Kinase Signaling and Neurological Disorders

Kinase Signaling

  • Protein kinases are a large family of enzymes that catalyze the transfer of a phosphate group from an ATP molecule to a substrate.

  • This reaction is reversible via protein phosphatases.

  • Kinases are implicated in neurological disorders like autism, schizophrenia, and Parkinson's (LRRK2, PINK1).

  • Kinases are highly conserved, making specific small molecule inhibitor development challenging.

Phosphorylation Process

  • Phosphorylation is reversible and spatially/temporally precise.

  • Steps:

    • ATP binding to kinase.

    • Recognition of specific substrate.

    • Transfer of gamma phosphate from ATP to substrate.

    • Release of phosphorylated substrate.

    • Release of ADP.

Consequences of Phosphorylation

  • Modulates substrates:

    • Changes protein conformation.

    • Induces binding to other molecules.

    • Modulates protein turnover or degradation.

    • Controls enzyme activity.

    • Affects localization (e.g., nucleus).

    • Induces other post-translational modifications (e.g., acetylation after phosphorylation).

Kinase Domain Structure

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  • Highly conserved structure across protein kinases.

  • N-terminal lobe: Contains beta sheets.

  • C-terminal lobe: Composed of alpha helices; determines substrate specificity.

  • Hinge region: Connects N and C lobes; ATP molecule binds here.

  • Magnesium ion required for ATP catalysis.

Inhibitor Design Challenges

  • High structural conservation makes selective inhibitor design difficult.

  • Inhibitors aim to displace ATP, inhibiting the kinase.

  • Inhibitors are designed as ATP mimetics.

Types of Kinase Inhibitors

  • Type 1: ATP-competitive; binds to the active kinase conformation; reversible.

  • Type 2: ATP-competitive; binds to the inactive kinase conformation; reversible.

  • Type 3 & 4: Allosteric inhibitors (non-ATP competitive).

    • Type 3: Binding site near ATP binding site.

    • Type 4: Binding site away from ATP binding site.

  • Type 5: Bivalent inhibitors spanning two regions (e.g., ATP site and allosteric site); can be ATP competitive or not depending on binding sites.

  • Type 6: Covalent inhibitors; non-ATP competitive; irreversible.

Antibody Inhibition

  • Limited to receptor tyrosine kinases (transmembrane domain).

  • Strategies:

    • Ligand-binding antibodies (sponge up ligands).

    • Receptor-binding antibodies (prevent homodimerization/cross-phosphorylation).

Summary of Kinase Targeting

  • Kinases essential for neuronal development/function.

  • Dysfunction implicated in genetic disorders.

  • Targeted therapeutically via small molecule inhibitors or antibodies.

Chronic Myeloid Leukemia (CML) Case Study

  • Slow-growing white blood cell cancer in bone marrow.

  • Myeloid stem cells give rise to red blood cells, platelets, neutrophils, monocytes.

  • Lymphoid stem cells give rise to B and T lymphocytes.

  • CML impacts lymphoblast stem cells, causing over-proliferation and crowding of normal blood cells.

  • Incidence: ~5,000 new cases/year in the US.

  • Risk factor: Exposure to radiation.

  • Caused by Philadelphia chromosome.

Discovery of Philadelphia Chromosome

  • David Hungerford (graduate student) found a short chromosome in CML patients' cells (1950s).

  • 95% of CML patients had the Philadelphia chromosome.

  • Later identified as a translocation between chromosomes 9 and 22.

  • Chromosomes break and swap material creating a fusion gene.

BCR-ABL Fusion Gene

  • Nora Heisterkamp identified the fusion of BCR and ABL genes as the cause.

  • ABL is a protein kinase with regulatory domains;

  • ABL: Internal domain auto-inhibits the kinase domain.

  • BCR-ABL lacks the auto-inhibition capability; constitutively active.

GLEEVEC (Imatinib) Development

  • Brian Druker (physician) and Nick Leiden (medicinal chemist) collaborated.

  • Leiden developed STI571 (imatinib/GLEEVEC), which killed CML cells in vitro.

  • GLEEVEC binds to the ABL kinase active site, stabilizing the inactive conformation.

  • It is an ATP-competitive inhibitor and is highly specific (IC50 = 250 nM).

GLEEVEC Mechanism

  • Normal: BCR-ABL active, causing uncontrolled cell proliferation.

  • GLEEVEC: Binds ABL, blocks activity, stops proliferation.

Resistance to GLEEVEC

  • Mutations arose in ABL kinase, blocking imatinib binding.

  • T315I was a key mutation resistant to first-generation drugs.

  • These mutations occur within the ATP-binding domain.

Second-Generation Drugs

  • Derivatives of imatinib were developed, but most didn't work for the T315I mutation.

  • T315I a "gatekeeper" mutation important for catalysis.

Allosteric Inhibitor Discovery

  • Asinanib: Mimics the myristoylated N-terminal and binds an allosteric site.

Asinanib

  • Does not bind the ATP binding site.

  • Effective against the T315I mutation.

  • Recently FDA-approved.

  • Traditional inhibitors target the ATP binding site.

CML Timeline

  • 1960s: Philadelphia chromosome discovery.

  • 1973: Translocation of chromosomes 9 and 22.

  • 1980s: BCR-ABL fusion identified.

  • 1992-1996: Preclinical development of imatinib.

  • 1999: Clinical trial results presented.

  • 2000: Crystal structure of kinase inhibitor with imatinib.

  • 2001: FDA approval of imatinib.

  • 2001: First cases of imatinib resistance.

  • 2021: FDA approval of asimanib (allosteric inhibitor).

Clinical Usage

  • Allosteric inhibitors are not used as first line of defense in order to prolong survival and prevent aquired mutations against the allosteric inhibitors.
    First traditional ATP inhibitors are given, then allosteric inhibitors.

Kinases in Parkinson's Disease

  • Parkinson's: Loss of dopaminergic neurons, Lewy body accumulation.

  • LRRK2 and PINK1 are key genetic causes.

LRRK2 vs. PINK1

Feature

LRRK2

PINK1

Inheritance

Autosomal dominant

Autosomal recessive

Onset

Late

Early

Kinase Activity

Increased

Loss of function

Therapeutic Aim

Kinase inhibitors

Kinase activators

LRRK2

  • Autosomal dominant; late onset (40-60 years).

  • G2019S mutation (3-7% of familial PD cases).

  • Classic alpha-synuclein positive Lewy bodies.

  • Large protein (~2500 amino acids).

  • Contains structural (ankyrin repeats, heat repeats, mucin-rich repeats) and catalytic motifs (kinase domain, Roc domain [GTP binding/hydrolysis]).

  • Mutations in kinase, GTP binding, and C-terminal of Roc domains. Mutation in kinase increases kinase activity.

  • Lifetime risk of PD increases with kinase activity.

LRRK2 Activation Hypothesis

  • Mutations increase kinase activity.

  • Exists as a dimer; dimerization activates the kinase domain due to GDP to GTP exchange, causing conformational change.

  • Mutations in GTPase domains can indirectly impact kinase activity.

LRRK2 Cellular Function

  • Acts at the lysosome which degrades misfolded proteins.

  • LRRK2 binds LAMP2 (lysosomal receptor) and chaperon protein HSC70. Leads to alpha synuclein degradation

  • Mutations prevent lysosomal degradation, leading to protein accumulation.

Denali's LRRK2 Inhibitor (DNL201)

  • Potent, selective, CNS-penetrant, orally bioavailable.

  • Inhibitor can improve aberrant lysosomes, preventing build-up of alpha synuclein.
    Inhibiting LRRK2 improves symptoms of Parkinson's even without mutations.

  • Phase 3 clinical trials (Lighthouse for LRRK2 mutation carriers, LUMA for non-carriers).

PINK1

  • Autosomal recessive; early onset (30s-50s).

  • Loss of kinase function.

PINK1 Mechanism

  • Healthy mitochondria: PINK1 gets inserted, cleaved by proteases. Because this is cleaving PINK1, the Mitochondria does not accumulate PINK1 and stays healthy.

  • Damaged mitochondria: Changes in membrane potential prevent cleavage. PINK1 accumulates, self-phosphorylates, recruits Parkin which causes the mitochondrial membrane to be ubiquitinated.

  • PINK1 acts as a sensor for mitochondrial health; degraded if healthy, accumulates and tags for autophagy if damaged.

  • Lack of PINK1 prevents degradation of damaged mitochondria, leading to neuronal death. It will keep accumulating the mitochondria, but will not auto phosphorylate.

Nick Hertz's Discovery

  • Found substrates of PINK1.
    PINK1 mutation (G309D) decreases kinase activity.
    Adds an ATP analog that increases activity.
    Analog (Kinetin) is cell-permeable, rescues PINK1 function, allows for mitochondrial autophagy.

MitraClinen (acquired by AbbVie)

  • Increasing PINK1 activity can improve pathology in non-mutation carriers.
    Increasing PINK1 activity can promote neuronal health because the Analog is able to ubiquitinate and induce the removal of bad Mitochondria.

Summary for LRRK2 and PINK1

  • Parkinson's mutation in LRRKQ increases its kinase activity and increases dysosomal function due to too little degradation. This promotes inhibiting LARQ

  • Parkinson's mutation in mitochondrial TIM is STINK one plus last loss of function, and so you you prevent the the autophagy of damaged mitochondria. And so resting PINK one activity by the ADT analog is beneficial for neuronal health by removal of damaged mitochondria. And these drugs are now under investigation for improving neuronal health.