mTOR inhibitors are a class of drugs used to treat several human diseases, including cancer, autoimmune diseases, and neurodegeneration. They function by inhibiting the mammalian target of rapamycin (mTOR) (also known as the mechanistic target of rapamycin), which is a serine/threonine-specific protein kinase that belongs to the family of phosphatidylinositol-3 kinase (PI3K) related kinases (PIKKs). mTOR regulates cellular metabolism, growth, and proliferation by forming and signaling through two protein complexes, mTORC1 and mTORC2. The most established mTOR inhibitors are so-called rapalogs (rapamycin and its analogs), which have shown tumor responses in clinical trials against various tumor types.
History The discovery of mTOR was made in 1994 while investigating the mechanism of action of its inhibitor, rapamycin. Rapamycin was first discovered in 1975 in a soil sample from Easter Island of South Pacific, also known as Rapa Nui, from where its name is derived. Rapamycin is a macrolide, produced by the microorganism Streptomyces hygroscopicus and showed antifungal properties. Shortly after its discovery, immunosuppressive properties were detected, which later led to the establishment of rapamycin as an immunosuppressant. In the 1980s, rapamycin was also found to have anticancer activity although the exact mechanism of action remained unknown until many years later.
In the 1990s there was a dramatic change in this field due to studies on the mechanism of action of rapamycin and the identification of the drug target. It was found that rapamycin inhibited cellular proliferation and cell cycle progression. Research on mTOR inhibition has been a growing branch in science and has promising results.
Protein kinases and their inhibitors
In general, protein kinases are classified in two major categories based on their substrate specificity, protein tyrosine kinases and protein serine/threonine kinases. Dual-specificity kinases are subclass of the tyrosine kinases.
mTOR is a kinase within the family of phosphatidylinositol-3 kinase-related kinases (PIKKs), which is a family of serine/threonine protein kinases, with a sequence similarity to the family of lipid kinases, PI3Ks. These kinases have different biological functions, but are all large proteins with common domain structure.
PIKKs have four domains at the protein level, which distinguish them from other protein kinases. From the N-terminus to the C-terminus, these domains are named FRAP-ATM-TRAAP (FAT), the kinase domain (KD), the PIKK-regulatory domain (PRD), and the FAT-C-terminal (FATC). The FAT domain, consisting of four α-helices, is N-terminal to KD, but that part is referred to as the FKBP12-rapamycin-binding (FRB) domain, which binds the FKBP12-rapamycin complex. The FAT domain consists of repeats, referred to as HEAT (Huntingtin, Elongation factor 3, A subunit of protein phosphatase 2A and TOR1). Specific protein activators regulate the PIKK kinases but binding of them to the kinase complex causes a conformational change that increases substrate access to the kinase domain.
Protein kinases have become popular drug targets. They have been targeted for the discovery and design of small molecule inhibitors and biologics as potential therapeutic agents. Small-molecule inhibitors of protein kinases generally prevent either phosphorylation of proteins substrates or autophosphorylation of the kinase itself.
mTOR signaling pathway It appears that growth factors, amino acids, ATP, and oxygen levels regulate mTOR signaling. Several downstream pathways that regulate cell-cycle progression, translation, initiation, transcriptional stress responses, protein stability, and survival of cells are signaling through mTOR.
The serine/threonine kinase mTOR is a downstream effector of the PI3K/AKT pathway, and forms two distinct multiprotein complexes, mTORC1 and mTORC2. These two complexes have a separate network of protein partners, feedback loops, substrates, and regulators. mTORC1 consists of mTOR and two positive regulatory subunits, raptor and mammalian LST8 (mLST8), and two negative regulators, proline-rich AKT substrate 40 (PRAS40) and DEPTOR. mTORC2 consists of mTOR, mLST8, mSin1, protor, rictor, and DEPTOR.
mTORC1 is sensitive to rapamycin but mTORC2 is considered to be resistant and is generally insensitive to nutrients and energy signals. mTORC2 is activated by growth factors, phosphorylates PKCα, AKT and paxillin, and regulates the activity of the small GTPase, Rac, and Rho related to cell survival, migration and regulation of the actin cytoskeleton.
The mTORC1 signaling cascade is activated by phosphorylated AKT and results in phosphorylation of S6K1, and 4EBP1, which lead to mRNA translation.
mTOR signaling pathway in human cancer
Many human tumors occur because of dysregulation of mTOR signaling, and can confer higher susceptibility to inhibitors of mTOR. Deregulations of multiple elements of the mTOR pathway, like PI3K amplification/mutation, PTEN loss of function, AKT overexpression, and S6K1, 4EBP1, and eIF4E overexpression have been related to many types of cancers. Therefore, mTOR is an interesting therapeutic target for treating multiple cancers, both the mTOR inhibitors themselves or in combination with inhibitors of other pathways.
Upstream, PI3K/AKT signalling is deregulated through a variety of mechanisms, including overexpression or activation of growth factor receptors, such as HER-2 (human epidermal growth factor receptor 2) and IGFR (insulin-like growth factor receptor), mutations in PI3K and mutations/amplifications of AKT. Tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a negative regulator of PI3K signaling. In many cancers the PTEN expression is decreased and may be downregulated through several mechanisms, including mutations, loss of heterozygosity, methylation, and protein instability.
Downstream, the mTOR effectors S6 kinase 1 (S6K1), eukaryotic initiation factor 4E-binding protein 1 (4EBP1) and eukaryotic initiation factor 4E (eIF4E) are related to cellular transformation. S6K1 is a key regulator of cell growth and also phosphorylates other important targets. Both eIF4E and S6K1 are included in cellular transformation and their overexpression has been linked to poor cancer prognosis.
Development of mTOR inhibitors Since the discovery of mTOR, much research has been done on the subject, using rapamycin and rapalogs to understand its biological functions. The clinical results from targeting this pathway were not as straight forward as thought at first. Those results have changed the course of clinical research in this field.
Initially, rapamycin was developed as an antifungal drug against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. A few years later its immunosuppressive properties were detected. Later studies led to the establishment of rapamycin as a major immunosuppressant against transplant rejection, along with cyclosporine A. Combining rapamycin with cyclosporine A, enhanced rejection prevention in renal transplantation. Therefore, it was possible to use lower doses of cyclosporine, which minimized toxicity of the drug.
In the 1980s the Developmental Therapeutic Branch of the National Cancer Institute (NCI) evaluated rapamycin and discovered it had an anticancer activity and was non-cytotoxic, but had cytostatic activity against several human cancer types. However, due to unfavorable pharmacokinetic properties, the development of mTOR inhibitors for the treatment of cancer was not successful at that time. Since then, rapamycin has also shown to be effective for preventing coronary artery re-stenosis and for the treatment of neurodegenerative diseases.
First generation mTOR inhibitors The development of rapamycin as an anticancer agent began again in the 1990s with the discovery of temsirolimus (CCI-779). This novel soluble rapamycin derivative had a favorable toxicological profile in animals. More rapamycin derivatives with improved pharmacokinetics and reduced immunosuppressive effects have since then been developed for the treatment of cancer. These rapalogs include temsirolimus (CCI-779), everolimus (RAD001), and ridaforolimus (AP-23573) which are being evaluated in cancer clinical trials. Rapamycin analogs have similar therapeutic effects as rapamycin. However they have improved hydrophilicity and can be used for oral and intravenous administration. In 2012 National Cancer Institute listed 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.
Rapalogs, which are the first generation mTOR inhibitors, have proven effective in a range of preclinical models. However, the success in clinical trials is limited to only a few rare cancers. Animal and clinical studies show that rapalogs are primarily cytostatic, and therefore effective as disease stabilizers rather than for regression. The response rate in solid tumors where rapalogs have been used as a single-agent therapy have been modest. Due to partial mTOR inhibition as mentioned before, rapalogs are not sufficient for achieving a broad and robust anticancer effect, at least when used as monotherapy.
Another reason for the limited success is that there is a feedback loop between mTORC1 and AKT in certain tumor cells. It seems that mTORC1 inhibition by rapalogs fails to repress a negative feedback loop that results in phosphorylation and activation of AKT. These limitations have led to the development of the second generation of mTOR inhibitors.
Rapamycin and rapalogs Rapamycin and rapalogs (rapamycin derivatives) are small molecule inhibitors, which have been evaluated as anticancer agents. The rapalogs have more favorable pharmacokinetic profile compared to rapamycin, the parent drug, despite the same binding sites for mTOR and FKBP12.
Sirolimus The bacterial natural product rapamycin or sirolimus, a cytostatic agent, has been used in combination therapy with corticosteroids and cyclosporine in patients who received kidney transplantation to prevent organ rejection both in the US and Europe, due to its unsatisfying pharmacokinetic properties. In 2003, the U.S. Food and Drug Administration approved sirolimus-eluting coronary stents, which are used in patients with narrowing of coronary arteries, or so-called atherosclerosis.
Recently rapamycin has shown effective in the inhibition of growth of several human cancers and murine cell lines. Rapamycin is the main mTOR inhibitor, but ridaforolimus/deforolimus (AP23573), everolimus (RAD001), and temsirolimus (CCI-779), are the newly developed rapamycin analogs.
Temsirolimus The rapamycin analog temsirolimus (CCI-779) is also a noncytotoxic agent which delays tumor proliferation.
Temsirolimus is a prodrug of rapamycin. It is approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), for the treatment of renal cell carcinoma (RCC). Temsirolimus has higher water solubility than rapamycin and is therefore administered by intravenous injection. It was approved on May 30, 2007, by FDA for the treatment of advanced RCC.
Temsirolimus has also been used in a Phase I clinical trial in conjunction with neratinib, a small-molecule irreversible pan-HER tyrosine kinase inhibitor. This study enrolled patients being treated for HER2-amplified breast cancer, HER2-mutant non-small-cell lung cancer, and other advanced solid tumors. While common toxicities included nausea, stomatitis, and anemia; responses were noted.
Everolimus
Everolimus is the second novel Rapamycin analog. Compared with the parent compound rapamycin, everolimus is more selective for the mTORC1 protein complex, with little impact on the mTORC2 complex. mTORC1 inhibition by everolimus has been shown to normalize tumor blood vessels, to increase tumor-infiltrating lymphocytes, and to improve adoptive cell transfer therapy.