Psilocybin, a hallucinogenic compound in magic mushrooms, primarily affects mood and behavior through agonistic interactions with the 5-HT2A receptor. This article explores the biochemical processes behind psilocybin's effects, focusing on its metabolism, protein interactions, and drug binding kinetics. Psilocybin is converted to psilocin via alkaline phosphatase and monoamine oxidase A, increasing lipid solubility and blood-brain barrier passage. Psilocin binds to the 5-HT2A receptor, a GPCR in the nervous system. Structural and kinetic studies reveal differential affinities between psilocybin and psilocin, highlighting the role of metabolic conversion in receptor binding and potential therapeutic applications.

Figure 1: The series of steps L-Tryptophan undergoes to form psilocybin.

Studies have also shown that this hallucinogen has a very similar structure to LSD, leading both to have similar chemical properties [1], [2]. Psilocybin's primary path of action is through the activation of the serotonin receptors that are located in the prefrontal cortex. According to a medical article [4], the consumption of psilocybin causes changes in mood, forms hallucinations, causes euphoria, dizziness, and several more side effects.

Figure 2 gives information about the structure of the protein. The functional groups present include a benzene ring that is responsible for any hydrophobic interactions, a tertiary amine that acts as a hydrogen bond acceptor, a secondary amine on the backbone responsible for interactions that form secondary structures, and a phosphate that makes the molecule highly polar and water-soluble. Moreover, it helps the protein act as a zwitterion at physiological pH, with the positive charge from the amine group and the negative charge from the oxygen, thus making the protein more water-soluble. However, this form of the protein is not its activated form, and it will only be able to enter the brain once and once it is activated. Psilocybin is dephosphorylated and converted into its activated ingredient, psilocin, by a metabolic reaction that uses two different enzymes: alkaline phosphatase and monoamine oxidase. Studies showed that the lack of the phosphate group makes psilocin more lipid soluble, and thus, it can easily cross the blood-brain barrier and bind to its receptor protein [5]. This research paper aims to comprehensively investigate the biochemical basis of psilocybin's effects, focusing on its metabolism, protein interactions, and drug-binding kinetics.

Figure 2: Shows the structures of psilocybin and tryptophan. The pink highlighted version (left) proves that psilocybin is a tryptophan derivative.

Psilocybin Metabolism and the Target Protein Structure

As mentioned in the introduction section, psilocybin must be converted to its active ingredient, psilocin, for it to pass the blood brain barrier; this conversion requires a series of chemical reactions. This process begins after the oral administration of psilocybin and under the strong acidic conditions of the stomach, psilocybin is dephosphorylated. This dephosphorylation not only occurs in the stomach but also in the intestine, the kidney, and the blood, where the alkaline phosphatase initiates and catalyses the reaction resulting in the generation of psilocin. Figure 3 shows the hydrolysis reaction of the dephosphorylation of psilocybin [6].

Figure 3: The dephosphorylation hydrolysis reaction of psylocibin with the presence of alkaline phosphatase that gives rise to psilocin and a phosphate.

Alkaline phosphatase is a quaternary protein that is made up of homodimer subunits. Each subunit is tightly bound to a Zn2+ ion, which gives the enzyme its structural integrity, and a loosely bound Zn2+ that helps the enzyme to transform into its active state and carry out its function [7]. Each subunit of the enzyme has a binding site for the Mg2+ ion. Once the ion binds to the active site of the enzyme, it stimulates enzymatic activity. The reaction mechanism involves the R166 and S102 amino acid residues, two Zn2+ ions, and a water molecule present in the active site of the enzyme. This nucleophilic ion is associated with the first Zn2+ ion that attacks the phosphoserine, resulting in a non-covalent interaction between the enzyme and the phosphate. In the last step of this reaction, the water molecule that is bound to Mg2+ acts as an acid and donates a proton to the oxygen of S102.

Another enzyme that aids the metabolism of psilocybin is monoamine oxidase A (MAO-A) which falls under the oxidoreductase class of the enzymes. Figure 4 shows the reaction that psilocin undergoes in the brain. The primary function of MAO-A is the catalysis of neurotransmitter amines and other therapeutic drugs like psilocin, that competes with serotonin to bind to this enzyme. MAO-A is located predominantly on the outer mitochondrial membrane of brain cells, where it prevents the accumulation of deleterious amines by converting them into imines [8]. These enzymes crystalise as a monomer, maintaining a tertiary structure that complements its function to metabolise amines. For MAO-A to carry out its function, it requires FAD and 8alpha-S-cysteinyl FAD as cofactors. FAD covalently binds to a cysteine residue to form 8alpha-S-cysteinyl FAD, which oxidises FAD during the deamination process. This oxidation of FAD allows MAO-A to carry out its function in a systematic manner. Additionally, of biological importance is the C-terminal transmembrane helix's functional role. It has also been demonstrated that the Lys-305, Trp-397, and Tyr-407 residues were involved in the non-covalent interactions between the enzyme and FAD. Specifically, the Tyr-407 and Tyr-444 residues form an aromatic hydrophobic sandwich that stabilises the active site, which in turn stabilises the binding [9].Figure 5 shows the cartoon of the rainbow format of the crystal structure of 5-HT2AR in a complex with risperidone. This protein is a GPCR that is expressed in the central nervous and peripheral nervous system. It consists of a primary amino acid linear sequence, with the first amino acid at the N terminus being glycine, located in the extracellular space, and the last amino acid (residue number 376) at the C terminus being lysine, present in the intercellular space. Since the N and C termini of this protein are on different sides of the membrane, this protein is classified as a type 1 integral membrane protein. Moreover, the primary structure of this protein allows us to find the transmembrane spanning domains of the polypeptide: 1(74-97), 2(110-136), 3(146-172), 4(192- 215), 5(233-256), 6(324-345), 7(359-383). Because these regions are packed with hydrophobic amino acids, they are present in the lipid section of the bilayer. The primary linear chain undergoes several changes, where the amino acid side chains form covalent bonds and fold, causing them to form 7 distinct alpha helices as a result of hydrogen bonding between the amino acids i and i+4. These secondary structures come together to form the tertiary structure of the protein, the helix bundle, with the help of the non-covalent interactions that form between the side chains of the amino acids. This type of folding, depicted in Figure 5, occurs due to the hydrophobic effect, which is the primary driving force in protein folding. The protein shown in Figure 5 is a monomer, and two identical monomers of this integral membrane protein interact together to form a dimer. This dimer is known as a homodimer and is the quaternary structure of the protein.

Figure 5: Cartoon of the rainbow format of the crystal structure of 5-HT2AR (protein code: 6A93) in complex with risperidone made with iCn3D.

The functional groups present in the amino acid side chains of the proteins allow the protein to interact with the ligands present. Figure 6 shows the ligand interaction of risperidone along with the 5-HT2AR receptor, and there are several non-covalent interactions that take place for the ligand to bind to the protein. The structure of 5-HT2AR exhibits two key features. The first is the bottom hydrophobic cleft in the ligand-binding pocket. The second feature is the side-extended cavity located between TM4 and TM5. These features are essential for the interaction with ligands in aminergic receptors. The highly hydrophobic amino acids I163 and F332 together form the first major hydrophobic cleft in the structure, forming the ligand binding protein W336 acts as a toggle switch in this scenario, and the conformation of these residues are incredibly significant for the GPCR activation. As seen in Figure 6, the hydrophobic ring of risperidone is sandwiched between the L228 residue and the V366 residue. The valine residue is important in this case because of its small hydrophobic side chain. While psilocybin acts as an agonist, increasing 5-HT2AR signaling and resulting in a variety of drug-related symptoms like hallucinations and others, risperidone acts as an antagonist. It blocks this signaling, generating no symptoms of drug ingestion and maintaining normal body response. This is why risperidone was chosen as the ligand, and not psilocybin. Since 5-HT2AR is a GPCR protein, it has 7 transmembrane spanning domains that are approximately 20 amino acids long which is illustrated in Figure 7.

The hydropathy plot in Figure 7 was proved to be right when Kimura et. al [10] described, “The overall structure of 5-HT2AR consists of the canonical G protein-coupled receptor (GPCR) structure with seven transmembrane helices (TM1–7) and an intracellular amphipathic helix H8,”. This confirms the presence of the seven blue-highlighted sections seen in Figure 7. The authors experimentally reconstructed the receptor protein, carried out the expression and purification processes on it, and then performed lipidic cubic phase crystallisation to prove that the 5-HT2A receptor has the typical structure of a GPCR — the 7 transmembrane spanning sections.

Figure 6: Binding site of the crystal structure of 5-HT2AR in complex with risperidone [10]

Drug Binding Kinetics

As discussed earlier, through a series of metabolic reactions, psilocybin is converted into its active form, psilocin, which is a lipid soluble molecule that can move through the blood-brain barrier and can bind to the serotonin receptor 5-HT2A. Upon further investigation and according to the PDSP Ki database, the Ki value for psilocin binding to 5-HT2A was 339.6 nM in Homo sapiens, indicating high affinity for the protein. Using the same database, the Ki value of psilocybin, however, was >10000.0 nM, indicating very low affinity for the protein in Homo sapiens. These two values suggest that psilocin can form a stronger interaction with the receptor due to its smaller inhibition constant, compared to psilocybin with its higher inhibition constant. This is one of the many reasons why psilocybin is converted into its active form (psilocin), to enable stronger interactions with the receptor protein. Psilocybin and psilocin bind to 5-HT2A receptors in the same way that LSD and other psychoactive drugs do. Figure 6 illustrates the two major ways that the two molecules interact. A π-π bond forms between psilocin and Phe340 on the receptor, as well as a salt bridge hydrogen bond forms between the amine group on psilocin and Asp155 on the receptor, helping stabilise the ligand. The ligand binding causes the stimulation of the signaling in the cell that results in the activation of phospholipase C (PLC), which catalyses the hydrolysis of phosphatidylinositol bisphosphate. This results in the production of inositol trisphosphate (IP3) and diacylglycerol (DAG), which activates PKC and Ca2+ to participate in diverse signaling pathways affecting neural activity, regulation of behavior, perception, and mood.

Conclusion

Psilocybin, a hallucinogenic polar protein found in magic mushrooms, is a controlled substance in many nations due to its agonistic effects on the 5-HT2A receptor, which alters human mood and behavior. Like the neurotransmitter serotonin, it is an indolylalkylamine molecule produced from L-tryptophan (Trp, W). The serotonin receptors of the prefrontal cortex are the primary mechanism of action, and their activation results in hallucinations, euphoria, dizziness, and other side effects. The biochemistry of the amino acid derivative provides insights into the metabolic reactions catalysed by MAO-A and alkaline phosphatase, which break down the molecule into its active form, enabling it to cross the blood-brain barrier. Once the protein enters the brain, it binds to the 5-HT2AR receptor with a specific binding affinity, which informs us about how readily the active molecule will bind and interact with the receptor's active site. Understanding the biochemical reactions and processes involved in this binding is cruical to gain insights on the physiological effect of the protein in humans.