Molecular Mechanisms of Drug Action

Molecular Mechanisms of Drug Action

• Chemotherapeutic drugs exert their effects by interacting with DNA or ribosomes.

Objectives

• Recognize the structural features of DNA and how nucleic acid can serve as a drug target
• Describe the structural features of drugs that target DNA biosynthesis, structure, and function.
• List some specific examples of drugs that act as alkylating agents, intercalators, and nucleoside analogs.
• Distinguish structural features of ribosomal protein synthesis and how this can act as a drug target.
• Recognize the structural features of drugs that target protein biosynthesis at various stages.
• List some specific examples of clinically useful drugs that act as protein synthesis inhibitors and understand their mechanism of action (MOA).

DNA Structure

• DNA has a helical structure, with major and minor grooves.
• The radius is 10Å and the diameter is 20Å.
• Nucleotide base pairs are spaced 3.4Å apart
• There is a 34Å distance per 360° turn.
• There are 10 nucleotide base pairs per 360° turn.

DNA Conformations

• A-DNA, B-DNA, and Z-DNA are different conformations of DNA.
• The parameters for A-, B-, and Z-DNA vary in terms of helix sense, base pairs per turn, axial rise, helix pitch, base pair tilt, twist angle, and helix diameter.
  • A-DNA: Right-handed helix, 11 base pairs per turn, 0.26 nm axial rise, 28° helix pitch, 20° base pair tilt, 33° twist angle, and 2.3 nm helix diameter.
  • B-DNA: Right-handed helix, 10 base pairs per turn, 0.34 nm axial rise, 34° helix pitch, -6° base pair tilt, 36° twist angle, and 2.0 nm helix diameter.
  • Z-DNA: Left-handed helix, 12 base pairs per turn, 0.45 nm axial rise, 45° helix pitch, 7° base pair tilt, -30° twist angle, and 1.8 nm helix diameter.

DNA as a Drug Target

• Nucleic acids play a crucial role in cellular processes like replication and transcription.
• Processes occur in both healthy and cancer cells.
• Small molecules can interact with or bind to DNA through various mechanisms:
  • Intercalators sandwich between DNA base pairs.
  • Alkylators chemically react with DNA.
  • DNA cleavage agents cause strand breaks.
  • DNA biosynthesis and catabolism inhibitors.

DNA-Intercalating Agents

• Intercalating agents: Actinomycin D and Doxorubicin

DNA Alkylating Agents

• Nitrogen mustards are a type of alkylating agent that crosslinks DNA.
• MOA: alkylating agents transfer alkyl groups to DNA bases, resulting in covalent bonding.
• Methanesulfonates are another class of alkylating agents.

Preferred Sites for DNA Alkylation

• N7 of guanine and N3 of adenine.

Platinum Complexes

• Cisplatin and carboplatin are anti-cancer agents that form strong platinum-nitrogen bonds with guanine and adenine bases.
• The cis configuration of these complexes leads to intra-strand cross-links that can cause helix unwinding, preventing transcription and leading to cell death.
• The trans isomer, trans-platin, is not an active anti-cancer agent because it cannot readily form intra-strand cross-links and tends to cross-link separate strands, which are more easily repaired.

DNA Cleavage Agents

• Bleomycin is a glycopeptide mixture with three domains:
  • Metal-binding [Fe(II)] domain
  • DNA-binding bithiazole domain
  • Carbohydrate domain thought to cause accumulation in cancer cells.
• Bleomycin binds tightly to guanine bases in DNA, particularly at G-T sequences (GGGGCTGGG) and G-C rich regions, leading to DNA chain cleavage.

Nucleic Acid Biosynthesis

• Sulfonamides prevent the synthesis of folate.
• Trimethoprim prevents the use of folate.
• DHFR inhibitors: Methotrexate and Pyrimethamine
• 5-Fluorocytosine is a prodrug that is converted to 5-Fluorouracil and then to 5-Fluoro-2'-deoxyuridylate, which inhibits DNA synthesis.
• The mechanism of action involves conversion to 5-Flucytosine (antimycotic) via cytosine deaminase.

DNA Strand Synthesis

• Involves the addition of nucleotides to a growing strand, releasing pyrophosphate.

Reactions Catalyzed by Topoisomerases

• Topoisomerases catalyze reactions such as relaxation/supercoiling, knotting/unknotting, and concatenation/decatenation.
• DNA gyrase introduces negative supercoils into closed circular DNA.
• Both DNA gyrase and topoisomerase IV can remove supercoils.
• Supercoiled DNA is catenated by DNA gyrase and decatenated by both DNA gyrase and topoisomerase IV.

Nucleic Acid Biosynthesis as Target

• Acyclovir lacks a 3' hydroxyl group for chain elongation.
• Cytarabine contains an arabinose sugar instead of deoxyribose.
• Nalidixic acid and Ciprofloxacin are DNA gyrase (Topoisomerase II) inhibitors.
• Azidothymidine is a reverse-transcriptase inhibitor.

Nucleic Acid Catabolism as Target

• Allopurinol is a xanthine oxidase inhibitor.
• Pentostatin is a 2-deoxycoformycin.

Ribosomal Protein Synthesis

• Requires Ribosomal RNA (rRNA), Initiation factors (IF), Elongation factors (EF) and Termination factors (TF).

Prokaryotic vs Eukaryotic Ribosomes

• Prokaryotic Ribosomes: 70S ribosomes composed of 50S (large) and 30S (small) subunits
• Eukaryotic Ribosomes: 80S ribosomes composed of 60S (large) and 40S (small) subunits

The Process of Translation

• All 3 phases of translation (initiation, elongation, termination) require "factors" that aid in the translation process
• Both initiation and chain elongation require energy provided by the hydrolysis of GTP.

Protein Synthesis

• Protein synthesis inhibitors can target the 30S or 50S ribosomal subunits.
  • Macrolides, chloramphenicol, and clindamycin target the 50S subunit.
  • Aminoglycosides and tetracyclines target the 30S subunit.

Nonribosomal Protein (NRP) Synthesis

• Synthesized without ribosomes and not genome encoded.
• Short peptides with unusual structures (cyclic, branched) and modified amino acids (D-forms, methylated etc.).
• Created by massive synthetases (enzyme complex).
• Many useful pharmaceutical agents, such as Cyclosporine and Penicillin and vancomycin are created in this manner.

Drugs Targeting Ribosomal Protein Synthesis

• Chloramphenicol binds to the 50S portion and inhibits the formation of peptide bonds.
• Erythromycin binds to ribosomal RNA at the 50S site and blocks movement along the mRNA.
• Aminoglycosides (e.g., streptomycin) change the shape of the 30S portion, causing the code on mRNA to be read incorrectly.
• Tetracyclines interfere with the attachment of tRNA to the mRNA-ribosome complex.

Aminoglycosides

• Bind to the 30S subunit, causing a misreading of the genetic code.
• Amino sugars are linked glycosidically.
• Stable at pH 2-11.
• Resistance can occur through adenylation and phosphorylation of OH groups, or acetylation of NH2 groups.
• Exhibit synergism with penicillin.
• Examples: Kanamycin B, Gentamicin C, Amikacin, Tobramycin

Macrolides

• Contain a 12, 14, or 16-member lactone ring (macrolide).
• Possess a ketone group.
• Feature a glycosidically linked amino sugar (desosamine), potentially with a neutral sugar (cladinose).
• Bind to the 50S subunit.
• Resistance: Resistance can occur by specific methylation of an adenine at the erm-binding site on the ribosome.
• Blocks translocation step (A→P) and thus exits of the peptide.
• Example: Erythromycin

Clarithromycin and Azithromycin

• Clarithromycin and Azithromycin are derivatives of Erythromycin
• Clarithromycin: 6-methyl ether of erythromycin - increases the acid stability and oral bioavailability.
• Azithromycin -- nitrogen containing 15-membered ring macrolide known as azalide - removal of the 9-keto group and addition of a weakly basic tertiary amine function at 10-position of the macrolide ring increases acid stability and also increases lipid solubility. Tissue levels far exceed plasma levels - prolonged elimination half-life up to 5 days.

Fidaxomicin

• A relatively new class of macrolide antibiotics. It inhibits bacterial RNA polymerase, preferentially resulting in the death of Clostridium difficile without altering the other bacterial species in the gut

Acid-Catalyzed Ketal Formation

• Formation of ketal in acid leads to inactivation.

Ketolides

• Telithromycin is effective against macrolide-resistant bacteria
• Binds at two sites at the bacterial ribosome
• Has a structural modification that makes it a poor substrate for efflux-pump mediated resistance.

Tetracyclines

• Bind to the 30S subunit.
• Prevent the binding of aminoacyl t-RNA.
• Contain an octahydronaphthacene ring system.
• Resistance: Efflux and Ribosomal protection
• Chelation with metal ions.

Glycylcyclines (Tigecycline)

• Inhibit protein translation in bacteria by binding to the 30S ribosomal subunit and blocking entry of amino-acyl tRNA molecules into the A site of the ribosome.
• This prevents incorporation of amino acid residues into elongating peptide chains.
• Tigecycline carries a glycylamido moiety attached to the 9-position of minocycline.
• Tigecycline is not affected by the two major tetracycline resistance mechanisms, ribosomal protection and efflux.

Newer Tetracyclines

• Omadacycline approved in October 2018 for acute skin infections and community acquired bacterial pneumonia.
• Eravacycline halogenated synthetic tetracycline that is indicated for the treatment of complicated intra-abdominal infections (cIAI).

Review Points

• Key points to remember when studying drug mechanisms of action:
  • Overall DNA structure and regions important for drug binding.
  • Chemical structures and features of drugs that target DNA.
  • Key steps in ribosomal peptide synthesis.
  • Broad classes of antibiotics that target ribosomes.
  • Chemical structures and characteristics of specific, clinically useful drugs that target protein synthesis.