## 3 Results and Discussion
### Main Complexes Formed with Microcystins with High Affinity Scores
#### 3.1.2 Interactions Related to Inflammatory Proteins and Neuroprotective Alteration  
The clear virtual links among MC-LA, MC-LR, and MC-LY with butylcholinesteraseterase (BChE) and \(\beta\)-secretase 1 (BACE1) could explain another route by which MCs generate neurotoxic effects. The altered behavior of the BChE is related to neurodegenerative dis  
Figure 2: Three-dimensional view of CD38 in complex with MC-LY (magenta) (**A**), Atomic level view showing interactions between contact residues on the binding site and the ligand, as predicted by Ligand Scout (**B**). Atoms are colored with the CPK scheme. eases and similar to BACE1, and has been related to the generation of beta amyloid-1 protein plaques in the brain [39; 40]. The function and regulation of BACE1 at the brain level have not been fully elucidated [41], and there are no reports of the association of MCs with this protein. However, based on the virtual results, we suggest that the interaction between MCs and BACE1 should be investigated experimentally. The \(\beta\)-secretase 1 showed the highest affinities with MC-LY and MC-LA with scores of \(-10.9\) kcal/mol and \(-10.5\) kcal/mol, respectively. The neurotoxic effects of the MCs are manifested as neuronal loss and morphological changes by hyperphosphorylation of the axon cytoskeleton [42]. Many of these effects have been associated with the obstruction generated by MCs in PP1 and PP2, which leads to oxidative stress, damage to the hippocampus, and hyperphosphorylation of tau protein, the main protein in the neuronal microtubules, thus favoring the development of some neurogenerative diseases [32; 33; 34]. However, these neurotoxic responses could also be explained as a positive regulation by MCs of the abundant proteins BACE1 and BChE, and not exclusively to the widely referred inhibition of PP1 and PP2. Alterations in the activity of BChE lead to the release of proinflammatory cytokines [43; 44]. In addition, BChE participates in the development of hepatic adiposity that is elevated in type I and II diabetes and hepatocellular carcinoma [45]. The interaction of MCs with this enzyme could also explain the negative effect that MCs generate in the liver as they induce increased hepatic steatosis and binucleation of the hepatocyte, generating strong inflammation and irreversible damage [35; 37]. Toll-like receptors (TLRs) were the main representatives of the interaction of MCs with inflammatory proteins (Table 1). TLR receptors are involved in adaptive immune responses and pathogen recognition [46]. The interaction of MCs with these proteins has been reported previously, specifically with TLR4, which generates persistent inflammation through the TLR/MyD88 pathway and the TLR4/NF-k\(\beta\) pathway [47; 48], and therefore can induce hypertension through vascular inflammation, renal and CNS damage, and insulin resistance [49]. We found that in addition to TLR4, the TLR8 receptor generates strong binding with MC-LY and MC-LA (Table 1 and Figure 3). TLR8 contributes to the production of IL-6, IL-12p70, NF-k\(\beta\), and TNF-\(\alpha\), which are predominantly expressed on macrophages, dendritic cells, neutrophils, and monocytes [50]. The inflammatory shock produced by MCs may have been caused by the synergistic effect of TLR4 and TLR8, in addition to the disruption of inducible nitric oxide synthase (iNOS), which showed virtual couplings of \(-11.5\) kcal/mol with MC-LA and MC-LY and whose interaction with MC-LR induced genotoxicity [51]. Angiotensin-I converting enzyme (ACE) is another enzyme that showed strong interactions with the four MC variants analyzed (Figure 4). ACE is a Zn\({}^{2+}\) and Cl\({}^{-}\) dependent metalloprotein responsible for the metabolism of angiotensin I, whose imbalance alters blood pressure and induces congestive heart failure [52]. Microcystins showed superior affinities to this metallopeptide (Table 1), which has also been demonstrated experimentally, generating a variety of mechanisms that affect renal and cardiac function.