Targeting GPCRs for Cancer Therapy 03/11/2014
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1 Targeting GPCRs for Cancer Therapy 03/11/2014 G Protein Coupled Receptors Actually 4% of the human genome GPCR dysfunction contributes to some of the most prevalent human diseases GPCRs represent 60% of all currently used therapeutic agents Agonist Binds to the receptor and triggers a response in the cell Mechanism When a receptor binds an agonist, causes phosphorylation of the receptor via GPCRK, usually GRK2. Phosphorylation can be in the c tail and it causes recruitment of beta arrestin molecules and binds the receptor. Class A GPCR beta arrestin instantly falls off Class B GPCR beta arrestin remains bound This targets clathrin coated pits which internalize and pinch off by dynein and this vesicle is now called an endosome. Antagonist Binds to the receptor and blocks an action GPCRs in Cancer Stimulate Growth Survival Invasion and Metastasis Angiogenesis
2 GPCR Activation Desensitization decline in the response to continuous or repeated application of agonist; mechanism for protecting cells against receptor overstimulation 1. Phosphorylation (seconds minutes) from GPCRKs 2. Sequesterization (min) receptors are sequestered in the endosome 3. Downregulation (minuteshours) persistent loss of receptors after long term exposure to agonist occurs L i g a n d B i n d i n g A c t i v a t i o n G p r o t e i n C o u p l i n g E f f e c t o r 2 n d M e s s e n g e r D e s e n s i t i z a t i o n ( G p r o t e i n U n c o u p l i n g ) P h o s p h o r y l a t i o n ( s e c o n d s - m i n u t e s ) S e q u e s t r a t i o n ( m i n u t e s ) D o w n - R e g u l a t i o n ( m i n u t e s - h o u r s ) during long term administration of receptor agonist (tolerance). Receptors can be degraded (lysosomes) or decreased synthesis of the receptor. The Hypothalamic Pituitary Gonadal Axis GnRH: a key regulator of the reproductive system; hormone is secreted from the hypothalamus but the receptor is located on the pituitary A decapeptide produced by hypothalamic neurons Pulsatile secretion of GnRH controls synthesis and pulsatile secretion of the gonadotropins in the pituitary gland Lutenizing hormone (LH) Follicle stimulating hormone (FSH) No t e : Ho r m o ne : GnRH I GPCR: GnRH R LH/FSH regulate the function of the testes/ovaries Pulsatile secretion from hypothalamus also controls the number of GnRH Rs in the pituitary gland Continuous stimulation by exogenous GnRH agonist (vs. pulsatile) desensitizes and down regulates GnRH Decreases LH and FSH Decrease in circulating sex hormones Decrease in steroid hormones GnRH R/GnRH I Expressed in the brain, breast, placenta, uterus, testes, ovaries
3 Type I Human Gonadotropin Releasing Hormone Receptor (GnRH R) Unique feature lack of cytoplasmic tail (common to all GPCRs) Cytoplasmic tail sequences are required for rapid desensitization and internalization of GPCRs; this means it can t be desensitized in the usual way Molecular Regulation GnRH R internalizes via clathrin coated pits in a beta arrestin and dynamin independent manner Usually, beta arrestin is cytosolic and in response to other receptors will translocate to the plasma membrane In the case of GnRH, beta arrestin is not translocated Activated GnRH R is coupled to multiple G alpha proteins Gq/11 activation Gs pathway Structure Decapeptide that has a glycine at aa 6 and glycine at aa 10 Endogenous GnRH is rapidly degraded in the blood; 2 4 minutes by enzymatic cleavage at aa 6 or aa 10 or both These amino acids are substituted to enhance GnRH activity Summary GnRH = decapeptide Pulsatile secretion of GnRH from the hypothalamus controls the number of GnRH Rs in the pituitary gland Mammalian GnRH R lacks a cytoplasmic tail (common to GPCRs) so it is internalized in a beta arrestin and dynamin independent manner How do we target GnRH R for cancer therapy?
4 Elevated expression of GnRH R in many hormone dependent diseases Prostate cancer Metastasizes to brain, lung, liver, bone marrow Older treatment is DES Diethylstilbestrol: lots of adverse side effects like N/V, thromboembolism and breast enlargement) Primary treatment now is androgen ablation Surgical castration Chemical castration (GnRH agonists) 80 90% response rate since cancer is androgen sensitive, response duration is months Hormone refractory prostate cancer; tumor becomes androgen resistant and patients are not longer sensitive to androgen ablation by GnRH analog treatments Risk Factors Age Race Family history of prostate cancer Diet Symptoms Urinary frequency Slow urinary flow Painful urination
5 Blood in urine/semen Impotence Lower back and thigh pain Early detection is the answer PSA prostate specific antigen (blood test) Glycoprotein produced by the prostate gland Trypsin like serine protease Found in ejaculate in small amounts, liquefies semen and allows sperm to swim Increases in 95% of patients with prostate cancer but also increases in other maladies of the prostate (infection, enlargement) Involved in the degradation of ECM proteins (facilitates invasion and metastasis) Digital rectal examination (physical) GNRH Agonists Bind GnRH receptor as the naturally occurring GnRH Primarily act on the PITUITARY Stimulate production of LH and FSH and initially cause flare up phenomenon or worsening of disease symptoms Results in desensitization of PITUITARY CELLS to GnRH, ultimately resulting in dramatically reduced LH and FSH and testosterone Eventually, the GnRH receptor becomes densensitized and internalized, suppresseing gonadotropic and testosterone production (chemical castration) Elimininates the mitogenic effects of testosterone on the cancer cells GnRH Superagonists Ex. Leuprolide
6 200x more potent than natural GnRH Prolonged activity resistant to enzymatic degradation Ex. Buserelin Side Effects Tumor flare Nasal irritation Hot flashes Breast tenderness and fullness Weight gain Loss of libido GnRH Anatagonists Binds to pituitary GnRH receptors, causing immediate suppression of LH and FSH within hours Turn off GnRH receptor immediately by persistently blocking it and avoiding testosterone surge and clinical flare First 3 residues are replaced by D amino acids in most antagonists Drawbacks Insufficient potency Lack of solubility Action is short lived and daily injections are necessary Unlike the GnRH agonists, long acting forms are not currently available; cannot be used for long term treatment Ex. Abarelix
7 First GnRH antagonist FDA approved Used in Germany Blocks GnRH R Sustained release formula (allows for monthly dosing) Many side effects (allergic reaction the most serious) A g o nis t s vs A nt ag o nis t s : Clinic al Ef f e c t s (Pat ie nt ) A g o nis t s I nit ial s t im ulat io n o f LH pr o d uc t io n and t e s t o s t e r o ne s ur g e c aus ing t um o r f lar e May c aus e t e m po r ar y r is e in PS A le ve ls S id e e f f e c t s inc lud e h o t f las h e s, ac c e le r at e d b o ne r e s o r pt io n, im po t e nc e, lo s s o f lib id o, lo s s o f m us c le m as s A nt ag o nis t s I m m e d iat e, c o m ple t e s uppr e s s io n o f LH and t e s t o s t e r o ne No r is k o f t e s t o s t e r o ne s ur g e ie us e f ul in ad vanc e d d is e as e wh e r e t e s t o s t e r o ne s ur g e and c linic al f lar e m ig h t b e d ang e r o us PS A s uppr e s s e d m o r e q uic kly His t am ine r e le as e
8 Both drugs are sufficient in achieving and maintaining castration at day 85 Abarelix achieved testosterone fall much faster (no flare up) Abarelix also caused PSA to decline rapidly vs. Leuprolide, however by day 57 85, the two treatment groups appeared to be similar *Good alternative to surgical castration Mechanism of Action: Cellular Uptake of Free/Conjugated Drug into GnRH R Containing Tumor Cells
9 Tumor cells expressing GnRH-R Normal cells do not express GnRH-R Drug is taken up by the GnRH R expressing cells and released in the acidic environment of the lysosome in which case it is free to the kill the cancer cells Targeted Chemotherapy: Cytotoxic GnRH Analogs GnRH mechanism GnRH receptors are overexpressed in various human cancers including prostate cancer Inhibit the mitogenic signal transduction of growth factor receptors (ex. EGFR) by coupling to Gi Coupling to Gi activates PTP (protein tyrosine phosphatase) which blocks EGFR by removing a phosphate Induces apoptosis
10 Inhibits proliferation GnRH analog: AN 152 (containing doxorubicin) Currently in Phase I/II clinical trials Mechanisms of GnRH Analogs Primarily works on the PROSTATE to take advantage of the unexplainable coupling of the GnRH R to the Gi pathway in cancer cells Reduces proliferation, blocks growth factor associated signal transduction pathways, leading to limited cancer cell growth and induces apoptosis Can mimic effects of superagonists at the pituitary GnRH s ig nalling in pr o s t at e c anc e r c e lls
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