AI3SD Video: Finding new in silico-based therapeutic strategies for IAHSP

Abstract

Infantile-onset ascending spastic paralysis (IAHSP) is a neurodegenerative autosomic recessive rare disease which affects less than 50 people worldwide. The pathogenesis starts in early childhood, with a progressive degeneration of the upper spinal motoneuron, progressively hindering deambulation until spread to the upper limbs and to the involuntary musculature(1). As it often occurs for rare diseases, although few interest from the pharma compartment, some information regarding this condition are available from case reports: key events responsible for this condition are mutations to the gene ALS2, which encodes for the cell trafficking-related protein alsin. Nevertheless, the relatively broad mutational landscape and the low number of reported cases still make a complete understanding of the physiopathology and the search for suitable therapeutic strategies pretty challenging. The majority of mutations described in literature result in a truncated form of alsin which is reputed to be degraded, thus depicting a scenario of loss-of-function pathogenesis. Nevertheless, some patients report missense mutation, leading to non-degraded, mutated forms. In those cases, the majority of amino-acid (aa) substitutions occur in the N-terminal RLD domain, essential for alsin localization to the plasma membrane and eventually to early and late endosomes upon activation of the RAC1 pathway. In endosomes, alsin binds to the small GTPase Rab5 and performs a guanosin-exchange factor activity (GEF) through its C-terminal VPS9 domain2. This pathway is reputed to be the major strategy that mammalian cells follow, in order to assemble endosomes and exchange materials within the cell architecture. In dimensionally important cells such as motoneurons, coordinated and efficient cell trafficking results crucial for correct development and function maintenance. Alsin exists in cytoplasmic solution as tetramer, firstly assembled by parallel dimerization through the VPS9 domain and subsequently by interaction of two dimers through their DH/PH domain, located upwards of the VPS9 region2. The first challenge that such a broad mutational landscape offers is that different mutations correspond to different multimers. These states do not just affect stability and solubility, but also subcellular localization and GEF activity. To make this situation more challenging, there is no experimentally-resolved 3D structure of alsin and a homology modeling effort to build the whole protein seems questionable because of the lack of a reliable template. In contrast with the majority of reports, here we present a patient case harboring two alsin mutations in the C-terminal region: one allele translates a frame-shifted, truncated form which gets degraded. The other allele is harboring the R1611W aa substitution in the VPS9 domain. With the aid of in silico computational tools, we managed to predict the 3D structure of normal and mutated forms of this domain. Moreover, we characterized physiologic and pathologic dimerization modes, discovering that mutated VPS9 preferentially forms an antiparallel dimer by interacting with the aforementioned RLD domain. We could link this discovery to the experimentally-determined loss of tetrameric aggregation and, more important, to the incorrect endosome localization. This finding corroborates and gives a mechanistic explanation for the experimentally-characterized reduced Rab5 GEF endosomal activity. Furthermore, we performed an in silico virtual screening, repurposing an already commercialized drug which is able to shield the pathologically-acquired hydrophobic moiety. In our hypothesis, this mechanism of action re-establishes physiological dimerization mode, subcellular localization and Rab5 activity in R1611W-mutated patients. The candidate is currently under pre-clinical testing in an alsin R1611W cellular model. Our hope and the scope of our effort is duplex: first, we want to provide a reliable treatment for alleviating symptoms and disease progression to our patient. Second, we would like to broaden the knowledge in the field and, by integrating in silico and in vitro procedures, establish a lean research pipeline that might once serve as mutation-based platform for individual drug repurposing for the treatment of alsin-related diseases.

References:

Lesca, G. et al. Infantile ascending hereditary spastic paralysis (IAHSP): Clinical features in 11 families. Neurology 60, 674–682 (2003).
Sato, K. et al. Altered oligomeric states in pathogenic ALS2 variants associated with juvenile motor neuron diseases cause loss of ALS2-mediated endosomal function. J. Biol. Chem. 293, 17135–17153 (2018).

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Identifiers

ORCID for Jeremy G. Frey: orcid.org/0000-0003-0842-4302

ORCID for Samantha Kanza: orcid.org/0000-0002-4831-9489

ORCID for Mahesan Niranjan: orcid.org/0000-0001-7021-140X

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