Activity and mechanisms of action of novel organosulfur derivatives of the HDAC inhibitor Valproic Acid in human experimental models of non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) represents the leading cause of cancer death worldwide, and 5-year survival is about 16% for patients diagnosed with advanced lung cancer and about 70-90% when the disease is diagnosed and treated at earlier stages. Treatment of NSCLC is changed in the last years with the introduction of targeted agents, such as gefitinib and erlotinib, that have dramatically changed the natural history of NSCLC patients carrying specific mutations in the EGFR gene, or crizotinib, for patients with the EML4-ALK translocation. However, such patients represent only about 15-20% of all NSCLC patients, and for the remaining individuals conventional chemotherapy represents the standard choice yet, but response rate to thise type of treatment is only about 20%. Development of new drugs and new therapeutic approaches are so needed to improve patients outcome. In this project we aimed to analyse the antitumoral activity of two compounds with the ability to inhibit histone deacethylases (ACS 2 and ACS 33), derived from Valproic Acid and conjugated with H2S, in human cancer cell lines derived from NSCLC tissues. We showed that ACS 2 represents the more promising agent. It showed strong antitumoral and pro-apoptotic activities, by inducing membrane depolarization, cytocrome-c release and caspase 3 and 9 activation. It was able to reduce the invasive capacity of cells, through inhibition of metalloproteinases expression, and to induce a reduced chromatin condensation. This last characteristic is probably responsible for the observed high synergistic activity in combination with cisplatin. In conclusion our results highlight the potential role of the ACS 2 compound as new therapeutic option for NSCLC patients, especially in combination with cisplatin. If validated in in vivo models, this compound should be worthy for phase I clinical trials.

Mechanisms Contributing to Tyrosin Kinase Inhibitor Resistance in GISTs: Toward a Personalized Therapy

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the gastrointestinal tract. This work considers the pharmacological response in GIST patients treated with imatinib by two different angles: the genetic and somatic point of view. We analyzed polymorphisms influence on treatment outcome, keeping in consideration SNPs in genes involved in drug transport and folate pathway. Naturally, all these intriguing results cannot be considered as the only main mechanism in imatinib response. GIST mainly depends by oncogenic gain of function mutations in tyrosin kinase receptor genes, KIT or PDGFRA, and the mutational status of these two genes or acquisition of secondary mutation is considered the main player in GIST development and progression. To this purpose we analyzed the secondary mutations to better understand how these are involved in imatinib resistance. In our analysis we considered both imatinib and the second line treatment, sunitinib, in a subset of progressive patients. KIT/PDGFRA mutation analysis is an important tool for physicians, as specific mutations may guide therapeutic choices. Currently, the only adaptations in treatment strategy include imatinib starting dose of 800 mg/daily in KIT exon-9-mutated GISTs. In the attempt to individualize treatment, genetic polymorphisms represent a novelty in the definition of biomarkers of imatinib response in addition to the use of tumor genotype. Accumulating data indicate a contributing role of pharmacokinetics in imatinib efficacy, as well as initial response, time to progression and acquired resistance. At the same time it is becoming evident that genetic host factors may contribute to the observed pharmacokinetic inter-patient variability. Genetic polymorphisms in transporters and metabolism may affect the activity or stability of the encoded enzymes. Thus, integrating pharmacogenetic data of imatinib transporters and metabolizing genes, whose interplay has yet to be fully unraveled, has the potential to provide further insight into imatinib response/resistance mechanisms.

Funktionelle und strukturelle Charakterisierung des Ovastacins in vivo und in vitro

Die Metalloprotease Ovastacin, ein Vertreter der Astacin-Familie, wurde erstmals 2004 beschrieben. Im Ovar von Säugetieren ist Ovastacin-mRNA im Zeitfenster vom Stadium der Sekundärfollikel bis kurz nach der Befruchtung der Eizelle zu finden. Der Expressionsort und -zeitpunkt sowie die Sequenzähnlichkeit von über 60% mit sogenannten „Schlüpfenzymen“ (engl. hatching enzymes), die man in den Eizellen und Zygoten niederer Wirbeltiere und Wirbelloser gefunden hatte, ließen die Vermutung aufkommen, es könnte sich hier um das Säugerhomolog dieser Proteasen handeln. Generell lösen hatching Enzyme die derben embryonalen Hüllstrukturen (bei Säugern die Zona pellucida, ZP) beim Schlüpfvorgang auf. Die essentielle Bedeutung des Ovastacins für die Befruchtung wird durch die um ca. 30% reduzierte Fruchtbarkeit von Ovastacin defizienten Mäusen belegt. Hochinteressant war in diesem Zusammenhang die Entdeckung des Ovastacins in den Cortikalgranula der Oocyten sowie seine Fähigkeit, das Zona pellucida Protein 2 zu schneiden. Die dadurch bewirkte Verhärtung der Zona pellucida verhindert das Eindringen weiterer Spermien, das heißt sie baut eine Barriere gegen Polyspermie auf. Ziel dieser Arbeit war es, Belege für die physiologische Funktion des Ovastacins zu finden. Vor allem galt es, potentielle Aktivatoren zu identifizieren, da das Enzym wie alle Astacine als inaktive Vorstufe gebildet wird, die proteolytisch aktiviert werden muss. Zu diesem Zweck exprimierte ich rekombinantes Pro-Ovastacin in Insektenzellen. Aktivierungsstudien in vitro zeigten, dass ein saures Milieu zu einer Aktivierung führt, ohne die Abspaltung des Propeptids zu bewirken. Sequenzalignments und ein homologes Strukturmodell des Ovastacins wiesen auf Trypsin- oder Elastase-ähnliche Serinproteasen als potentielle Aktivierungsenzyme hin. Tatsächlich konnte mit diesen beiden Proteasetypen zum ersten Mal aktives Ovastacin aus Pro-Ovastacin erzeugt werden. Trypsin kommt als physiologischer Aktivator allerdings nicht in Betracht, da es bisher in keinem der Gewebe nachgewiesen werden konnte, in dem Ovastacin exprimiert wird. Die neutrophile Elastase dagegen konnte in der Leber, im Herz sowie im Blutplasma nachgewiesen werden. Mit Hilfe spezifischer Antikörper konnte das Herz als Expressionsort für Ovastacin bestätigt werden. Somit wäre Elastase ein potentieller physiologischer Aktivator von Ovastacin. Die Identifikation des Ovastacins in Geweben wie Leber, Herz, Nabelschnur und im Blutplasma weist auf eine Rolle der Protease in proteolytischen Netzwerken außerhalb der Spermien-Ei-Interaktion hin. Die Bedeutung der biologischen Kontrolle des Ovastacins bei der Befruchtung der Säugereizelle wird durch die Beobachtung untermauert, dass das Leberprotein Fetuin B als physiologischer Ovastacininhibitor fungiert und dadurch eine vorzeitige Verhärtung der Zona pellucida verhindert, die andernfalls die Penetration von Spermien prinzipiell verhindern würde.

Mechanisms of resistance of tumor cells in response to treatment with the vacuolar H+-ATPase inhibitor archazolid B

Resistance of cancer cells towards chemotherapy is the major cause of therapy failure. Hence, the evaluation of cellular defense mechanisms is essential in the establishment of new chemotherapeutics. In this study, classical intrinsic and acquired as well as new resistance mechanisms relevant in the cellular response to the novel vacuolar H+-ATPase inhibitor archazolid B were investigated. Archazolid B, originally produced by the myxobacterium Archangium gephyra, displayed cytotoxicity in the low nanomolar range on a panel of cancer cell lines. The drug showed enhanced cytotoxic activity against nearly all cancerous cells compared to their non-cancerous pendants. With regards to ABC transporters, archazolid B was identified as a moderate substrate of ABCB1 (P-glycoprotein) and a weak substrate of ABCG2 (BCRP), whereas hypersensitivity was observed in ABCB5-expressing cells. The cytotoxic effect of archazolid B was shown to be independent of the cellular p53 status. However, cells expressing constitutively active EGFR displayed significantly increased resistance. Acquired drug resistance was studied by establishing an archazolid B-resistant MCF-7 cell line. Experiments showed that this secondary resistance was not conferred by aberrant expression or DNA mutations of the gene encoding vacuolar H+-ATPase subunit c, the direct target of archazolid B. Instead, a slight increase of ABCB1 and a significant overexpression of EGFR as well as reduced proliferation may contribute to acquired archazolid B resistance. For identification of new resistance strategies upon archazolid B treatment, omics data from bladder cancer and glioblastoma cells were analyzed, revealing drastic disturbances in cholesterol homeostasis, affecting cholesterol biosynthesis, uptake and transport. As shown by filipin staining, archazolid B led to accumulation of free cholesterol in lysosomes, which triggered sterol responses, mediated by SREBP-2 and LXR, including up-regulation of HMGCR, the key enzyme of cholesterol biosynthesis. Furthermore, inhibition of LDL uptake as well as impaired LDLR surface expression were observed, indicating newly synthesized cholesterol to be the main source of cholesterol in archazolid B-treated cells. This was proven by the fact that under archazolid B treatment, total free cholesterol levels as well as cell survival were significantly reduced by inhibiting HMGCR with fluvastatin. The combination of archazolid B with statins may therefore be an attractive strategy to circumvent cholesterol-mediated cell survival and in turn potentiate the promising anticancer effects of archazolid B.

Synthese, Charakterisierung und Testung von Phenylacrylsäure-Derivaten, Benzothiazolen und Diarylthioethern als potentielle Inhibitoren der Dengue-Virus-Typ-2-NS2B-NS3-Protease

Dengue-Fieber ist eine durch Stechmücken der Gattungen Aedes aegypti und Aedes albopticus übertragene, virale Infektionskrankheit des Menschen, welche eine zunehmende Bedrohung für die Weltbevölkerung darstellt; das Infektionsrisiko betrifft vorwiegend Menschen, die in tropischen und subtropischen Gebieten der Erde (Asien, Afrika, Amerika) leben. Bei dem Erreger handelt es sich um ein Flavivirus, bestehend aus einer positiv polarisierten Einzelstrang-RNA, welches in vier verschiedenen Serotypen existiert. Eine Infektion mit Dengue-Viren zeigt sich durch drei mögliche Krankheitsbilder: Klassisches Dengue-Fieber (DF), hämorrhagisches Dengue-Fieber (DHF) oder Dengue-Schock-Syndrom (DSS). Das Dengue-Virus-Genom codiert eine Serin-Protease mit einer klassischen katalytischen Triade, bestehend aus den Aminosäuren His51, Asp75 und Ser135. Die Funktion der Dengue-Virus-Protease besteht in der post-translationalen, proteolytischen Prozessierung des viralen Polyprotein-Vorläufers, womit sie essentiell für die Virus-Replikation ist und damit einen wichtigen therapeutischen Ansatz für die Entwicklung neuer Wirkstoffe gegen Dengue-Fieber darstellt. Die Ziele der vorliegenden Arbeit bestanden darin, neue potentielle Inhibitoren der Dengue-Virus Typ 2 NS2B-NS3 Protease (DEN-2 NS2B-NS3pro) zu synthetisieren, deren Hemmwirkung sowie den Inhibitionstyp mithilfe fluorimetrischer Enzym-Assays zu bestimmen, Struktur-Wirkungs-Beziehungen (u.a. mithilfe von Molecular Docking-Rechnungen) zu analysieren und die erhaltenen Leitstrukturen zu optimieren. In der vorliegenden Arbeit wurden zwei Substanzklassen und damit zwei Teilprojekte behandelt: Phenylacrylsäureamide im ersten Teilprojekt, Benzothiazole und Diarylthioether zusammen im zweiten Teilprojekt. Im ersten Teilprojekt zeigten einige Phenylacrylsäureamide eine schwache Hemmung der DEN-2 NS2B-NS3pro zwischen ca. 50 und 61 % bei einer Inhibitorkonzentration von 50 µM sowie eine nicht-kompetitive Hemmung, welche jedoch durch vielfältige Derivatisierung kaum verändert oder verbessert werden konnte. Darüber hinaus wurden die endogenen Serin-Proteasen alpha-Chymotrypsin und Trypsin durch einige Phenylacrylsäureamide erheblich stärker gehemmt als die DEN-2 NS2B-NS3pro. Das zweite Teilprojekt befasste sich mit der Synthese und Testung von Diarylthioethern mit hydroxy-substituierten Benzothiazol-Bausteinen sowie der Testung einiger methoxy-substituierter Synthese-Vorstufen der Endverbindungen, um die Relevanz und den Einfluss der einzelnen Bausteine auf die Hemmung der DEN-2 NS2B-NS3pro zu untersuchen. Der in der vorliegenden Arbeit synthetisierte, potenteste Inhibitor der DEN-2 NS2B-NS3pro (Hemmung: 90 % [50 µM]; IC50 = 3.6 +/- 0.11 µM) und der DEN-3 NS2B-NS3pro (Hemmung: >99 % [100 µM]; IC50 = 9.1 +/- 1.02 µM), SH65, ein Diarylthioether-Benzothiazol-Derivat, entstand aufgrund der Vorhersage zweier möglicher Bindungsmodi (kompetitiv und nicht-kompetitiv) mithilfe von Molecular Docking-Experimenten an der Röntgen-Kristall-struktur der DEN-3 NS2B-NS3pro (PDB-Code: 3U1I). Nach experimenteller Bestimmung der IC50-Werte bei unterschiedlichen Substratkonzentrationen erwies sich SH65 jedoch als nicht-kompetitiver Inhibitor der DEN-2 NS2B-NS3pro. Trypsin wurde von SH65 vergleichbar stark gehemmt (96% [50 µM]; IC50 = 6.27 +/- 0.68 µM) wie die beiden getesteten Dengue-Virus-Proteasen, nicht jedoch alpha-Chymotrypsin (nur 21% Hemmung bei 50 µM), wodurch diesem Inhibitor zumindest eine relative Selektivität gegenüber Serin-Proteasen zugeschrieben werden kann. SH65 zeigte lediglich Protease-Hemmung in den Enzym-Assays, jedoch keine antivirale Aktivität bei der Testung an Dengue-Virus-infizierten Zellen, was aber wiederum bei der synthetisierten Vorstufe von SH65, welche anstelle der beiden Hydroxy-Gruppen über Methoxy-Gruppen verfügt, der Fall war. Diarylthioether mit mehrfach hydroxy-substituiertem Benzothiazol-Baustein stellen hiermit eine neue, vielversprechende Wirkstoffgruppe zur Hemmung sowohl der Dengue-Virus Typ 2- als auch der Dengue-Virus Typ 3 NS2B-NS3 Protease dar.

Brugada syndrome ECG provoked by the selective serotonin reuptake inhibitor fluvoxamine

A patient with an SCN5A p.W822X nonsense mutation, localized in the transmembrane region DII-S4 of the Na(v)1.5 sodium channel and leading to a non-expression of the mutant allele, was prescribed the selective serotonin reuptake inhibitor (SSRI) fluvoxamine (Floxyfral), 100 mg per day. His normal baseline ECG changed to a characteristic Brugada-Type-1-ECG pattern. To investigate whether fluvoxamine may reduce the cardiac sodium current, the effect of this drug was studied on the wild-type voltage-gated cardiac sodium channel Na(v)1.5 stably expressed in HEK293 cells. Patch-clamp recording showed a 50% inhibition of the current at a concentration of 57.3 microM. In our patient, no arrhythmia occurred but the proarrhythmic potential of SSRI in patients with SCN5A mutations cannot be excluded. Therefore, we advise 12-lead ECG control after administering SSRI in these patients.

Differential inhibition sensitivities of MET mutants to the small molecule inhibitor SU11274

Point mutations emerge as one of the rate-limiting steps in tumor response to small molecule inhibitors of protein kinases. Here we characterized the response of the MET mutated variants, V1110I, V1238I, V1206L and H1112L to the small molecule SU11274. Our results reveal a distinct inhibition pattern of the four mutations with IC(50) values for autophosphorylation inhibition ranging between 0.15 and 1.5muM. Differences were further seen on the ability of SU11274 to inhibit phosphorylation of downstream MET transducers such as AKT, ERK, PLCgamma and STAT3 and a variety of MET-dependent biological endpoints. In all the assays, H1112L was the most sensitive to SU11274, while V1206L was less affected under the used concentration range. The differences in responses to SU11274 are discussed based on a structural model of the MET kinase domain.

Reversal of established lupus nephritis and prolonged survival of New Zealand black x New Zealand white mice treated with the topoisomerase I inhibitor irinotecan

Systemic lupus erythematosus is a chronic autoimmune disorder that predominantly affects women of childbearing age. Lupus-associated glomerulonephritis is a major cause of mortality in these patients. Current treatment protocols for systemic lupus erythematosus include cyclophosphamide, prednisolone, azathioprine, and mycophenolate mofetil. However, in mice none of these agents alone or in combination were shown to reverse established proteinuria. Using New Zealand Black x New Zealand White F1 mice, we report that administration of the topoisomerase I inhibitor irinotecan from week 13 completely prevented the onset of proteinuria and prolonged survival up to at least 90 wk without detectable side effects. Furthermore, application of irinotecan to mice with established lupus nephritis, as indicated by grade 3+ (> or =300 mg/dl) and grade 4+ (> or =2000 mg/dl) proteinuria and, according to a median age of 35 wk, resulted in remission rates of 75% and 55%, respectively. Survival was significantly prolonged with 73 wk (grade 3+ and 4+ combined) versus 40 wk for control animals. Although total IgG and anti-dsDNA Abs in the serum and mesangial IgG deposits in the kidneys were not reduced in irinotecan-treated mice, subendothelial immune deposits were considerably diminished, suggesting a prevention of glomerular basement membrane disruption. This effect was accompanied by increased rates of ssDNA breaks and inhibition of renal cell apoptosis being different to what is known about irinotecan in anticancer therapy. In conclusion, our data provide evidence that irinotecan might represent an entirely new strategy for the treatment of systemic lupus erythematosus.

The stem cell gene "inhibitor of differentiation 1" (ID1) is frequently expressed in non-small cell lung cancer

Inhibitor of differentiation 1 (ID1) plays a role in cellular differentiation, proliferation, angiogenesis and tumor invasion. As shown recently, ID1 is positively regulated by the tyrosine kinase SRC in lung carcinoma cell lines and with that appears as a potential new therapeutic target in non-small cell carcinoma (NSCLC). To substantiate this hypothesis we examined ID1, SRC and matrix metalloproteinase-9 (MMP-9) immunohistochemically in human NSCLC specimens.

Bilateral total knee arthroplasty in a patient with hemophilia A, high inhibitor titre and aneurysma spurium of the popliteal artery. A case report

The authors report on bilateral simultaneous knee arthroplasty in a 40-year-old male patient with haemophilia A, high inhibitor titre and an aneurysma spurium of the right popliteal artery. Both knees showed a fixed flexion deformity of 20 degrees. To build up haemostasis, treatment with activated prothrombin complex concentrate (APCC) and recombinant activated factor seven (rFVIIa) was initiated preoperatively. A tourniquet was used on both sides during the operation and factor VIII (FVIII) was administered to further correct coagulopathy. On the eleventh postoperative day the patient complained of increasing pain and pressure in the right knee. An ultrasound suggested aneurysm, which was confirmed by substraction angiography. Under the protection of rFVIIa the aneurysm could be coiled and further rehabilitation was uneventful. At one year post-op the patient presented a range of motion of 90/5/0 degrees for both knees and had returned to full time office work. This case indicates that haemophiliacs with high antibody titre and destruction of both knees can be operated on in one session in order to diminish the operative risk of two consecutive surgical procedures, thus allowing an effective rehabilitation programme. Because of the significant frequency of popliteal aneurysms, preoperative angiography is recommended.

A novel mode of action of the putative sphingosine kinase inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKI II): induction of lysosomal sphingosine kinase 1 degradation

Sphingosine kinase 1 (SK1) is a key enzyme in the generation of sphingosine 1-phosphate (S1P) which critically regulates a variety of important cell responses such as proliferation and migration. Therefore, inhibition of SK-1 has been suggested to be an attractive approach to treat tumor growth and metastasis formation.

Recurrent non-melanoma skin cancer: remission of field cancerization after conversion from calcineurin inhibitor- to proliferation signal inhibitor-based immunosuppression in a cardiac transplant recipient

Non-melanoma skin cancers (NMSCs) are the most common malignancies after solid organ transplantation. Their incidence increases with time after transplantation. Calcineurin-inhibitors (CNIs) and azathioprine are known as skin neoplasia-initiating and -enhancing immunosuppressants. In contrast, increasing clinical experience suggests a relevant antiproliferative effect of mammalian target of rapamycin inhibitors, also named proliferation signal inhibitors (PSIs). We report the case of a cardiac allograft recipient with an impressive and consolidated reduction of recurrent NMSC, observed after conversion from CNI-therapy to a PSI-based protocol.

Fetuin-A and cystatin C are endogenous inhibitors of human meprin metalloproteases

Meprin and , zinc metalloproteinases, play significant roles in inflammation, including inflammatory bowel disease (IBD), possibly by activating cytokines, like interleukin 1 , interleukin 18, or tumor growth factor . Although a number of potential activators for meprins are known, no endogenous inhibitors have been identified. In this work, we analyzed the inhibitory potential of human plasma and identified bovine fetuin-A as an endogenous meprin inhibitor with a K(i) (inhibition constant) of 4.2 × 10(-5) M for meprin and a K(i) of 1.1 × 10(-6) M meprin . This correlated with data obtained for a fetuin-A homologue from carp (nephrosin inhibitor) that revealed a potent meprin and inhibition (residual activities of 27 and 22%, respectively) at a carp fetuin concentration of 1.5 × 10(-6) M. Human fetuin-A is a negative acute phase protein involved in inflammatory diseases, thus being a potential physiological regulator of meprin activity. We report kinetic studies of fetuin-A with the proteolytic enzymes astacin, LAST, LAST_MAM, trypsin, and chymotrypsin, indeed demonstrating that fetuin-A is a broad-range protease inhibitor. Fetuin-A inhibition of meprin activity was 40 times weaker than that of meprin activity. Therefore, we tested cystatin C, a protein structurally closely related to fetuin-A. Indeed, cystatin C was an inhibitor for human meprin (K(i) = 8.5 × 10(-6) M) but, interestingly, not for meprin . Thus, the identification of fetuin-A and cystatin C as endogenous proteolytic regulators of meprin activity broadens our understanding of the proteolytic network in plasma.

The protease inhibitor alpha-2-macroglobulin-like-1 is the p170 antigen recognized by paraneoplastic pemphigus autoantibodies in human

Paraneoplastic pemphigus (PNP) is a devastating autoimmune blistering disease, involving mucocutaneous and internal organs, and associated with underlying neoplasms. PNP is characterized by the production of autoantibodies targeting proteins of the plakin and cadherin families involved in maintenance of cell architecture and tissue cohesion. Nevertheless, the identity of an antigen of Mr 170,000 (p170), thought to be critical in PNP pathogenesis, has remained unknown.