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Proteasomes inhibitor


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proteosomes inhibitor, by Dr Suyash Bharat MD Pharmacology GMC Haldwani.

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Proteasomes inhibitor

  1. 1. Proteasomes Inhibitor Dr Suyash Bharat PG-JR 3rd pharmacology SEMINAR – 28-10-2015
  2. 2. Intracellular traffic & sorting of Protein
  3. 3. Figure representing RER branch of Protein Sorting.
  4. 4. How the proteasome acts ?
  5. 5. Proteasome inhibition in live cells: • Up to 80% of cell proteins are processed by the proteasome • Proteasome inhibition in mammalian cells induces apoptosis and sensitizes cells to pro- apoptotic agents • Rapidly dividing cells are more sensitive than slowly dividing cells • MALIGNANT CELL LINES ARE MUCH MORE SENSITIVE
  6. 6. The Ubiquitin-Proteasome System • The proteasome is the main nonlysosomal endoprotease enzyme complex present in the cytoplasm and nucleus of all eukaryotic cells. • It plays a critical role in the degradation of most short-lived intracellular proteins that control cellular events such as cell cycle, transcription, DNA repair, cell death, signal transduction, metabolism, morphogenesis, differentiation, antigen presentation and neuronal function. • The proteasome is also responsible for protein quality control by eliminating damaged and abnormal proteins.
  7. 7. • The proteasome is a large hollow and cylindrical 26S enzymatic complex of at least 66 proteins • It is composed of the catalytic 20S core and two 19S or 11S regulatory units at either ends. • The catalytic 20S core is organized into a stack of four seven-subunit rings, with the top and bottom rings formed by seven polypeptides, termed the α-subunits, and the two inner rings of seven β-subunits.
  8. 8. • Poly-ubiquitination drives the interaction between the 19S (11S) and 20S particles.  It requires the activity of three enzymes: 1. ubiquitin-activating enzyme (E1), 2. ubiquitin-conjugating enzyme (E2) 3. ubiquitin-protein ligase (E3) • Polyubiquitination each Ub is added sequentially to the growing chain of target protein . • 19S regulatory particle has an affinity for and recognizes these UBL domains. • The substrate protein is unfolded by hydrolases so that it may enter the narrow gate of the 20S particle and then be degraded.
  9. 9. • The 19S regulatory particle is divided into 2 subcomplexes called the BASE and the LID. • The base consists of 6 AAA+ ATPases & 3 non- ATPase polypeptide chains. • The lid includes at least 9 non-ATPase polypeptide chains that help remove ubiquitin from the substrates. • The lid and base connection is stabilized by the Rpn10 subunit. • The base of hexameric ring of 6 ATPases in the 19S regulatory particle facilitates the opening of the 20S gate and is responsible forsubstrate recognition, deubiquitination, unfolding & translocation into the core particle.
  10. 10. • Proteolytic chamber (20S core) - 3 types of catalytic activities: • chymotrypsin-like  β5 • trypsin-like  β2 • caspase-like β1  In immune cells the β1, β2, and β5 subunits, which are constitutively expressed, are replaced by β1i, β2i, β5i induced subunits to compose the Immunoproteasome. • Immunoproteasome has increased chymotrypsin- like and trypsin like activities which assists in Antigen Processing . • Instead of the 19S regulatory particle, the immunoproteasome utilizes the 11S regulatory particle.
  11. 11. Proteasome Inhibitors Peptide aldehydes (MG132)  first proteasome inhibitors  act against serine and cysteine proteases. • Peptide aldehyde inhibitors are rapidly oxidized into inactive acids in cells and transported out of the cell by the multidrug resistance (MDR) carrier system
  12. 12. Peptide boronates- Bortezomib • Peptide boronates bind with the hydroxyl group of the N- terminal threonine residue in the proteasome by a non- covalent bond. • The boron atom can receive the oxygen lone pair of the N- terminal threonine residue  stable tetrahedral intermediate . • BORTEZOMIB • A dipeptide that contains a boronic acid instead of a carboxylic acid at the C-terminus. • Bortezomib’s boronic acid reversibly binds to the chymotrypsin-like β5 subunit of the catalytic chamber of the 20S particle and inhibits proteasome function
  13. 13. Mechanism of action
  14. 14. • Bortezomib -> bind to β 5 (20S)  disrupts i/c signaling cascade  l/t apoptosis • NFkB (cytosol) bound to IkB  ubiquitinated degrade proteosomes. • Under stress IkB gets ubiquitinated  degrade  release NFkB  enter nucleus  increase transcription of cell survival gene (cell adhession pr, E selectin , ICAM 1, VCAM 1), Proliferative Pr (cyclin D1), anti apoptotic molecule (CIAPs, BCL2) • NFkB expressed in Tx cell  help Tx cell to survive hypoxia, chemotherapy
  15. 15. • Bortezumib also disrupts  UPS degradation of P21,P27, P53 / initiator of apoptosis/ other key regulator  unfolding increase + apoptosis • It also sensitize to other cytotox(alk agents/ anthracycline) • Dose  1.3 mg/m2  IV bolus • Day 1, 4, 8 & 21 (21 day cycle) (10 day gap/ cycle) • T1/2 – 5.5hr
  16. 16. Toxicity • Thrombocytopenia (28%) • Fatigue (12%) • Peripheral neuropathy (12%)
  17. 17. Resistance to bortezomib • Either inherit or acquire mechanisms 1. Mutated or overexpressed (β5) 2. Increased Levels of downstream effectors (chaperone pr- BIP) 3. Heat shock proteins (resistance to apoptosis )  overexpressing HSP27, 70, and 90 &T cell factor . 4. Constitutive NF-ĸB activity 5. Failing to accumulate pro-apoptotic proteins 6. Increase the levels of anti-apoptotic proteins, induce autophagy, and increase the levels of anti- oxidants .
  18. 18. Peptide epoxyketone inhibitors Carfilzomib • α,β-epoxyketone moiety  adduct with the N- terminal threonine residue  inactivates proteasome function • Carfilzomib is an irreversible inhibitor of the chymotrypsin-like subunit of the proteasome and immunoproteasome. • Undergoing phase II and III trials. • Carfilzomib is used to treat recurrent multiple myeloma, non-Hodgkin’s lymphoma and few solid tumors.
  19. 19. Β-lactone-γ-lactam inhibitors Marizomib • Irreversible inhibitor of the chymotrypsin-like, caspase-like, and trypsin-like activities of the immunoproteasome. • In phase Ib • It is used to treat recurrent multiple myeloma, solid tumors, lymphomas, and leukemias
  20. 20. Thank you