Structural Analysis of Tacrolimus Binding FKBP12
This Jmol Exploration was created using the Jmol Exploration Webpage Creator from the MSOE Center for BioMolecular Modeling.
AJ, a 45 year old male, presents to the transplant center at Froedtert Hospital because he got the call his kidney was ready. A first year pharmacy resident is assigned to the case upon AJ's admission to the transplant floor. The resident is fresh on the job and, being rusty on his transplant pharmacotherapy, asks the pharmacist for a review of what type of immunosuppressive drug should be used for AJ's solid organ kidney transplant anti-rejection therapy. The pharmacist replies that there are several options available for maintenance therapy but one drug that is ideal for this case. The pharmacist lists antiproliferative drugs, corticosteroids, and calcineurin inhibitors as potential choices. The floor pharmacist also asks the resident to look up appropriate monitoring, drug-drug interactions, and toxicities for his choice of therapy for education purposes.
The resident selects a calcineurin inhibitor for maintenance therapy. After researching, the resident identified that of the two options for calcineurin inhibitors (cyclosporine and tacrolimus) tacrolimus has demonstrated better graft survival rates when compared in clinical trials1.
The resident reports to the pharmacist that the patient should be counseled on the importance of avoiding omeprazole, a proton pump inhibitor that competitively inhibit the cytochrome P450 3A4 and 2C19 liver enzymes that metabolize tacrolimus. Inhibition of these liver enzymes would cause tacrolimus to accumulate in excess leading to various toxicities such as nephrotoxicity2. The resident recommends targeting tacrolimus serum trough levels of 10-15 ng/mL for the first year of therapy followed by 5-10 ng/mL thereafter. Monitoring for side effects of hyperglycemia, opportunistic infection, and hair loss should be done routinely throughout the lifelong course of therapy.
The resident looked further into recommended treatment plans based on the KDIGO Guideline for Kidney Transplant Recipients. For kidney transplantation, the resident recommends starting tacrolimus for renal transplant rejection prophylaxis: begin with 0.1 mg/kg orally once in the 12 hours prior to the transplant operation. Following transplantation, administer 0.2 mg/kg/day by mouth once daily. Monitor this regimen closely and titrate this dose based on the patient response keeping in mind target trough goal (10-15 ng/mL).
The pharmacist is satisfied by the resident's answer and suggests that looking at the structure and binding site for tacrolimus will help the resident's understanding of how the drug reduces the immune response mounted against a foreign organ and why it is important to do so in post-transplant maintenance therapy.
The immune system is an intricate defense mechanism of the body; its role is to protect against substance that may cause harm to the body, such as germs or viruses. These harmful substances have proteins coating their surfaces called antigens. As soon as these antigens enter the body, the immune system recognizes that these antigens are not from the body, labels them as 'foreign' and attacks them. When transplant patients receive an organ from another person, their body's immune system may recognize the transplanted organ as 'foreign' due to the antigens identified from the organ. This response can lead to an immune response mounted against the organ further progressing to organ rejection.
To help prevent this immune reaction, surgeons match the organ donor to the patient receiving the organ. The better the match, meaning the more similar the antigens are between the donor and recipient, the less likely the organ will be rejected. Transplant medications are also used to suppress the recipient's immune system. The goal is to prevent the immune system from attacking the newly transplanted organ when the organ. If these medications are not use, the body will almost always launch an immune response and destroy the 'foreign' organ.
Tacrolimus is a macrolide antibiotic produced by the organism Streptomyces tsukubaensis3. The drug is an immunosuppressive agent used in organ transplant patients to prevent or treat graft rejection. Tacrolimus comes in oral capsules, intravenous solution, and topical ointments. Intravenous tacrolimus is only indicated for transplant patients, but oral tacrolimus can also be used to treat myasthenia gravis. Topical tacrolimus may be used to treat arthritis and atopic dermatitis4.
Tacrolimus has two distinct mechanisms to impair immune function: inhibiting FKBP12 and binding with FKBP12 to form a complex that can inhibit calcineurin. The first mechanism involves inhibiting an immunophilin protein called FKBP12. Normally functioning FKBP12 inhibits transforming growth factor - (TGF) receptors on immune T cells preventing a rise in p21, the Cdk2/cyclin E complex inhibitor. Ultimately, FKBP12 allows a T cell to enter the synthesis phase of the cell cycle6. When tacrolimus is bound to FKBP12, immune T cells will arrest in the G1 phase of the cell cycle preventing cell proliferation. The action of tacrolimus causes a decrease in T cell counts; this is how the immune system is suppressed. With fewer T cells, our immune system is not as efficient or effective at fighting off infections or identifying foreign antigens. This suppression is desired for a patient receiving a foreign organ as the immune response will not be sufficient to damage the newly introduced organ allowing the body to accept the transplanted organ. Tacrolimus decreases the number of T cells to suppress the immune response preventing the body from identifying foreign antigens as non-self.
For the second mechanism, tacrolimus binds to FKBP12 and the complex that forms from both molecules inhibits the enzyme calcineurin7. The calcium-dependent serine threonine phosphatase enzyme called calcincurin naturally activates T cells after they have been presented foreign antigen8. By preventing T cell activation, tacrolimus further suppresses the organ recipient's immune response.
Now we will look into how these molecules interact with one another to cause immune suppression as described above.
Tacrolimus (seen in Figure 1) is an immunosuppressive drug that binds to the protein called FKBP12 or tacrolimus bindin protein (seen in Figure 2). The drug is considered a macrolide due to its large lactone ring. Tacrolimus interacts with FKBP12 by hydrogen bonds and van der Waals interactions. The active site of FKBP12 is found near the center of the molecule. In Figure 3 we can see Tacrolimus bound to FKBP12.
Figure 3: Tacrolimus Bound to ProteinFigure 3, an image of tacrolimus bound to the active site, helps depict the interaction of His87. His87 provides a hydrophobic region that helps a methyl group stay in the correct conformation, but more importantly it helps stabilize the Asp37 and brings Asp37 closer to tacrolimus to form a hydrogen bond. The Asp37 hydrogen binds to a hydroxyl group in close proximity to the methyl group in conjunction with His87. The Phe46 and Trp59 in the active site of FKBP12 provide a localized dense hydrophobic region to stabilize the piperidine group found in tacrolimus (Figures 3 and 4).
Figure 4: Tacrolimus in its Binding Site With InteractionsThe Tyr82 found in the active site provides a hydroxyl group for a hydrogen bond and the benzene ring on Tyr82 provides a hydrophobic region for a methyl ether group to bind (Figure 4). This interaction causes tacrolimus to fold because the hydrogen bond is between a ketone adjacent to the piperidine and the methyl ether on the opposite side of the molecule3. This is easier to see in Figure 1.
In Figure 5 we can see the hydrophobic interactions between tacrolimus and tacrolimus binding protein. Figure 6 shows polar interactions between tacrolimus and tacrolimus binding protein.
Figure 5: Hydrophobic InteractionsFigure 7 shows the second protein structure of the tacrolimus binding protein. Shown in yellow are beta sheets and in blue are alpha helices. These secondary structures aid in hydrogen bond stabilization7.
Figure 7: Secondary Structure of Tacrolimus Binding ProteinTacrolimus, as stated earlier, can be used to suppress the immune system in organ transplant patients. Tacrolimus has high affinity for the protein FKBP12 and when bound, Tacrolimus inhibits FKBP12 function. Inhibition of this protein causes p21 values to rise preventing immune T cells from entering into the synthesis phase of the cell cycle6. Another mechanism through which tacrolimus can suppress the immune system is by calcineurin inhibition2,11. The most important medicinal chemistry interactions that cause this inhibition involve Tyr82 and the effector loop protrusion out of the tacrolimus protein structure. The interaction involving Tyr82 causes tacrolimus to fold over on itself, and the portion extending from tacrolimus interacts with calcineurin. This interaction how the complex formed when tacrolimus binds FKBP12 inhibits calcineurin function. Calcineurin inhibition decreases phosphatase activity which results in inhibition of transcription for the genes that code a variety of interleukins8. The main interleukin of focus in this case is IL-2 which is important in the process of T cell activation. Both of the aforementioned mechanisms decrease the amount of T cells and therefore prevent the immune system from working to its full potential.
Tacrolimus has a high probability of causing adverse events in patients because of its extensive role in suppressing the immune system. The immune system is a part of the whole body; it is not limited to just one area. Tacrolimus not only makes the body more prone to infection from immune suppression, but also may cause problems such as nephrotoxicity, insulin resistance, and increased QTc interval2,11. Of further concern is metabolism of tacrolimus by liver enzymes increasing the likelihood of drug-drug interactions. The drug is highly lipophilic and is metabolized by cytochrome P450 (CYP) 3A subfamily. A majority of the drug-drug interactions with tacrolimus occur through CYP3A4 induction or inhibition8. Altering CYP3A4 function changes the amount of tacrolimus within the body. This presents as a problem because tacrolimus has a very narrow therapeutic index. It can be deduced that a patient will likely experience adverse effects from toxicity caused by CYP3A4 inhibition and the consequent increase in plasma concentration of tacrolimus. Adversely, the end result of CYP3A4 induction is a loss of tacrolimus efficacy and possibly organ rejection.
Back to our case, when caring for a patient receiving a kidney transplant and initiating tacrolimus therapy, it is important to analyze every detail within the patient's chart. The pharmacist must use critical thinking in order to determine dose increases, dose decreases, alternative therapies, and drug discontinuations for each patient as an individual. Attention to detail is especially important in this case since tacrolimus decreases immune system function. A weakened immune system places the patient at higher risk of infection. Medications with narrow therapeutic indices, like tacrolimus, require strict therapeutic drug monitoring in order to keep patients safe. Therapeutic drug monitoring for tacrolimus is done by an enzyme immunoassay using the monoclonal antibody raised against tacrolimus in order to obtain trough levels. An example of additional parameters to monitor for a drug such as tacrolimus are kidney function, liver function, glucose levels, and potassium levels. Pharmacists encountering future metabolism cases need to make note of the variables that can affect metabolism such as race, medications, disease states, and genetic differences when dosing patients in practice.
1. MedLine Plus: Transplant rejection [internet]. Bethesda, MD: A.D.A.M. Quality Inc.; 1997. [cited 2013 Dec 2]; [first screen]. Available from: http://www.nlm.nih.gov/medlineplus/ency/article/000815.htm.
2. Tacrolimus. In: DRUGDEX System [Internet database]. Greenword Village, CO: Thomas Reuters Inc. Updated periodically. [cited 2013 Dec 1] Available from: http://0-www.micromedexsolutions.com.topcat.switchinc.org/micromedex2/librarian.
3. PDB ID: 1FKJ. Wilson KP, et al. Comparative x-ray structures of the major binding protein for the immunosuppressant FK506 (tacrolimus) in unliganded form and in complex with FK506 and rapamycin. Biolog Crystallography. 1995 Jul:51(4)511-521.
4. Iwasaki, K. Metabolism of tacrolimus (FK506) and recent topics in clinical pharmacokinetics. Drug Metab Pharmacokinet. 2007 Oct;22(5):328-35.
5. Griffith JP, et al. X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex. Cell. 1995 Aug;82(3):507-22.
6. Aghdasi B, et al. FKBP12, the 12-kDa FK506-binding protein, is a physiologic regulator of the cell cycle. Proc Natl Acad Sci USA. 2001 Feb;98(5):2425-30.
7. Ivery MT, Weiler L. Modeling the interaction between FK506 and FKBP12: a mechanism for formation of the calcineurin inhibitory complex. Bioorg Med Chem. 1997 Feb;5(2):217-32.
8. Brazelton TR, Morris RE. Molecular mechanisms of action of new xenobiotic immunosuppressive drugs: tacrolimus (FK506), sirolimus (rapamycin), mycophenolate mofetil and leflunomide. Curr Opin Immunol. 1996 Oct;8(5):710-20.
9. DrugBank: Tacrolimus [internet]. Canada: Genome Canada and Genome Alberta; 2005. [cited 2013 Nov 26]; [Metabolism section]. Available from: http://www.drugbank.ca/drugs/DB00864.
10. Itoh S, Navia MA. Structure comparison of native and mutant human recombinant FKBP12 complexes with the immunosuppressant drug FK506 (tacrolimus). Protein Science. 1995 Oct:4:2261-2268.
11. Lexi-Comp [Internet]. Hudson, OH: Lexi-Comp, Inc. [cited 2013 Dec 3]. Available from: http:// 0-online.lexi.com.topcat.switchinc.org/.
12. Jmol [an open-source Java viewer for chemical structures in 3D]. [cited 2013 Nov 15] Available from: http://www.jmol.org/.