Inhibition and detection of 15-lipoxygenase-1

Humaan 15-lipoxygenase-1 (15-LOX-1) is een belangrijke lipoxygenase in zoogdieren, welke een cruciale rol speelt in de biosynthese van inflammatoire signaalmoleculen. 15-LOX-1 wordt hierdoor gezien als een regulerend enzym in verschillende inflammatoire longziekten zoals astma, chronic obstructive pulmonary disease (COPD) en chronische bronchitis. Recentelijk is er ook een rol voor dit enzym beschreven in ziektes van het centraal zenuwstelsel zoals Alzheimer, Parkinson en beroerte. Nieuwe, krachtige remmers en moleculaire detectiemethodes voor 15-LOX-1 activiteit zijn dringend nodig om de rol van het enzym verder te onderzoeken en om de ontwikkeling van nieuwe geneesmiddelen mogelijk te maken. Dit proefschrift bestaat uit een deel A en een deel B met aanverwante, maar verschillende, onderwerpen. In deel A is de ontwikkeling van nieuwe remmers van 15-LOX-1 beschreven. Deze remmers zijn ontwikkeld door de toepassing van een nieuwe benadering voor fragment gebaseerde screening. Deze werkwijze maakt gebruik van divers gesubstitueerde moleculen voor een benadering die wordt aangeduid als substitution oriented screening (SOS). De moleculen die het enzym bleken te remmen zijn geoptimaliseerd met behulp van computermodellen op basis van de moleculaire structuur van de remmers en het enzym 15-LOX-1. Hierdoor konden verschillende potente remmers ontwikkeld konden worden. De beste remmers werden geëvalueerd in celgebaseerde onderzoeken en in ex vivo studies op precisie gesneden plakjes longweefsel van muizen. In deze studies bleken de ontwikkelde remmers ontstekingsremmende en neuroprotectieve effecten te hebben. In deel B is de ontwikkeling van een nieuwe detectiemethode om het actieve enzym 15-LOX-1 te bestuderen beschreven. Met een zogenoemde activity-based probe bleek het mogelijk te zijn om gezuiverd 15-LOX-1 te labelen op basis van de activiteit van dit enzym. Het bleek ook mogelijk om deze methode in te zetten om de 15-LOX-1-activiteit aan te tonen in cellysaten en weefsel. Deze methode is ontwikkeld door moleculen te maken die het natuurlijke substraat van dit enzym nabootsen maar die bij binding aan het enzym covalent binden aan het actieve centrum van het enzym op basis van de enzymactiviteit. Deze moleculen zijn voorzien van een terminale alkeen als chemisch label dat gebruikt kan worden voor bioorthogonale koppeling van een detecteerbare biotine functionaliteit via de oxidatieve Heck reactie

Long-term radiotherapy related complications in children with head and neck cancer: Another era for pediatric oncologic pathology

Long-term radiotherapy-related complications in children with head and neck cancer have been frequently reported, especially facial growth disorders and dental abnormalities. We report on two male children (8 and 14 years old) with head and neck cancer, who were successfully treated with chemoradiotherapy and presented with growth deficiency of middle face and mandible hypoplasia, eight years and one year later, respectively. These severe growth complications attributed to chemoradiotherapy, while the patients survived primary malignancy. Patient age at irradiation was significantly correlated with the severity of disorders. We consider late sequelae in children with head and neck cancer due to chemoradiotherapy another era for pediatric oncologic pathology for prevention, if possible, or to manage them efficiently

Long term chemoradiotherapy-related dental and skeletal complications in a young female with nasopharyngeal carcinoma

We describe the long-term complications six years after chemoradiotherapy in a 20-year old woman with nasopharyngeal carcinoma. We wanted to know whether the radiation dose was constant throughout the oral cavity, and thus uniformly affecting the corresponding dental and skeletal structures. Clinical and radiologic findings are described six years after chemoradiotherapy based on a two-dimensional computerized treatment planning system. This revealed radiation caries limited only to posterior teeth, proximal caries in the anterior teeth, limited but continuous salivary flow, mild periodontal infection, mild xerostomia, and a regenerative capacity of bones and the developmental process. The quantitative assessment of radiation delivered to the mandible revealed a high radiation dose in the posterior area and a minimal dose in the anterior area. This explains the differences in caries manifestation between the anterior and posterior teeth. According to the present study, individualized radiation fields, using a two-dimensional treatment planning system, result in restriction of severe damage of the dental and skeletal structures, which usually follows chemoradiotherapy. Orthodontic treatment could be initiated according to individual patient needs

Photoactivation provides a mechanistic explanation for pan-assay interference behaviour of 2-aminopyrroles in lipoxygenase inhibition

Human 15-lipoxygenase-1 (h-15-LOX-1) is a promising drug target in inflammation and cancer. In this study substitution-oriented screening (SOS) has been used to identify compounds with a 2-aminopyrrole scaffold as inhibitors for h-15-LOX-1. The observed structure activity relationships (SAR) proved to be relatively flat. IC50's for the most potent inhibitor of the series did not surpass 6.3 μM and the enzyme kinetics demonstrated uncompetitive inhibition. Based on this, we hypothesized that the investigated 2-aminopyrroles are pan assay interference compounds (PAINS) with photoactivation via a radical mechanism. Our results demonstrated clear photoactivation of h-15-LOX-1 inhibition under UV and visible light. In addition, the investigated 2-aminopyrroles decreased viability of cultured human hepatocarcinoma cells HCC-1.2 in a dose-dependent manner with LD50 ranging from 0.55 ± 0.15 μM (21B10) to 2.75 ± 0.91 μM (22). Taken together, this indicates that photoactivation can play an important role in the biological activity of compounds with a 2-amino-pyrrole scaffold as investigated here

Small Multitarget Molecules Incorporating the Enone Moiety

Chalcones represent a class of small drug/druglike molecules with different and multitarget biological activities. Small multi-target drugs have attracted considerable interest in the last decade due their advantages in the treatment of complex and multifactorial diseases, since "one drug-one target" therapies have failed in many cases to demonstrate clinical efficacy. In this context, we designed and synthesized potential new small multi-target agents with lipoxygenase (LOX), acetyl cholinesterase (AChE) and lipid peroxidation inhibitory activities, as well as antioxidant activity based on 2-/4- hydroxy-chalcones and the bis-etherified bis-chalcone skeleton. Furthermore, the synthesized molecules were evaluated for their cytotoxicity. Simple chalcone b4 presents significant inhibitory activity against the 15-human LOX with an IC50 value 9.5 µM, interesting anti-AChE activity, and anti-lipid peroxidation behavior. Bis-etherified chalcone c12 is the most potent inhibitor of AChE within the bis-etherified bis-chalcones followed by c11. Bis-chalcones c11 and c12 were found to combine anti-LOX, anti-AchE, and anti-lipid peroxidation activities. It seems that the anti-lipid peroxidation activity supports the anti-LOX activity for the significantly active bis-chalcones. Our circular dichroism (CD) study identified two structures capable of interfering with the aggregation process of Aβ. Compounds c2 and c4 display additional protective actions against Alzheimer's disease (AD) and add to the pleiotropic profile of the chalcone derivatives. Predicted results indicate that the majority of the compounds with the exception of c11 (144 Å) can cross the Blood Brain Barrier (BBB) and act in CNS. The results led us to propose new leads and to conclude that the presence of a double enone group supports better biological activities

A nexus of intrinsic dynamics underlies translocase priming

The cytoplasmic ATPase SecA and the membrane-embedded SecYEG channel assemble to form the Sec translocase. How this interaction primes and catalytically activates the translocase remains unclear. We show that priming exploits a nexus of intrinsic dynamics in SecA. Using atomistic simulations, smFRET, and HDX-MS, we reveal multiple dynamic islands that cross-talk with domain and quaternary motions. These dynamic elements are functionally important and conserved. Central to the nexus is a slender stem through which rotation of the preprotein clamp of SecA is biased by ATPase domain motions between open and closed clamping states. An H-bonded framework covering most of SecA enables multi-tier dynamics and conformational alterations with minimal energy input. As a result, cognate ligands select preexisting conformations and alter local dynamics to regulate catalytic activity and clamp motions. These events prime the translocase for high-affinity reception of non-folded preprotein clients. Dynamics nexuses are likely universal and essential in multi-liganded proteins.</p

Evaluation of the Possible Transmission of BSE and Scrapie to Gilthead Sea Bream (Sparus aurata)

In transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative disorders affecting many species, the key event in disease pathogenesis is the accumulation of an abnormal conformational isoform (PrPSc) of the host-encoded cellular prion protein (PrPC). While the precise mechanism of the PrPC to PrPSc conversion is not understood, it is clear that host PrPC expression is a prerequisite for effective infectious prion propagation. Although there have been many studies on TSEs in mammalian species, little is known about TSE pathogenesis in fish. Here we show that while gilthead sea bream (Sparus aurata) orally challenged with brain homogenates prepared either from a BSE infected cow or from scrapie infected sheep developed no clinical prion disease, the brains of TSE-fed fish sampled two years after challenge did show signs of neurodegeneration and accumulation of deposits that reacted positively with antibodies raised against sea bream PrP. The control groups, fed with brains from uninfected animals, showed no such signs. Remarkably, the deposits developed much more rapidly and extensively in fish inoculated with BSE-infected material than in the ones challenged with the scrapie-infected brain homogenate, with numerous deposits being proteinase K-resistant. These plaque-like aggregates exhibited congophilia and birefringence in polarized light, consistent with an amyloid-like component. The neurodegeneration and abnormal deposition in the brains of fish challenged with prion, especially BSE, raises concerns about the potential risk to public health. As fish aquaculture is an economically important industry providing high protein nutrition for humans and other mammalian species, the prospect of farmed fish being contaminated with infectious mammalian PrPSc, or of a prion disease developing in farmed fish is alarming and requires further evaluation