Farmakologi merupakan salah satu ilmu yang mempelajari mengenai obat, baik secara farmakodinamika dan farmakokinetika.
Pada kuliah ini akan dijelaskan mengenai prinsip aksi obat lebih menekankan pada bagaimana obat bisa memberikan efek pada tubuh manusia.
Obat dapat memberikan efek/respon berdasarkan dari mekanisme aksi obat tersebut didalam tubuh.
Obat harus berinteraksi dengan targetnya (secara spesifik dengan protein makromolekul) dan mengaktifkan sinyal regulasi dalam tubuh yang nantinya akan memberikan efek/respon.
Target aksi obat didalam tubuh seperti : enzim, kanal ion, reseptor maupun molekul pembawa.
potency sauté obat adalah jumlah dosis yang dibutuhkan untuk menimbulkan efek
efikasi adalah efek maksimal yang dihasilkan suatu obat
ENZIM
Sisi aktif enzim (active site) adalah bagian dari molekul enzim tempat berikatannya substrat, untuk membentuk kompleksenzim substrat, dan selanjutnya membentuk pro- duk akhir. Sisi aktif suatu enzim berbentuk tiga dimensi, sering berupalekukan pada permukaan protein enzim, tempat substrat berikatan secara lemah.
note :
-Setiap enzim memiliki nilai Kmyang berbeda-beda untuk suatu subtrat, dan ini dapat menunjukkan seberapa kuatnyapengikatan substrat ke enzim.
– kcat, yang merupakan jumlah molekul substrat yang dapat ditangani oleh satu tapak aktif per detik.
-Pada kelajuan yang maksimum (Vmax), semua tapak aktif enzim akan berikatan dengan substrat, dan jumlah kompleksES adalah sama dengan jumlah total enzim yang ada
-Setiap enzim memiliki nilai Kmyang berbeda-beda untuk suatu subtrat, dan ini dapat menunjukkan seberapa kuatnyapengikatan substrat ke enzim.
– kcat, yang merupakan jumlah molekul substrat yang dapat ditangani oleh satu tapak aktif per detik.
-Pada kelajuan yang maksimum (Vmax), semua tapak aktif enzim akan berikatan dengan substrat, dan jumlah kompleksES adalah sama dengan jumlah total enzim yang ada
Sisi aktif enzim (active site) adalah bagian dari molekul enzim tempat berikatannya substrat, untuk membentuk kompleksenzim substrat, dan selanjutnya membentuk pro- duk akhir. Sisi aktif suatu enzim berbentuk tiga dimensi, sering berupalekukan pada permukaan protein enzim, tempat substrat berikatan secara lemah.
Ikatan obat/inhibitor dengan enzim à substrat tidak dapat berikatan dengan sisi aktif enzim
salah satu obat sebagai produg pada enzim adalar prednison
Prednison dan prednisolon menunjukkan aktivitas antiaterogenik dengan melindungi makrofag dari akumulasi lipid danpembentukan sel busa
Prednisone mengurangi inflamasi dengan cara menginhibisi migrasi sel polimorfonuklear (PMN) dan mengurangipeningkatan permeabilitas kapiler.
Metabolisme terjadi di hati dengan cara hidroksilasi menjadi metabolit aktif, prednisolon. Proses metabolisme oleh enzim CYP3A4 di hati/usus.
these data took from paper : Putri DD, Kawasaki T, Murase M, Sueyoshi T, Deguchi T, Ori D, Suetsugu S, Kawai T. PtdIns3P phosphatases MTMR3 and MTMR4 negatively regulate innate immune responses to DNA through modulating STING trafficking. Journal of Biological Chemistry. 2019 May 24;294(21):8412-23.
Cover glasses were prepared on a 24-well plate and were treated with Poly-L-lysine solution (Sigma) for seeding cells. The cells were stimulated with ligand cGAS-STING (ISD (1μg/ml)-lipofectamine 2000), or several TLR ligand, for various time points. Then, cells were washed with PBS and fixed with 4% paraformaldehyde (Nacalai Tesque) for 20 minutes at room temperature. After washing three times with 0.02% Triton X-100 in PBS, 100 mM glycine in 0.02% Triton X-100/PBS was added at room temperature for 30 minutes. Cells were washed 3 times with 0.02% Triton X-100 in PBS and were blocked with 10% FBS, 0.02% Triton X-100/PBS for 1h at room temperature. Cells were incubated with primary antibodies diluted with 10% FBS 0.02% Triton X-100 at 4 °C overnight. After washing three times with 0.02% Triton X-100, cells were applied with the secondary antibodies diluted with 10% FBS in 0.02% Triton X-100 and were incubated for 1 hour. After three times washing with 0.02% Triton X-100, Hoechst 33342 (Dojin) was diluted with 10% FBS in 0.02% Triton X-100 and incubated for 10 minutes. After 3 times washing with 0.02% in Triton X-100, samples were enclosed on slide glasses with Fluoro-KEEPER Antifade Reagent, Non-Hardening Type (Nacalai Tesque). The prepared samples were observed with confocal fluorescence microscope LSM 700 (ZEISS), and images were processed with ZEN software. Dilution ratios of the primary and secondary antibodies are shown below.
note : the primary and secondary antibodies were optimised by dewi in her experiment.Cellular localization x gene on HEK293 cells were transfected with x-flag was stained with Hoechst 33342 (blue), anti-FLAG (green) and the indicated antibodies; TOM (red). Data was representative of at least two independent experiments. Scale bar 10 µm. (data belongs to dewi)Cellular localization x gene on RAW 264.7 cells were transfected with x-flag was stained with Hoechst 33342 (blue), anti-FLAG (green) and the indicated antibodies; GM130 (red). Data was representative of at least two independent experiments. Scale bar 10 µm. (data belongs to dewi)
paper publication that I wrote as reference, feel free to discuss and citation.
Various regulatory mechanisms for cGAS and STING pathway have been shown (Cai et al., 2014; Chen and Chen, 2016; Liu et at., 2015; Liu and Wang 2016; Hu et al., 2016). Sumoylation and Mono-ubiquitination of cGAS at Lys355 that are induced by E3 ubiquitin ligase TRIM56 is important for its DNA sensing activity, resulting in increased cGAMP production (Xiong et al., 2018; Sun et al., 2013; Seo et al., 2018). STING moves from ER to Golgi apparatus via the translocon-associated protein (TRAP) complex TRAPb(SSR2) that is recruited by inactive rhomboid protein 2 (iRhom2) and these complex reaches to Sec-5 containing perinuclear microsome or cytoplasmic punctate structures to assemble with TBK1 and IKK complex (Ishikawa et al., 2008; Abe and Barber, 2014; Luo et al, 2016). RAB2B-GARIL5 (Golgi-associated RAB2B interactor-like 5) complex is a regulator of STING in Golgi apparatus and promotes IFN response through regulating phosphorylation of IRF3 by TBK1. The autophagy-related protein such as autophagy-related protein (Atg), ULK1 (a homologue of Atg1), microtubule-associated protein 1 light chain (LC)3 (homologue of yeast Atg8) and Atg9 negatively regulate STING signaling through interfering with STING-TBK1-IRF3 signaling (Saitoh et al., 2009, Tooze et al., 2010; Konno et al., 2013). Atg-mediated degradation modulates baseline of STING protein level, but it does not impact the trafficking of STING. The function of STING is regulated by post-translational mechanism such as TRIM32 and TRIM56, that conjugate K63-linked polyubiquitination on STING and promote the recruitment of TBK1 (Tsuchida et al., 2010). ER-associated E3 ligase, AMFR, catalyzes the K27-linked polyubiquitination of STING together with INSIG1 (Wang et al., 2014). K27-linked polyubiquitination on STING induces TBK1 recruitment and activation. iRhom2, which recruits de-ubiquitination enzyme EIF3S, maintains the stability STING through removal of its K48-linked polyubiquitin chains. STING translocates from ER to Golgi apparatus and then moves to late endosome/lysosome (Dobbs et al., 2015). Helix aa281-297 of STING is shown to be degraded through V-ATPase in endosome/lysosome. The blockade of V-ATPase suppresses STING degradation, which potentially leads to enhance STING signaling (Gonugunta et al., 2017).
onse through regulating phosphorylation of IRF3 by TBK1. The autophagy-related protein such as autophagy-related protein (Atg), ULK1 (a homologue of Atg1), microtubule-associated protein 1 light chain (LC)3 (homologue of yeast Atg8) and Atg9 negatively regulate STING signaling through interfering with STING-TBK1-IRF3 signaling (Saitoh et al., 2009, Tooze et al., 2010; Konno et al., 2013). Atg-mediated degradation modulates baseline of STING protein level, but it does not impact the trafficking of STING. The function of STING is regulated by post-translational mechanism such as TRIM32 and TRIM56, that conjugate K63-linked polyubiquitination on STING and promote the recruitment of TBK1 (Tsuchida et al., 2010). ER-associated E3 ligase, AMFR, catalyzes the K27-linked polyubiquitination of STING together with INSIG1 (Wang et al., 2014). K27-linked polyubiquitination on STING induces TBK1 recruitment and activation. iRhom2, which recruits de-ubiquitination enzyme EIF3S, maintains the stability STING through removal of its K48-linked polyubiquitin chains. STING translocates from ER to Golgi apparatus and then moves to late endosome/lysosome (Dobbs et al., 2015). Helix aa281-297 of STING is shown to be degraded through V-ATPase in endosome/lysosome. The blockade of V-ATPase suppresses STING degradation, which potentially leads to enhance STING signaling (Gonugunta et al., 2017).
STING-mediated DNA sensing signaling pathway. cGAS binds to DNA and synthesizes cGAMP which activates STING. AMFR-INSIG1 catalyzes K27-linked ubiquitination on STING and activates TBK1. iRhom2 and TRAPbmediate the trafficking of STING to Golgi. RNF26, TRIM32, and TRIM56 modulate ubiquitination on STING. ATG9a controls the translocation of STING. V-ATPase regulates Rab7-mediated translocation of STING to lysosome and degradation of STING. Inhibition of V-ATPase by BafA1 suppresses STING degradation and enhances innate immune response. (original pict drawn by dewi)
references :
Cai, X., Chiu, Y. H., & Chen, Z. J. (2014). The cGAS-cGAMP-STING pathway of cytosolic DNA sensing and signaling. Molecular cell, 54(2), 289-296.
Chen, Q., Sun, L., & Chen, Z. J. (2016). Regulation and function of the cGAS–STING pathway of cytosolic DNA sensing. Nature immunology, 17(10), 1142.
Liu, X., & Wang, C. (2016). The emerging roles of the STING adaptor protein in immunity and diseases. Immunology, 147(3), 285-291.
Hu, M.M., Yang, Q., Xie, X.Q., Liao, C.Y., Lin, H., Liu, T.T., Yin, L. and Shu, H.B. (2016). Sumoylation promotes the stability of the DNA sensor cGAS and the adaptor STING to regulate the kinetics of response to DNA virus. Immunity, 45(3), pp.555-569.
Xiong, M., Wang, S., Wang, Y. Y., & Ran, Y. (2018). The Regulation of cGAS. Virologica Sinica, 1-8.
Sun, L., Wu, J., Du, F., Chen, X., & Chen, Z. J. (2013). Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science, 339(6121), 786-791.
Seo, Gil Ju, et al. “TRIM56-mediated monoubiquitination of cGAS for cytosolic DNA sensing.” Nature communications 9.1 (2018): 613.
Ishikawa, H., & Barber, G. N. (2008). STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature, 455(7213), 674.
Abe, T., & Barber, G. N. (2014). Cytosolic DNA-Mediated, STING-Dependent Pro-Inflammatory Gene Induction Necessitates canonical NF-κΒactivation Through TBK1. Journal of virology, JVI-00037
Luo, W. W., Li, S., Li, C., Lian, H., Yang, Q., Zhong, B., & Shu, H. B. (2016). iRhom2 is essential for innate immunity to DNA viruses by mediating trafficking and stability of the adaptor STING. Nature immunology, 17(9), 1057.
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Tooze, S.A., Jefferies, H.B., Kalie, E., Longatti, A., Mcalpine, F.E., Mcknight, N.C., Orsi, A., Polson, H.E., Razi, M., Robinson, D.J. and Webber, J.L., (2010). Trafficking and signaling in mammalian autophagy. IUBMB life, 62(7), pp.503-508.
Konno, H., Konno, K., & Barber, G. N. (2013). Cyclic dinucleotides trigger ULK1 (ATG1) phosphorylation of STING to prevent sustained innate immune signaling. Cell, 155(3), 688-698.
Tsuchida, T., Zou, J., Saitoh, T., Kumar, H., Abe, T., Matsuura, Y., Kawai, T. and Akira, S., (2010). The ubiquitin ligase TRIM56 regulates innate immune responses to intracellular double-stranded DNA. Immunity, 33(5), pp.765-776.
Wang, Q., Liu, X., Cui, Y., Tang, Y., Chen, W., Li, S., Yu, H., Pan, Y. and Wang, C., (2014). The E3 ubiquitin ligase AMFR and INSIG1 bridge the activation of TBK1 kinase by modifying the adaptor STING. Immunity, 41(6), pp.919-933.
Dobbs, N., Burnaevskiy, N., Chen, D., Gonugunta, V. K., Alto, N. M., & Yan, N. (2015). STING activation by translocation from the ER is associated with infection and autoinflammatory disease. Cell host & microbe, 18(2), 157-168.
Gonugunta, V. K., Sakai, T., Pokatayev, V., Yang, K., Wu, J., Dobbs, N., & Yan, N. (2017). Trafficking-Mediated STING Degradation Requires Sorting to Acidified Endolysosomes and Can Be Targeted to Enhance Anti-tumor Response. Cell reports, 21(11), 3234-3242.
ddewipputri 