Mechanisms Underlying Direct Cardiac Sarcomere Modulators That Target Troponin

• Author / Creator

  Fangze Cai

• Heart muscle contraction is regulated by calcium binding to cardiac troponin C. This induces troponin I (cTnI) switch region binding to the regulatory domain of troponin C (cNTnC). Compounds that directly modulate the interface of cNTnC and cTnI switch region have potential for treating heart diseases.
  In chapter 2, we identified that some small molecules bind tightly to cNTnC-cTnI chimera (cChimera) with KD ~ 10 μM. NMR structure of cChimera faithfully represents the native interface between cNTnC and cTnI, making cChimera a great tool for structure, dynamics, and drug binding studies. NMR structure of cChimera-3-mDPA reveals that the small molecule binds deeply in the hydrophobic pocket of cNTnC and minimal structural perturbation is observed.
  In chapter 3, we determined the structure of sarcomere inhibitor W7 bound to cChimera. The structure shows that W7 does not perturb the overall structure of the cNTnC-cTnI interface. The naphthalene ring of W7 sits in the hydrophobic pocket of cNTnC, while the positively charged amine tail extends into the solvent. We characterized the role of electrostatics and the precise location of the charged amino group in the mechanism of sarcomere inhibitor W7 in chapters 4 and 5. A7, where the amino group of W7 was replaced with a carboxyl group, decreased the binding affinity of cTnI to cNTnC significantly less than W7 and has a much weaker negative inotropic effect than W7 on ventricular trabeculae. The short negatively charged A6 and positively charged W6 do not change the calcium sensitivity of ventricular trabeculae. The structures show that naphthalene rings of W7, W6 and A7 sit in the same hydrophobic pocket, but the tails of W7 and W6 go to the same rout while the tail of A7 takes a different route to the surface of the complex. These results indicate that both electrostatic repulsion and the precise location of the charged amino group are important in the negative inotropic effect of W7.
  In chapter 6, we reported a strong cardiac sarcomere inhibitor, DN-F01, targeting the cNTnC-cTnI interface. ATPase activity studies show that DN-F01 has a strong inhibiting effect on cardiomyofibrils. The NMR titrations reveal that the binding position of DN-F01 bound covalently or non-covalently to the cNTnC-cTnI chimera might be different.
  In chapter 7, we found that the cardiac-specific N-terminal extension of cTnI (residues 1–37) remains largely disordered while it interacts electrostatically with cNTnC. This interaction does not directly affect the calcium binding affinity of cNTnC, but indirectly increases calcium affinity by positioning cNTnC to bind the cTnI switch peptide.

• Subjects / Keywords

  • sarcomere modulator
  • nuclear magnetic resonance spectroscopy
  • drug development
  • cardiac troponin

• Graduation date

  Spring 2021

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-azy3-9p95

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.