Proteasomes Inhibitor

Dr Suyash BharatPG-JR 3rd pharmacology

SEMINAR – 28-10-2015

Intracellular traffic & sorting of Protein

Figure representing RER branch of Protein Sorting.

How the proteasome acts ?

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

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.

• 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.

• 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.

• 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.

• 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.

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

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

Mechanism of action

• 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

• 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

Toxicity

• Thrombocytopenia (28%)• Fatigue (12%)• Peripheral neuropathy (12%)

Resistance to bortezomib• Either inherit or acquire mechanisms1. 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 activity5. Failing to accumulate pro-apoptotic proteins6. Increase the levels of anti-apoptotic proteins, induce

autophagy, and increase the levels of anti-oxidants .

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.

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

Thank you