User:Lybbar12/Drug discovery and development of mTOR inhibitors

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History

The discovery of mammalian target of rapamycin (mTOR) was made about 20 years ago while investigating the mechanism of action of its inhibitor, called rapamycin. (1) Rapamycin was first discovered in 1975 in a soil sample from Easter Island of South Pacific, also known as Rapa Nui, from were its name is derived. (2) Rapamycin is a macrolide that was produced by the microorganism Streptomyces hygroscopius and had antifungal properties. A few years after its discovery, immuosuppressive properties were discovered, which later led to the establishment of rabamycin as a immunosuppressant. In the 1990s, rapamycin was also found to have anticancer activity although the exact mechanism of action was not known until many years later. (1) For 20 years after the discovery of rapamycin there were only about dozen papers published related to the subject. However, the field experienced a dramatic turn of fortune in the 1990s with studies on the mechanism of action of rapamycin and the identification of the drug target. (2) It was found that the rapamycin inhibited cellular proliferation and cell cycle progression. (1) The substrate for rapamycin was identified as TOR and the mammalian analouge was designated mTOR. (3) With growing research on rapamycin, the clinical interest also renewed. Several rapamycin analogs have since then also been synthesized and tested in clinical trials. Studies have also given us insight into the molecular structure (architecture?) of the mTOR pathway. The role of the mTOR pathway as a key prosess in many human disease has either been discovered or confirmed . Rapamycin and rapamycin analogs (rapalogs) are currently in clinical trials for several condisions, especially for cancer. (2)

mTOR signaling

Discovery

mTOR was discovered in the 1990s, with investigation on rapamycin, and since then a lot of research has been done on the subject. The drug development against mTOR began when limited was known about its function. The clinical results from targeting this pathway were not as straight forward as thought at first. These results changed the course of clinical research in this field. (5)

Development

Initially, rapamycin was developed as an antifungal drug against Candida albicanis, Aspergillus fumigatus and Cryptocossus neoformans. (7) Few years later, its immunosuppressive properties were discovered and after many later studies that led to the establishment of rabamycin as a major immunosuppressant against transplant rejection, along with cyclosporine A. (1) This discovery stimulated the exploration of the mechanism of action of rapamycin. It inhibits T-cell proliferation and also inhibits proliferative responses induced by several cytokines, including interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-6, IGF, PDGF and colony-stimulating factors (CSFs). Using rapamycin in combination with cyclosporin A it enhanced the rejection prevention in renal transplantation, therefore it was possible to use lower doses of cyclosporine which minimized the toxicity. In the 1980s rapamycin was evaluated by the Developmental Therapeutic Branch of the National Cancer Institude (NCI). It was discovered that rapamycin had a anticancer activity. It was identified as a non-cytotoxic agent that had cytostatic activity against several human cancers. (7) However, due to unfavorable pharmacokinetic properties, the development of mTOR inhibitors for the treatment of cancer was not successful at that time. (3) The development of rapamycin as an anticancer agent began again in the 1990s with the discovery of CCI-779. This was a novel soluble rapamycin derivative that had a safe toxicological profile in animals. More rapamycin derivatives with improved pharmacokinetics and reduced immunosuppressive effect have since then been developed for the treatment of cancer. (7) These rapalogs include CCI-779 (temsirolimus), RAD001 (everolimus), and AP-23573 (deforolimus) and are being evaluated in cancer clinical trials. (6) Rapamycin analogs have similar therapeutic effect to rapamycin. However they have improved hydrophilicity and can be used for oral and intravenous administration. (2) The U.S. National Cancer Institude lists more than 200 clinical trials testing the anticancer activity of rapalogs both as monotherapy or as a part of combination therapy for many cancer types. (9)

First generation mTOR inhibitors

Rapalogs, which are the first generation mTOR inhibitors, have proven effective in a range of preclinical models. (8) These inhibitors form a complex with the intracellular immunophilin FKBP12 and the complex that is formed then inhibits mTOR. This effect restores the proper control of the activated PI3K/AKT/mTOR signaling pathway in the cancer cells. (7) The success in clinical trials can however be limited to only a few rare cancers. These cancer types include mantle cell lymphoma, renal cell carcinoma and endometrial cancer. It has since then been discovered that mTOR forms at least two functional multiprotein complexes, named mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). (8) These two complexes have a separate network of protein partners, feedback loops, substrates and regulators that is very complex. Rapamycin and rapalogs have an incomplete inhibition of mTORC1 and are inactive against mTORC2 but mTORC2 is also important for anticancer effect. (5) However, several studies have found that although rapalogs do not directly inhibit mTORC2, prolonged exposure may affect the mTORC2 assembly and AKT signaling. (1)(10) Another reason for the limited success is that there is a feedback loop between mTORC1 and AKT in certain tumor cells. That means that in some cases, treatment with rapalogs can cause elevated AKT activity through insulin substrate 1 (IRS-1) which enhances cell survival when mTORC1 in inhibited. (8) These limitations have led to the development of the second generation of mTOR inhibitors.

Second generation mTOR inhibitors

The second generation of mTOR inhibitors are known as ATP-competitive mTOR kinase inhibitors (TKIs). (9) A small molecule designed to compete with ATP in the catalytic site of mTOR, inhibits all of the kinase-dependent functions of mTORC1 and mTORC2 and therefore blocks the feedback activation of PI3K/AKT signaling, unlike rapalogs that only target mTORC1. (9) (11) Many TKIs have been developed and several of them are being tested in clinical trials. TKIs, like rapalogs, decrease protein translation, attenuate cell cycle progression and inhibit angiogenesis in many cancer cell lines and also in human cancer. In fact they have been proven to be more potent than rapalogs. (9) Theoretically, the most important advantages of TKIs is the considerable decrease of AKT phosphorylation on mTORC2 blockade and also the a better inhibition on mTORC1. (5) However thera are also some drawbacks, even though TKIs have been effective in rapamycin-insensitive cell lines, they have only shown limited success in KRAS driven tumors. This suggests that combinational therpy may by necessary for the treatment of these cancers. Another drawback is also their potential toxicity. These fact have raised concerns about the long term efficacy of these type of inhibitors. (9) Concerns with resistance to TKIs because of feedback activation of PI3K/Akt led to the development of dual PI3K/mTOR inhibitors as well as the close interaction of mTOR with the PI3K pathway. (9) Compared with drugs that inhibit either mTORC1 or PI3K, these drugs have the benefit of inhibiting mTORC1, mTORC2 and all the catalytic isoforms of PI3K. By targeting both kinases at the same time, it reduces the upregulation of PI3K that with inhibition on mTORC1 is typicall produced. (5) The inhibition of the PI3K/mTOR pathway has been shown to potently block proliferation in by inducing G1 arrest in different tumor cell lines. Strong induction of apoptosis and autophagy has also been seen. Despite good promising results, there are preclinical evidence that some type of cancers may be insensitive to this dual inhibition. The dual PI3K/mTOR inhibitors are also likely to have increased toxicity. (9) Significant progress has been made in the understanding of mTOR signlaing pathway in the last few years. (5) Number of cancers respond to monotherapy treatment with rapalogs, TKIs and dual PI3K/mTOR inhibitors. However, resistance remains a major concern. (9)

New insights into mTOR biology will be made in time, with the study of the mTOR pathway. This will help with the development of more effective therapeutic strategies for treating mTOR-related diseases, especially for the treatment of cancer. (12) limitation for the development of mTOR inhibition therapy is that biomarkers are not presently available to predict whick patient will respond to them. A better understanding of the molecular mechanisms that are involved the the response of cancer cells to mTOR inhibitors are still required so this can by possible. (9)

A way to overcome the resistance and improve efficacy of mTOR targeting agents may be with stratification of patients and selection of drug combination therapies. This may lead to a more effective and personalized cancer therapy. (9) (12)

Mechanism of action

Rapamycin and rapalogs crosslinks the immunophilin FK506 binding protein, FKBP-12, through its methoxy group . The rapamycin-FKBP12 complex interacts with and can inhibit mTOR (Vignot, et al., 2005). That inhibition blocks the binding of the acccessory protein Raptor (regulatory-associated protein of mTOR) to mTOR, but that is necessary for downstream phosphorylation of S6K1 and 4EBP1 (Faivre, et al., 2006). As a consequence, S6K1 dephosphorylates, which reduces protein synthesis and therefore decreases cell motality and size. Rapamycin induces dephosphorylation of 4EBP1 as well, resulting in an increase in p27 and a decrease in cyclin D1 expression, which will lead to late blockage of G1/S cell cycle. Rapamycin has shown to induce cancer cell death by inducing autophagy or apoptosis, but the molecular mechanism of adoptosis in cancer cell has not yet been fully resolved. One suggestion between mTOR inhibition and apoptosis might be through the downstream target S6K1, which can phosphorylate BAD, a pro-apoptotic molecule, on Ser136. That reaction breaks the binding of BAD to BCL-XL and BCL2, a mitochondrial death inhibitors, resulting in inactivation of BAD (Faivre, et al., 2006) and decreased cell survival (Vignot, et al., 2005). Rapamycin has also shown to induce p53-independent apoptosis in certain types of cancer. Molecular interaction between FKBP12 , mTOR, and Rapamycin can last for about three days (72 hours) (Faivre, et al., 2006). There are some evidence that extended therapy with Rapamycin may have effect on Akt and mTORC2 as well (Strimpakos, et al., 2009).

Rapalogs

The rapalogs (Rapamycin derivatives) have the same molecular structure as Rapamycin but with substitution of the lactonic macrocycle. They have the same binding sites for mTOR and FKBP12. The semi-synthetic modifications made results in few structural changes, mostly located at the C40 hydoxyl group outsite the FKBP12 and mTOR binding domains (Faivre, et al., 2006).

Chemical structures and names of rapalogs

Sirolimus

Rapamycin (also known as Sirolimus) is a natural antibiotic (Vignot, Faivre, Aguirre, & Raymond, 2005)produced by Streptomyces hygroscopius and was originally developed as an antifungal medication to treat Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans (Faivre, Kroemer, & Raymond, 2006; Vignot, et al., 2005). It is a carboxylic lactone-lactam macrolid(Simamora, Alvarez, & Yalkowsky, 2001). Rapamycin has shown different effects, e.g. as immunosuppressive agent, preventing coronary artery re-stenosis, and in the treatment of neurodegenerative diseases. The studies of Rapamycin as immunosuppressive agent enabled us to understand its mechanism of action. Recently Rapamycin has shown effective in the inhibition of growth of sveral human cancer and murine cell lines (Faivre, et al., 2006). Rapamycin is the main mTOR inhibitor, but deforolimus (AP23573), everolimus (RAD001), and temsirolimus (CCI-779) are the newly developed rapamycin analogs (Strimpakos, Karapanagiotou, Saif, & Syrigos, 2009). Rapamycin contains no functional groups that ionize in the pH range 1-10 and is therfore rather insoluble in water (Simamora, et al., 2001). Despite its effectiveness in preclinic cancer models, its poor solubility in water, stabilty, and the long half-time elimination made its parenteral use difficult but the development of soluble rapamycin analogs vanquised various barriers (Strimpakos, et al., 2009)

Temsirolimus

The Rapamycin analog, Temsirolimus (CCI-779 or Tosirel™, Wyeth)(Strimpakos, et al., 2009) is also a noncytotoxic agent which delays tumour proliferation. It is an ester derivative of rapamycin (Vignot, et al., 2005), and its first derivative (Strimpakos, et al., 2009), increasing the water-solubility of Rapamycin and making it available as intravenous formulation (Vignot, et al., 2005). Although, it is rather insoluble in water but soluble in alcohol. It was approved in May 30, 2007, by FDA for the treatment for advanced renal cell carcinoma (RCC)

Everolimus

Everolimus is the second novel Rapamycin analog (Strimpakos, et al., 2009). From March 30, 2009 to May 5, 2011 the FDA approved Everolimus (Afinitor®, Afinitor Disperz®) for the treatment of advanced Renal Cell Circinoma after Failure of Treatment with Sunitinib or Sorafenib, Subependymal Giant Cell Astrocytoma (SEGA) associated with Tuberous Sclerosis (TS), and Progressive Neuroendocrine Tumors of Pancreatic Origin (PNET). In July and August 2012, two new indications were approved, for advanced Hormone Receptor-positive, HER2-negative Breast Cancer in Combination with Exemestane, and Pediatric and Adult Patients with Subependymal Giant Cell Astrocytoma (SEGA)

Ridaforolimus

Ridaforolimus (AP23573, MK-8669), or Deforolimus, is the newest Rapamycin analog and it is not a prodrug (Strimpakos, et al., 2009). The C43 secondary alcohol moiety of the cyclohexyl group of Rapamycin was substituted with phosphonate and phosphinate groups, preventing the high-affinity binding to mTOR and FKBP. Computional modelling studies helped the synthesise of the compound (Vignot, et al., 2005). It is not on market, since FDA wanted more more human testing on it due to its effectiveness and safety in June 2012

Structure activity relationship

Sirolimus binding sites: The green domain represents the pipecolate region of rapamycin that binds to FKBP12 and the purple domain represents mTOR binding site

Rapamycin is the macrocyclic lactone that has binding activity to the intracellular immunophilin FK506 binding protein 12 (FKBP12) and creates complex and interferes with FRB domain of mTOR (ballau 2011) that inhibits its activity (tanneeru 2011). The pipecolate region of rapamycin structure seems necessary for rapamycin-binding to FKBP12, and this step is required for further binding of rapamycin to the mTOR kinase, which is the key enzime in many biological actions of rapamycin. (Ritacco) -Ritacco et. al observed incorporation of couple of different sulfur-containing analogs og L-pipecolate. Results show that by adding sulfur atom to the pipecolate/thiazine ring of rapamycin increases IC50 and that associates much weaker binding activity of the product. (ritacco ) – Eigum við að hafa þetta The X-ray crystal structure study of the FKBP12-rapamycin-FRB complex shows that rapamycin binds FKBP12 binding sitesby occupying two different hydrobphobic binding pockets and FRB domain which brings two proteins close together. Although there are few interactions between these two proteins. (ballau 2011) The X-ray crystal structure [65] of rapamycin bound to FKBP12 revealed a number of key hydrogen bonds that explain rapamycin´s high affinity to this protein. The key contacts for this binding between FKBP12 binding-pocket, rapamycin and temsirolimus (FKB 506) are pipecolic acid, tricarbonyl region from c-8 to c10 and the lactone functionalities. The most impotant hydrogen bonds include the lactone carbonyl oxygen at C-1 to the backbone H of Ile56, amide carbonyl at C-8 to the phenolic group on the sidechain of Tyr82 and the hydroxyl proton at the hemiketal carbon, C-10, to the sidechain of Asp37. The key to rapamycin strong binding affinity to the FRB domain of mTOR are several hydropobic contacts with the 340 Å^2 of solvent accessible surface area of rapamycin,with the absence of hydrogen bond-based interactions. These hydrophobic contacts occur principally between the triene region of C-16 to C-23 of rapamycin and several aromatic residues in the FRB domain. Also a number of methyl groups make hydrophobic contacts to the FRB domain, notably methoxy group at C-16 and methyl groups at C23 and C29 and C31. (drug discovery – bók í zotero) Luengo et al reported that modification to the C-7 center of rapamycin results in deviation of biological effects of rapamycin and its analogs as well as their binding ability to FKBP12. It appears that C-7 substituent might form of the FRAP-binding surface, and it may lie in an interfacial space between FKBP12 and FRAP. Rapamycin and tacrolimus have similar chemical structures and bind to FKBP12, though their mechanism of action differs. (ballau 2011) (mynd) Temsirolimus that is a dihydroxymethyl proponic acid ester of rapamycin, is more water soluble so it can be given peripherally. (ballau2011) Everolimus has O-2 hydroxyethyl chain substitution and deforolimus has a phosphine oxide substitution at position C-40 in the lactone ring of rapamycin.(ballau 2011)

Limitations of rapalogs

As mentioned before rapalogs are selective mTORC1 inhibitors, and it is the one of reasons for their failure.(wander 2011) mTOR kinase also participates mTORC2 protein that activates AKT oncogene through phosphorylation of AKT Ser473, and can provide cell survival through other cell mechanisms. It seems that mTORC1 inhibition by rapalogs fails to repress a negative feedback loop that results in phosphorylation and activation of AKT. (Sutherlin 2011)Animal and clinical studies show that rapalogs are primarily citostatic, and therefore effective as disease stabilizators rather than for regression.(Wander 2011) However the clinical use of rapalogs has been limited to few rare cancers. The response rate in solid tumors where rapalogs have been used as a single-agent therapy, have been modest, due to partial mTOR inhibition, rapalogs are not sufficient for acheving a broad and robust anticancer effect, at least when used as monotherapy. (Zhang 2011)(brachmann 2009) Rapalogs are well tolerated, but clinical trials show that these compounds seem to be less effective then expected at fist. In particular, they have promising activity in patients with advanced renal cell carcinomas (RCC), that have failed in prior therapy as well as in patients with tuberous sclerosis (TSC) who harbor renal giomyolipomas. These results confirm the obligating science linking mTOR activation with von Hippel-Lindau (VHL) mutations in RCC or TSC. (brachmann)

Biomarkers

Identification of predictive biomarkers of efficacy for tumor types that are sensitive to mTOR inhibitors remains a major issue (debaldo, populo) Possible predictive biomarkers for tumor response to mTOR inhibitors, as have been described in glioblastoma, breast and prostate cells, may be the differential expression of mTOR pathway proteins, PTEN, AKT and S6. (populio) Thus, this data is based on preclinical assays, based on in vitro cultured tumor cell lines, suggest that the effects of mTOR inhibitors may be more pronounced in cancers displaying loss of PTEN functions or PIK3CA mutations.(DELBADO2011) However the use of PTEN, and PIK3CA mutations and AKT –phospho status for predicting rapalog sensitivity has not been fully validated in clinic. To date, attempts to identify biomarkers of rapalog response have been unsuccessful. (wander2011) mTOR is ubiquitously expressed in both tumor and healthy tissues and resistance or sensitivity cannot be predicted upon the presence oft the target alone. Several molecular patterns are emerging for sensitivity as well as resistance of rapalogs. The main parameter of this pathway assessed in tumor models as biomarkers of sensibility or resistance are loss of PTEN function, AKT phosphorylation and PI3K mutations. High levels of AKT and reduced PTEN expressions showed that renal carcinoma cell lines were sensitive to mTOR inhibition. However a high level of p-AKT might also result from the feedback loop induced by mTORC2, characterized by rapamycin insensitive mTOR activity as well as from induced upstream signal pahtway. Therefore high levels of p-AKT might not be used as predictor of mTORC1 responce.(Delbaldo 2011) Many other potentional biomarkers as p-S6, p-4E-BP-1, hTERT and telomere length have been explored, but their effectiveness in discrimination of which tumors types will respond to the antiproliferative effects of rapamycin was show to be deficient. (Má segja þetta svoan) (delbaldo 2011) It seems that analyses of p-S6K1 and p-4EBP1 can be used to monitor the biologically active doses of mTOR inhibitors that have effects on healthy cells as well as tumor cells, but it might be need for development of more accurate molecular biomarkers. (faivre). There is a great need for indetification of predictive biomarkers of response to able selection of patients bearing tumors which may respond and benefit from mTOR inhibition therapies. (populio)

Sensitivity

Clinical and translational data suggests that tumor types that are sensitive and have adequate parameters and functional apoptosis pathways, might not need high doses of mTOR inhibitors to trigger apoptosis. In most cases, cancer cells might only be partially sensitive to mTOR inhibitors due to redundant signal transduction or lack of functional apoptosis signaling pathways. In situations like this high doses of mTOR inhibitors might be required. In recent study in patients with RCC, resistance to Temsirolimus was associated with low levels of p-AKT and p-S6K1, that play the key role in mTOR activation. These data strongly suggests that there is a number of tumors with an activated PI3K/AKT/mTOR signaling pathway that does not respond to mTOR inhibitors. For the future studies it is recommended to exclude patients with low or negative p-AKT levels from trials with mTOR inhibitors. (faivre 2006) Current data is insufficient to predict sensitivity of tumors to rapamycin. However the existing data allows us to characterize tumors that might not respond to rapalogs. (faivre)

Future

The limitations of currently available rapalogs have led to new approaches to mTOR targeting. Studies suggest that mTOR inhibitors may have anticancer activity in many cancer types. Those are for example RCC, neuroendocrine tumors, breast cancer, hepatocellular carcinoma, sarcoma and large B-cell lymphoma. (Yuan 2009)

Other mTOR inhibitors

mTor inhibitors can be devided in two groups. As mentioned before one group is rapamycin and its derivates and second group are small molecules that are mTOR kinase inhibitors, and they bind to the ATP-binding site of the mTOR kinase domain that is required for the functions of both mTOR complexes, respectively. (populio 2012) Due to PI3K and mTORC2 ability to regulate AKT phosphorylation, these two compounds play a key role in minimizing the feedback activation of AKT. (Zhang april 2011) Several so called mTOR/PI3K dual inhibitors (TPdIs) have been developed. Their development has been benefited from previous studies with PI3K-selective inhibitors. Several TPdIs have promising results in early-stage clinical trials. Novartis has developed compound NVPBE235 that was reported to inhibit tumor growth in many preclinical models, and enhances antitumor activity of several other drugs such as vinchristine.(ZHAN 2011) The activity of these small molecules from rapalog activity differs in the way that they block both mTORC1-dependent phospholylation of S6K1 and mTORC2-dependent phosphorylation of AKT Ser473 recidue.it has been reported in preclinical studies that anticancer effect of these compounds is superior to rapalogs.(populio 2012) A new generation of mTOR-specific inhibitors came forth from screening and drug discovery efforts. These compounds block activity of both mTOR complexes and are called mTORC1/mTORC2 dual inhibitors. (zhang) Limitations of new generation mTOR inhibitors Although the new generation mTOR inhibitors hold great promise for anticancer therapy and are rapidly moving into clinical trials, are many important issues that determine their success in the clinic. First of all predictable biomarkers for benefit of these inhibitors are not available. It appears that genetic determinants predispose cancer cells to be sensitive or resistant to these compounds. Tumors that depend on PI3K/mTOR pathway should respond to these agents but it is unclear if compounds are effective in cancers with distinct genetic lesions. (zhang) PI3K inhibitors bind to ATP- binding pocket reversibly and inhibits p110∂ lipid kinase and has negligible potency against alfa and beta p110 isoforms. (Brachmann)