Alzheimer’s disease (AD) is one of the major medical challenges of our century, affecting 55 million patients worldwide [1]. This progressive, as yet incurable, neurodegenerative disorder, primarily affects people over the age of 60 years. In the brain of an AD patient, the formation and breakdown of amyloid beta protein (Aβ) are out of balance, leading to increased aggregation of Aβ to the point where normal cognitive functions are obstructed. This usually manifests with changes in memory, behaviour and ultimately interferes the control over the body functions [2].
Passive immunotherapy, that is directed against Amyloid beta, has so far proven to be the most promising therapy option. Accelerated approval of Aduhelm (Aducanumab), a monoclonal antibody targeting Aβ [3], was recently granted by the U.S. FDA [4]. Showing undesirable side effects and minor effectiveness, the use of this drug is controversial [5]. One reason is presumably the physiological function of Aβ. Therefore, the search continues for alternative approaches that address only the pathological aggregated Aβ. Here, post-translationally modified Aβ forms are of great interest, since they are often specific found in deposited Aβ.
The 3-nitrotyrosine modification at position 10 (3NY10) of Aβ is suspected to be involved in the pathogenic course of AD [6]. Two highly specific 3NY10-Aβ antibodies are now generated, which can be used as tool for further investigation and beyond shall be developed into a drug to fight Aβ deposits in AD patient’s brain, while leaving the physiological Aβ unaffected. In context of these work, the antibodies will be characterised by a variety of biochemical and kinetic analyses (ITC, ELISA, TEM, Aggregation studies). For a better understanding and evaluation of the interaction between the antibody and its target, the macromolecular structure shall furthermore be determined by X-ray crystallography.
So far, one of two antibodies was co-crystalised using the responding Fab-fragment and the 3NY10-Aβ target-peptide. After initially no crystals could be obtained by the sitting drop vapor diffusion method using commercial available random matrix screens at 96-well format, precipitate from one of the former screenings was used as a microseed. Suddenly, about 20 % of the tested commercial available crystallisation conditions were suitable for crystal growth. Hence obtained stretched hexagonal crystals have been tested for their diffraction pattern and the condition with the best crystal was selected for further optimisation. Due to change to hanging drop vapor diffusion in a 15-well format and variation of the seeding concentration, crystal size and thickness could be controlled. This slightly improved a problem of the crystal: different planes of the crystal are slightly staggered in their orientation, making it difficult to collect only one set of diffraction data at a time. Currently the diffraction data obtained are used in building and refinement of the structure.
We thank the Deutsche Forschungsgemeinschaft (DFG) for supporting the cooperation project with Prof. Steffen Roßner from the Paul-Flechsig-Institute for brain research, department Molecular Imaging in Neurosciences.