By Killian Robinson
Alzheimer’s is a terrible disease that causes neurological deterioration which can often lead to other diseases like dementia. There are currently 50 million cases of Alzheimer’s worldwide which is a startling figure and is estimated to almost double every decade, with 82 million cases projected in 2030 and 152 million in 2050 (ADI, 2021). Since Alzheimer’s is so prevalent, it is very important that we investigate how we can better treat this disease, and multi-target drug ligands (MDTL’s) could offer a potential therapeutic solution which we will discuss in this article.
There were two main theories to explain the pathogenesis of Alzheimer’s – the “tau” and the “amyloid” hypotheses. The “amyloid” hypothesis involves the production of Aβ (amyloid-β) plaques during abnormal processing via the aspartyl protease, BACE1, which can cause neuronal cell death, possibly via the induction of an immune response where the plaques build up (Brightfocus, 2009). Interestingly, small Aβ plaques known as ‘seeds’, have been observed to accelerate plaque formation in a prion like manner, which makes the pathogenesis much faster and hence deadlier (Olsson, Klementieva & Gouras, 2018). On the other hand, the “tau” hypothesis suggests that as we age and GSK-3β levels increase, this causes elevated GSK-3β activity leading to the hyperphosphorylation of tau proteins found on microtubules at serine residues. Tau proteins have roles in microtubule stabilization, however subsequent to hyperphosphorylation, the tau proteins can dissociate from the microtubules they are bound to and form a helical filaments with other dissociated tau proteins. This aggregation of helical filaments can eventually lead to neurofibrillary tangle formation which can contribute to neural degeneration (Liu, Xie, Meng & Kang, 2019).
Figure 1 – Diagram showing the amyloid hypothesis (upper) and the tau hypothesis (lower), as it turns out, these hypotheses may not be mutually exclusive at all, but all part of a large network of pathways, ultimately leading to Alzheimer’s. Highlighted in blue are drugs that have been terminated in phase 3 clinical trials that target solely the amyloid hypothesis (Liu, Xie, Meng & Kang, 2019).
Despite the research into the separate hypotheses, drugs targeting GSK-3β and BACE1 independently have not been very successful. Newer research suggests that these hypotheses are not mutually exclusive and are connected in one large network, all the pathways of which acting to cause amyloid deposition and subsequent neurodegeneration (Simone et al., 2021). Hence, new drug development is focused on creating drugs that consider multiple targets, known as multi-target drug ligands (MTDL’s), in order to prevent Alzheimer’s progression. The impetus being that if multiple enzymes and pathways are utilized in Alzheimer’s progression, a drug targeting multiple enzymes and pathways must also be used to treat the disease.
Figure 2 – The role of GSK-3β and BACE1 in modulating Alzheimer’s progression. The bottom left shows are conventional tau hypothesis, and the upper left showing the “amyloid” hypothesis. However, it also shows how GSK-3β is involved in connecting pathways to each other, with roles in many networks. Additionally, it also has a role in processes undescribed by the hypotheses, such as neuroinflammation and upregulating Aβ formation. It does the latter by affecting the production of presenilin (PS1), a component of the γ-secretase enzyme required for Aβ production (Simone et al., 2021).
To investigate how MTDL’s can be used and how efficacious they are, we are going to focus on a dual GSK-3β/BACE1 inhibitor. To produce this MTDL, a fragment-based design must be applied to produce a molecule that can dock at both the GSK-3β and BACE1 catalytic sites. As shown in Figure 3, sections common to many BACE1 inhibitors were combined with sections common to GSK-3β (although not shown) to produce an inhibitor capable of the task required. Additionally, figure 3 shows a molecule with a fluorine R-group docked into the catalytic region of BACE1 and GSK-3β (Prati et al., 2014).
Figure 3 – The upper diagram is the fragment-based strategy employed to produce a dual GSK-3β/BACE1 inhibitor (Simone et al., 2021). The lower left diagram is such an inhibitor in BACE1’s catalytic site, with it having hydrogen bonds to aspartic acid (D32,228) and threonine (T231) residues, with a binding affinity of –7.291kcal/mol. The right diagram is the same molecule docked in the GSK-3β catalytic site, where it forms two hydrogen bonds with Valine (V135) and stacking interactions with Arginine (R141). The binding affinity for this was –6.732 kcal/mol (Prati et al., 2015)
Experiments into the pharmacodynamics of dual GSK-3β/BACE1 inhibitors with different R groups have been conducted. To probe whether dual inhibitors are worthy of further research, IC50 and ligand efficiency values were calculated and the effect on neuroglioma cells harboring large quantities Aβ were investigated.
Beginning with IC50 values, Inhibitor IV and SB415286 were used as reference (non-multi target) inhibitors for BACE1 and GSK-3β, their values being 0.02 μM and 0.05 μM respectively. The IC50 value for a promising compound (3) (shown in the bottom right of the upper diagram of figure 3) was 18.03±0.01μM for BACE1 and 14.67±0.78μM for GSK-3β which is a decent inhibitory profile for a dual inhibitor. Additionally, this compound had a higher ligand efficiency (0.32) than the independent drugs with inhibitor IV (0.19) and SB415286 (0.29) (Prati et al., 2015). Furthermore, it was shown that in neuroglioma cells harboring the Swedish mutation (which causes increase levels of secreted Aβ), there was a significant difference in Aβ levels when treated with compound 3 from untreated levels, although it was quite limited (Prati et al., 2015).
Figure 4 – Data for different compound effects on neuroglioma cells harboring the Swedish mutation (error bars indicating ±SD). For the graph on the left, using an MTT assay cell viability was measured using different compounds with SB216763 making a significant increase in viability **p < 0.01. Interestingly, despite compound 3 not making a significant increase in viability, there was no increase for inhibitor IV either. The graph on the right denotes the drugs effect on Aβ levels using an ELISA test, of which Inhibitor IV had a very significant difference, ***p < 0.01, with compound 3 also making a significant difference to Aβ levels (*p < 0.05), albeit small (Prati et al., 2015).
Further experiments investigated the inhibition of GSK-3β in inflammatory responses, as overactive GSK-3β can lead to the increased induction of inducible nitric oxide synthase (iNOS). iNOS produces increased levels of nitric oxide which can enhance neurodegenerative effects (Contestabile, Monti & Polazzi, 2012). LPS exposure can result in iNOS expression via GSK-3β, and therefore the effect of an inhibitor on this will tell us about how effective the inhibitor is against the inflammation in Alzheimer’s that can contribute to neurodegeneration (as described in Figure 2) (data shown in Figure 5).
Additionally, the cell surface receptor, TREM-2, was examined as it is involved in microglial phagocytic activity which acts clear the brain of toxins and debris (like Aβ plaques). By examining changes to its expression, it will help to elucidate the modulatory action of compound 3 in microglial phenotype. The results (shown in figure 5) display that there was no change to TREM-2 expression at 10μM – so no loss in neuroprotective function of microglia, but in addition the compound was able to induce a different microglial phenotype. It converted microglia from M1 to M2 – a much more anti-inflammatory phenotype which research suggests may enhance protective effects and diminish neurodegenerative effects (Boche, Perry & Nicoll, 2013). Here we can see that not only is the compound reducing the iNOS levels which contribute to Alzheimer’s progression, it is also enhancing and maintaining neuroprotective effects.
Figure 5 – The Figure displays western blots and bar graphs for both rat microglia and astrocyte cells to convey the effect of compound 3 on iNOS levels. The ratio of iNOS/β-actin represents its efficacy, the lower of which being a more effective inhibition of inflammatory activity. β-actin is used as a control here, with the data showing that compound 3 reduced iNOS expression, in a dose dependent manner, in both types of cells. TREM-2 was investigated and shown to not be affected by compound 3, but the compound did initiate conversion to M2 phenotypes and paired with a decreased iNOS expression shows this compound is not only anti-inflammatory, but also boasts increased neuroprotectivity (Prati et al., 2015).
To conclude, what is key here is that these compounds have potential. These drugs are clearly not perfect from the fact that compound 3 was not as efficacious as hoped against neuroglioma cells with increased Aβ levels (Figure 4), and the maximum cerebral concentration recorded was 0.62 μM, which is notably different from its IC50 value which it must reach to have the desired effects. However, it has a decent inhibitory profile, has been shown to induce some rather compelling effects on microglia associated with neuroprotective abilities (Figure 5) and has higher ligand efficiencies (Prati et al., 2015). Therefore, it is important to not dismiss the potential these compounds clearly hold. Furthermore, considering the projected prevalence of Alzheimer’s, approximately doubling every decade from now, it is paramount that we do as much research as possible into new therapeutic options. Further research into these drugs may just offer salvation to the millions worldwide suffering from this appalling disease.
- ADI – Dementia statistics. (2021) Available from: https://www.alzint.org/about/dementia-facts-figures/dementia-statistics/ [Accessed Jun 9, 2021].
- Amyloid Plaques and Neurofibrillary Tangles. (2015) Available from: https://www.brightfocus.org/alzheimers-disease/infographic/amyloid-plaques-and-neurofibrillary-tangles [Accessed Jun 9, 2021].
- Boche, D., Perry, V. H. & Nicoll, J. a. R. (2013) Review: activation patterns of microglia and their identification in the human brain. Neuropathology and Applied Neurobiology. 39 (1), 3-18. Available from: doi: 10.1111/nan.12011. [Accessed Jun 9, 2021].
- Contestabile, A., Monti, B. & Polazzi, E. (2012) Neuronal-glial Interactions Define the Role of Nitric Oxide in Neural Functional Processes. Current Neuropharmacology. 10 (4), 303-310. Available from: doi: 10.2174/157015912804143522. [Accessed Jun 9, 2021].
- De Simone, A., Tumiatti, V., Andrisano, V. & Milelli, A. (2021) Glycogen Synthase
Kinase 3β: A New Gold Rush
in Anti-Alzheimer’s Disease Multitarget Drug Discovery? Journal of Medicinal Chemistry. 64 (1), 26-41. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016207/. Available from: doi: 10.1021/acs.jmedchem.0c00931. [Accessed Jun 9, 2021].
- Gandy, S. (2005) The role of cerebral amyloid β accumulation in common forms of Alzheimer disease. The Journal of Clinical Investigation. 115 (5), 1121-1129. Available from: https://www.jci.org/articles/view/25100. Available from: doi: 10.1172/JCI25100.
- Liu, P., Xie, Y., Meng, X. & Kang, J. (2019) History and progress of hypotheses and clinical trials for Alzheimer’s disease. Signal Transduction and Targeted Therapy. 4 29. Available from: doi: 10.1038/s41392-019-0063-8. [Accessed Jun 9, 2021].
- Olsson, T. T., Klementieva, O. & Gouras, G. K. (2018) Prion-like seeding and nucleation of intracellular amyloid-β. Neurobiology of Disease. 113 1-10. Available from: doi: 10.1016/j.nbd.2018.01.015. [Accessed Jun 9, 2021].
- Palomo, V., Perez, D. I., Perez, C., Morales-Garcia, J. A., Soteras, I., Alonso-Gil, S., Encinas, A., Castro, A., Campillo, N. E., Perez-Castillo, A., Gil, C. & Martinez, A. (2012) 5-imino-1,2,4-thiadiazoles: first small molecules as substrate competitive inhibitors of glycogen synthase kinase 3. Journal of Medicinal Chemistry. 55 (4), 1645-1661. Available from: doi: 10.1021/jm201463v. [Accessed Jun 9, 2021].
- Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A. & Bray, F. (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: A Cancer Journal for Clinicians. 71 (3), 209-249. Available from: doi: 10.3322/caac.21660. [Accessed Jun 9, 2021].
- Prati, F., De Simone, A., Bisignano, P., Armirotti, A., Summa, M., Pizzirani, D., Scarpelli, R., Perez, D. I., Andrisano, V., Perez-Castillo, A., Monti, B., Massenzio, F., Polito, L., Racchi, M., Favia, A. D., Bottegoni, G., Martinez, A., Bolognesi, M. L. & Cavalli, A. (2015) Multitarget Drug Discovery for Alzheimer’s Disease: Triazinones as BACE-1 and GSK-3β Inhibitors. Angewandte Chemie International Edition. 54 (5), 1578-1582. Available from: https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.201410456. Available from: doi: 10.1002/anie.201410456. [Accessed Jun 9, 2021].