Xenobiotic-Target Reactions

• Xenobiotic-target reactions can be reversible or irreversible

Non-Covalent Interactions

• Hydrogen bonds, ionic interactions, Van der Waals forces, and hydrophobic interactions happen between the xenobiotic and target molecule
• Reversible interactions are common
• Knowing targeted molecules assists the development of therapies
• Tamoxifen, an antitumor drug, links with the estrogen receptor through non-covalent interactions
  • It occupies the site of the endogenous estrogen, preventing the action on tumor cells
  • It is used for estrogen-dependent breast tumors
• Saquinavir, an HIV protease inhibitor, binds to the active center through non-covalent interactions
  • It is one of the drugs used for AIDS therapy

Covalent Interaction

• It is very common with electrophilic xenobiotics
• They form adducts with nucleophilic groups in endogenous molecules, and are generally irreversible

Electrophilic Ligand Interaction

• Cysteine, serine, or threonine usually form covalent links through interaction with a reactive functional group
• This leads to the formation of covalent adducts
• Covalent reactions with nucleophilic xenobiotics are rare because electrophilic groups are not abundant in biomolecules
• Cisplatin forms covalent links with residues of guanine, generating intra-string (60% G-G, 25% A-G, 5% G-NG) bridges, that are difficult to repair
• Inter-string (<5%) that allow more reaction with proteins, RNA and other molecules

Example

• Benzopyrene generates an adduct that impedes correct DNA function
• When xenobiotics covalently bind to proteins it can trigger the immune system
  • Molecules that do this are called haptens and they form neoantigens that are recognized as foreign by the immune system
  • These molecules are only active after their formation

Halothane

• Halothane, a gas used as an anesthetic until the 90s leads to the production of trifluoroacetic acide
• Which can bind covalently to proteins to generate a very grave immune response against the liver, in approx. 1/35000 cases
• Covalent linking of a xenobiotic is more dangerous

Reactions

• Hydrogen abstraction
  • Neutral free radicals can capture H from endogenous compounds, converting them to reactive radicals
  • For example, radicals NO2 capture an atom of H from the lateral string of the tyrosine and then it is nitrosylated
• Electron transfer
  • Some compounds can oxide Fe (II) of hemoglobin to Fe (III), forming metahemoglobin that cannot transport O2
  • For example, benzocaine and sulfamides
• Catalysis
  • Certain toxins act like enzymes, catalyzing reactions
  • Botulinum acts as a Zn-dependent protease on proteins involved in the exocytosis of acetylcholine (neurotransmitter)
  • Breaking the proteins leads to a more durable effect
• Effects on the target
  • To elicit an effect on the organism, xenobiotics must alter the functionality of their target
  • Xenobiotics can activate or inhibit target molecules

Agonist vs Antagonist

• Agonist –> compound that can stabilize the active conformation of the receptor, responsible for the generation of biochemical responses
• Anatagonist –> compound that can stabilize a non active conformation of the receptor, either through direct action
  • Over the natural linking place of the receptor (competitive antagonism), or through action over a site separate from the linking center (non competitive or allosteric antagonism)
• An allosteric antagonist compound will cause a conformational change in the binding site of the substrate
  • An agonist ligand creates a productive conformation that allows to generate active answers
  • A competitive antagonist impedes the natural substrate linking, and generates an unproductive conformation

Xenobiotic Agonists and Antagonists

• Scopolamine and atropine block signals from muscarinic receptors, as they are antagonists and compete with the endogenous ligand, acetylcholine
• Morphine actives opioid receptors, like its endogenous ligand, endorphins, therefor it acts as an agonist of said receptors
• Doxorubicin, daunomycin, camptothecin and other compounds separate in the DNA interfering with replication
• As a result it produces more errors and, usually, apoptosis
• Doxorubicin blocks the DNA-topoisomerase I complex, an enzyme that relaxes the super windings from DNA for transcription during replication
  • It can also elevates the production of quinone type free radicals, contributing to its cytotoxic capacity
• Camptothecin blocks the DNA-topoisomerase I complex during replication. This stopping it and leading to one-string breaking
• Many xenobiotics inhibit enzymes, or can act as activators but this section emphasizes its inhibition capabilities

Enzyme Inhibitors

• Active Site Inhibitors
  • Reversible or irreversible "suicide substrates"
• Allosteric Site Inhibitors
• Can have competitive or noncompetitive inhibition
• In the specific case of Competitive Inhibition, Active Site Inhibition it can be reversible with an overload of the natural compound

Example Saquinavir

• This agent treats HIV by inhibiting protease activity
• Electrophilic compounds can unite with OH or SH groups from the side string of amino acids
• Disulfiram treats chronic alcoholism, and reacts with the thiol groups of the aldehyde dehydrogenase to form an adduct
  • This leads to the accumulation of acetaldehyde that produces diverse symptoms on alcohol consumption
• The 5-FU substitutes the uracil in the tymidilate synthase reaction, catalyzing the tymidilate synthesis reaction and metabolizing what the DNA requires
• Lastly aspirin, connects covalently to the catalyzing center of the cyclooxygenase (COX), stopping the synthesis of prostaglandins and tromboxanes

Non Competitive Inhibition

• Attaching to a different place of an enzyme separate from the already active site
• Tumor Drug: 6-mercaptopurine, to be inhibitory of the first step in the purine synthesis

Determining Xenobiotic-Target

• Indirectly, by determining the biomolecules or the ligand, with/without presence of either
• Directly, by detecting molecular level signaling derived from the biomolecule-ligand interaction

In Vitro Assays

• Saturation of ligand
  • Purified biomolecule from some cellular extract
  • The union without specificity, must be tested through an excessive amount of the already unlabeled compound
  • Must have some type of propriety that makes it detectable through a known technique: spectrography, radioactivity
• The fluoroscopy spectrography is the spectrography through which the variations can be found by varying concentration levels

Fluorescence Spectroscopy

• Intrinsic fluorescence, happens aromatic amino acids, but will have a different and more determined effect for each of them
• Extrinsic fluorescence due to chain introduced agents
• You must characterize the molecule to see the processes
• Al increase the amount of biomolecule, it is possible to achieve the affinity with the ligand

Spectroscopic Methods

• Circular Dichroism
  • It is used to study the secondary or tertiary structure of biomolecules
  • Determines the difference of the polarized light absorbed
  • The structures with pattern like alpha helix, Beta sheets, DNA double helix, offer a characterized Spectrums/Behavior
  • When the ligand connects, some change happens that affects said spectroscopic patterns
• Biophysical techniques
  • SPR
    • This technique detects deviation that happens on the union of the Ligand
    • The ligand is then inmobilized for a plate and it starts to pass
• Isothermal Titration Calorimetry, apt for DNA and amino acids
  • It only detects the warmth and energy that this biomolecule produces
  • It’s a technique that can measure the process, energy and parameters
• Electroforesis on Agarose, affects the mobility on such test

In Cellulo Assays

• Microscopy Through Luminescence
  • Adding Ligands With Luminescence Agents and probes

Cellular Effects and Functions

• Some processes depend on their proper functioning with biomolecules that form cells
• Unit of Basic Live: Structural Capacity to be Biological functions where Life is achieved
• Diene depends in where the Xenobiotic: can affect cell activity, gene alteration, and or change cell functionality
• Xenobiotics are also involved General Maintenance for Cell Equilibrium. Which also alters cell damage and apoptosis
• To understand its effect it is necessary to understand Cell Signalization and Equilibrium-which are mostly composed of ions, hormones peptides, and neurotransmitters
• Signals are usually transmitted from cell to cells which means Cell Transduction
• These signals can lead to the endocrine, paracrine, neuronal or a contact depending
• Cellular Surface capture by receptors is that are specific and that can be at the main area of (A) or can be Inter Cellular (B)
• Larger portion Receives signals through receptors such Ligands or the polar molecule kind. When union the receptor can change, and provoke reactions.
• Intercellular Receptors receive non polar signals or Non polar Molecular composition where in combination, the receptor creates a new confirmation of his initial condition.
• Union of signal and receptor leads to an answer and cell
• If the signal stops or is absent the cell will die, undergo apoptosis
• Other processes depend on chains, proteins and signalizations

Adenosine

• An example is the signal EGF that bonds to cell membrane, which chain the conformation on receptors. It’s the trigger for reactions inside the cell and their growing
• Acetylcholine (neurotransmitter) bonding in muscle cells changes conformation where the sodium can pass or chain from membrane, this will create the muscle-contraction that follows

Kinases and Signals

• These enzymes transfer phosphorus group starting from a molecule ATP towards the signal reaction
• signals from the cell membrane to intracellular enzymes alter the shape and expression of genes in DNA
• molecules transmit, amplify, integrate and contribute to different signals received throughout the cell starting when ligands connect to their receptors where the transfer/relay begins