Previous studies have shown that many bacteria can undergo a coordinated dispersal event in the presence of low concentrations of nitric oxide (NO), suggesting that NO could be used to initiate biofilm dispersal in chronic infections, enabling clearance of the more vulnerable planktonic cells. In this study, we describe efforts to create "all-in-one" cephalosporin-based NO donor prodrugs (cephalosporin-3'-diazeniumdiolates, C3Ds) that show both direct ß-lactam mediated antibacterial activity and antibiofilm effects. Twelve novel C3Ds were synthesized and screened against a panel of P. aeruginosa CF clinical isolates and other human pathogens. The most active compound, AMINOPIP2 ((Z)-1-(4-(2-aminoethyl)piperidin-1-yl)-2-(((6R,7R)-7-((Z)-2-(2-aminothiazol-4-yl)-2-(((2-carboxypropan-2-yl)oxy)imino)acetamido)-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl)methoxy)diazene 1-oxide)-ceftazidime 12, showed higher antibacterial potency than its parent cephalosporin and front-line antipseudomonal antibiotic ceftazidime, good stability against ß-lactamases, activity against ceftazidime-resistant P. aeruginosa in vitro biofilms, and efficacy equivalent to ceftazidime in a murine P. aeruginosa respiratory infection model. The results support further evaluation of AMINOPIP2-ceftazidime 12 for P. aeruginosa lung infections in CF and a broader study of "all-in-one" C3Ds for other chronic infections.

Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa.

OBJECTIVES: The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D ('DiEthylAmin-Cephalosporin-3'-Diazeniumdiolate') has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. METHODS: ß-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. RESULTS: DEA-C3D was confirmed to selectively release NO in response to contact with bacterial ß-lactamase. Despite lacking direct, cephalosporin/ß-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. CONCLUSIONS: DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

6-Substituted amiloride derivatives as inhibitors of the urokinase-type plasminogen activator for use in metastatic disease.

The oral K+-sparing diuretic amiloride shows anti-cancer side-activities in multiple rodent models. These effects appear to arise, at least in part, through moderate inhibition of the urokinase-type plasminogen activator (uPA, Ki = 2.4 µM), a pro-metastatic trypsin-like serine protease that is upregulated in many aggressive solid malignancies. In applying the selective optimization of side-activity (SOSA) approach, a focused library of twenty two 6-substituted amiloride derivatives were prepared, with multiple examples displaying uPA inhibitory potencies in the nM range. X-ray co-crystal structures revealed that the potency increases relative to amiloride arise from increased occupancy of uPA's S1ß subsite by the appended 6-substituents. Leading compounds were shown to have high selectivity over related trypsin-like serine proteases and no diuretic or anti-kaliuretic effects in rats. Compound 15 showed anti-metastatic effects in a xenografted mouse model of late-stage lung metastasis.

Introduction of a Fixed-Charge, Photolabile Derivative for Enhanced Structural Elucidation of Fatty Acids.

Fatty acids are a structurally diverse category of lipids with a myriad of biochemical functions, which includes their role as building blocks of more complex lipids (e.g., glycerophospholipids and triacylglycerols). Increasingly, the analysis of fatty acids is undertaken using liquid chromatography-mass spectrometry (LC-MS), due to its versatility in the detection of lipids across a wide range of concentrations and diversity of molecular structures and masses. Previous work has shown that fixed-charge pyridinium derivatives are effective in enhancing the detection of fatty acids in LC-MS workflows. Herein, we describe the development of two novel pyridinium fixed-charged derivatization reagents that incorporate a photolabile aryl iodide that is selectively activated by laser irradiation inside the mass spectrometer. Photodissociation mass spectra of fatty acids conjugated to 1-(3-(aminomethyl)-4-iodophenyl)pyridin-1-ium (4-I-AMPP+) and 1-(4-(aminomethyl)-3-iodophenyl)pyridin-1-ium (3-I-AMPP+) derivatives reveal structurally diagnostic product ions. These spectra feature radical-directed dissociation of the carbon-carbon bonds within the fatty acyl chain, enabling structural assignments of fatty acids and discrimination of isomers that differ in site(s) of unsaturation, methyl branching or cyclopropanation. These derivatives are shown to be suitable for hyphenated LC-MS methods, and their predictable photodissociation behavior allows de novo identification of unusual fatty acids within a biological context.

Prototyping kinase inhibitor-cytotoxin anticancer mutual prodrugs activated by tumour hypoxia: A chemical proof of concept study.

Amide- and ester-linked kinase inhibitor-cytotoxin conjugates were rationally designed and synthesised as prototype hypoxia-activated anticancer mutual prodrugs. Chemical reduction of an aryl nitro trigger moiety was shown to initiate a spontaneous cyclisation/fragmentation reaction that simultaneously released the kinase inhibitor semaxanib (SU5416) and the amine- or alcohol-linked cytotoxin from the prodrugs. Preliminary cell testing and reduction potential measurements support optimisation of the compounds towards tumour-selective mutual prodrugs.

The Urokinase Plasminogen Activation System in Rheumatoid Arthritis: Pathophysiological Roles and Prospective Therapeutic Targets.

Rheumatoid arthritis (RA) is a chronic and progressive inflammatory disease characterized in its early stages by synovial hyperplasia and inflammatory cell infiltration and later by irreversible joint tissue destruction. The plasminogen activation system (PAS) is associated with a wide range of physiological and pathophysiological states involving fibrinolysis, inflammation and tissue remodeling. Various components of the PAS are implicated in the pathophysiology of RA. Urokinase plasminogen activator (uPA) in particular is a pro-inflammatory mediator that appears to play an important role in the bone and cartilage destruction associated with RA. Clinical studies have shown that uPA and its receptor uPAR are overexpressed in synovia of patients with rheumatoid arthritis. Further, genetic knockdown and antibody-mediated neutralization of uPA have been shown to be protective against induction or progression of arthritis in animal models. The pro-arthritic role of uPA is differentiated from its haemodynamic counterpart, tissue plasminogen activator (tPA), which appears to play a protective role in RA animal models. This review summarises available evidence supporting the PAS as a critical determinant of RA pathogenesis and highlights opportunities for the development of novel uPAS-targeting therapeutics.