The potential of lumbrokinase and serratiopeptidase for the degradation of Aß 1-42 peptide - an in vitro and in silico approach.

BACKGROUND: Alzheimer's disease (AD) is diagnosed with the deposition of insoluble ß-amyloid (Aß) peptides in the neuropil of the brain leading to dementia. The extracellular deposition of the fibrillar Aß peptide on the neurons is known as senile plaques. Therefore, Aß degradation and clearance from the human body is a promising therapeutic approach in the medication of AD. METHODS: In the current study, the enzyme lumbrokinase (LK) was extracted and purified from earthworm and its activity was utilized toward Aß 1-42 amyloids degradation in vitro alongside with an additional enzyme serratiopeptidase (SP) considering nattokinase (NK) as a standard. RESULTS: The output of this study revealed that preformed Aß 1-42 amyloids was disintegrated by both LK and SP, as demonstrated from fluorescence assay using Thioflavin T dye. In addition, dynamic light scattering study revealed the lower size of the preformed fibrils Aß 1-42 at various time intervals after incubation with the enzymes LK and SP. Furthermore, in silico approach showed high affinity thermodynamically favorable interaction of LK as well as SP toward Aß 1-42 amyloid. Finally, the toxicity of degraded preformed Aß 1-42 amyloid was assessed by MTT assay which showed reduced toxicity of enzyme treated Aß 1-42 amyloid compared to only Aß 1-42 amyloid. CONCLUSION: The findings of the present study indicated that LK and SP, not only had Aß 1-42 amyloid degrading potential, but also could reduce the toxicity which can make them a suitable drug candidate for AD. Furthermore, the in vivo studies are needed to be executed in future.

Attenuation of subcutaneous insulin induced amyloid mass in vivo using Lumbrokinase and Serratiopeptidase.

The protein misfolded structure called amyloids is related with extensive range of pathologies like local amyloidosis and neurodegenerative diseases. Several studies have reported the potential of insulin to generate local amyloidosis under certain state. Reports also showed that fibrils of insulin generated local amyloid mass due to continuous subcutaneous injection in mouse as well as rat. The present study was designed to examine the consequence of insulin fibril injections in rats, as well as the ability of enzymes, Lumbrokinase (LK) and Serratiopeptidase (SP) in diminishing this amyloid mass progression. The results showed that insulin fibrils generated amyloid masses in rats after subcutaneous injection for two weeks which was significantly condensed in size for the groups injected with insulin fibrils combined with LK or SP. At higher doses of LK and SP, the absence of amyloid structure was observed in histopathological studies. Light microscopy, polarized microscopy as well as Lumia live in vivo imaging system was used to analyze the results. In conclusion, the overall outcome of this study showed the anti-amyloid potential of enzyme LK and SP in the attenuation of local amyloidosis.

The Potential of Serratiopetidase and Lumbrokinase for the Degradation of Prion Peptide 106-126 - an In Vitro and In Silico Perspective.

BACKGROUND: PrPC is a host-encoded prion protein, which gets post translationally modified into a transmissible, ß-sheet rich disease associated protein called PrPSc, responsible for the Prion disease including mad cow disease in cattle and CJD in humans. The PrP 106-126 region in PrPSc peptide initiates the conformational change in that protein leading to fibrillation. Any agent that can destabilize or disintegrate such proteins can be served as a potential drug candidate for Prion diseases. METHODS: In the present study, an enzyme Lumbrokinase (LK) was isolated from earthworm and its activity was exploited towards PrP 106-126 amyloids in vitro along with another enzyme Serratiopeptidase (SP) taking Nattokinase (NK) as a standard. RESULTS: The results showed that PrP 106-126 amyloid formation was inhibited by both LK and SP, as evidenced from Thioflavin T fluorescence assay. Further, the size of fibrils as estimated by dynamic light scattering, was also found to be lower at different time intervals after incubation of the prion amyloids with LK and SP. Additionally, the molecular dynamics simulation revealed the thermodynamically favorable interaction of PrP 106-126 with LK as well as with SP with high affinity. CONCLUSION: Finally, the toxicity of the disintegrated amyloids was assessed using PC12 cell lines which showed higher cell viability in case of LK and SP treated amyloids compared to only PrP 106- 126 amyloid treatment. Altogether, the study concluded that the serine proteases like LK and SP have the potential to disintegrate PrP 106-126 amyloids with improved cell viability. The in vivo studies are needed to be executed in future.

ZnO Nanoflower petals mediated amyloid degradation - An in vitro electrokinetic potential approach.

An electrokinetic potential (ζ-potential) based approach was introduced to address the amyloid degradation on ZnO-nanoflower platform. The hallmark of neurodegenerative disorders like Alzheimer's disease, Parkinson's disease (PD), Creutzfeldt-Jakob Disease (CJD), Prion- associated diseases, type-II diabetes, etc. is the deposition of misfolded protein aggregates predominantly ß-sheeted in structure and fibrillar morphology, known as amyloids, in the brain and different parts of the body. Agents that can degrade these amyloids can be potential candidate for the therapy of amyloidosis. Ultrasmall nanoparticles are gaining interest due to their ability to cross blood brain barrier (BBB) which is favorable for the treatment of neurodegenerative disorders. Considering the influence of Zn2+ in the formation of Aß aggregates instead of fibrillation, the present study was designed based on the ZnO nanoparticles (ZnO-NP) and ZnO nanoflowers (ZnO-NF) to compare the anti amyloid ability using a model huminsulin amyloid. Fluorescence study, atomic force microscopy (AFM), IR spectroscopy (FTIR) and reduction of fibril size using dynamic light scattering showed that ZnO-NF can degrade amyloids with a higher capacity than their nanoparticle counterpart. Significant reduction in magnitude of ζ-potential in ZnO-NF treated huminsulin amyloid supported the notion to come to the consensus and became the new indicator for anti-amyloidosis. The cell viability assay of ZnO-NP and ZnO-NF at a higher dose than that used for amyloid degradation using PC12 and HaCaT cell lines showed their biocompatibility in a safe manner. Thus, it can be suggested that ZnO-NF would be a better candidate for amyloid degradation compared to ZnO-NPs due to higher surface to volume ratio of the petals.

ZnO nanoflower based sensitive nano-biosensor for amyloid detection.

Zinc oxide (ZnO) is a semiconductor metal oxide nanoparticle with inherent optical properties. Among the different zinc oxide nanostructures, nanoflowers have greater surface area. Utilizing this property a reagentless biosensor has been developed for the detection of beta amyloids, a hallmark of neurodegenerative diseases like Alzheimer's disease, Creutzfeldt-Jakob Syndrome, insulin dependent type II diabetes etc. The poor fluorescence quantum yield and photobleaching effect of Thioflavin T (ThT) upon binding to the model insulin amyloid beta sheets in solution can be overcome by the present engineered biosensor where ThT acts as a target as well as a reporter to detect amyloids adsorbed on a solid template based on ZnO nanoflower. ThT was adsorbed on ZnO NFs grown over nano-silver thin film coated glass slide. The in vivo imaging system was used to detect and quantify the fluorescence intensity generated from the substrates upon binding with insulin amyloid. ZnO NFs have the waveguiding property which increases the local field intensity caused by a resonance between the guided fundamental mode and evanescent field associated with high- order modes. This resonance phenomenon reinforces the excitation of the fluorophores in close proximity of the NFs thereby exhibiting enhanced fluorescence like Fabry Pero't Resonator (FPR). Considering the engineering and sensitivity, the reported nanobiosensor developed on ZnO nanoflower can be treated as faster and cost effective amyloid sensor.

In vitro and in vivo insulin amyloid degradation mediated by Serratiopeptidase.

A transition of amyloidogenic protein by alternative folding pathway under certain conditions leads to the formation of protease resistant amyloid fibrils, having predominantly cross ß structure. These amyloids are related to various neurodegenerative diseases and clearance of such amyloids may be a therapeutic approach for amyloid-related diseases. Insulin, that can form amyloids, is widely used as a model amyloidogenic protein for the study of various amyloid related diseases. In this study, insulin amyloids were formed in vitro and the potential of Serratiopeptidase (SP), a fibrinolytic-like serine protease, towards the dissociation of insulin amyloids was explored. The dissociation of the amyloids was demonstrated using in vitro and in vivo using zebrafish model. The amyloid dissociation property was compared with a standard amyloid dissociating enzyme nattokinase (NK). SP shows better amyloid dissociation ability than NK and therefore, SP can be considered as amyloid dissociating agent with potential as a drug candidate for different amyloid related disorders.