Category: General Knowledge

Proteolysis targeting chimeric (PROTAC) technology is a recently discovered technique that has profound efficacy in cancer. This approach was first established in 2001. It is based on a ubiquitin-proteasome system that works to degrade target proteins via ubiquitination, thus suppressing the growth of tumors. Many kinds of research and clinical trials have been performed recently to determine the effect and feasibility of PROTAC technology in degrading endogenous proteins that have shown promising results. One of the most promising and encouraging results was found in oral PROTAC therapy in breast cancer and prostate cancer treatment. (1)

Mechanism of Action of PROTAC:

Different physiological and pathophysiological mechanisms work in our body for the degradation of proteins, including cell cycle control, gene transcription, apoptosis, etc. Among all these systems, PROTAC technology uses a ubiquitin-proteasome system (UPS) for the targeted degradation of protein in different cancerous and tumor cells. UPS depends on ATP for its activity and has two ordered steps, including:

• Polyubiquitination of the target protein
• Proteolysis of polyubiquitin by 26S proteolytic enzyme complex

PROTAC molecules attach E3 ligases to a target protein that initiate the degradation process by ubiquitination of target proteins by E3 ligases. Proteasome (26S) then degrades this ubiquitinated protein, and the activity of PROTACs is not dependent upon the kinase activity of target proteins. (2)

Effects of PROTAC During the Course of Cancer Causation:

Initiation and progression of cancer is a complex process, including different pathophysiological changes in the cells, including proliferative signaling, inhibiting growth suppression, induction of angiogenesis, countering cell death, and activation and local invasion and distant metastasis. Evidence-based research and clinical trials have explained the crucial role of overactivated and overexpressed proteins in the initiation and progression of cancer. These proteins are the focus of most of the therapeutic approaches used for the treatment and abolishment of cancer and different associated factors. (3) Here, we will précis about the PROTAC approach in cancer therapy.

Cancer Cell Proliferation:

PROTAC technology targets proteins that are hyperactivated or mutated and result in cell cycle progression that favors cancer cell proliferation. One main pathway of cell cycle regulation that is hyperactivated in proliferating cancer cells is the RAS-RAF-MEK-ERK pathway. Proteins act as inhibitors or accelerators for regulating the cell cycle by controlling CDK expressions. CDKs and their chaperones release transcription factor E2F by phosphorylation of retinoblastoma protein resulting in DNA replication. Growth signaling and progression also depend upon the RAS-RAF-MEK-ERK pathway, which plays a key role. Until now, PROTACs have been developed that target oncogenic proteins including BRD4, EGFR, CDK4/6, AURORA-A, CDK2/5, MEK1/2, HER, etc. (4)

Cancer Apoptosis:

Apoptosis is the programmed death of the cells that helps to maintain tissue hemostasis during DNA damage, immune surveillance, and cellular stress. In cancerous cells, proteins favoring cell death are downregulated, and those inducing cell survival are upregulated. It increases cell survival, resistance to endogenous and exogenous apoptotic factors, and cancer causation and recurrence. PROTACs target proteins that regulate apoptosis, thus improving the effects of anticancer factors and increasing the programmed death of the cells. Downregulation of antiapoptotic proteins, including Bcl-2, Bcl-xL, Bcl-6, and Mcl-1, and upregulation of proapoptotic proteins, including Bax and Puma, by PROTACs make them a better therapeutic choice for the treatment of different types of cancers. (5)

Cancer Angiogenesis:

Tumor cells require neurovascular supplies to get nutrients and excrete waste products essential for their growth and proliferation. Angiogenesis depends upon various factors, including VEGF (a principal factor), whose expressions are activated by hypoxia resulting in the induction of angiogenesis. PROTAC technology can target these growth factors that can result in the suppression of angiogenesis and proliferation of cancer cells. Growth factors that are the targets of PROTACs in the inhibition of angiogenesis include VEGFR2, PI3K, CBP/p300, etc. (6)

Cancer Immunity and Inflammation:

Tumor cells involve activated signaling of different regulatory cells, including T-cell receptor (TCR) and B-cell receptor (BCR), that cause reprogramming of cancerous microenvironment leading to immune evasion and inflammation. These processes are essential for tumor cell survival. Immunotherapies have significant roles in enabling immune-meditated clearance of cancer cells. However, some patients have resistance to these therapies. PROTACs can target the proteins that carry out different pathways, such as TCR, BCR, and JAK-STAT pathways. They have evidence-based activity against BTK, PD-L1, STAT3, ITK, JAK, etc., which are essential in carrying out TCR, BCR, and JAK-STAT pathways. (4)

Cancer Metastasis:

Cancerous cells can successfully disseminate and travel via blood or lymphatic circulation and colonize distant organs away from their primary site of occurrence. This process is called metastasis which is responsible for approximately 90% of cell deaths worldwide. Metastasis requires a key step called Epithelial-to-mesenchymal transition, which is dependent upon different cellular signaling pathways, including Integrin/FAK/PI3K/AKT axis. PROTACs have been developed in past few years to target proteins related to Epithelial-to-mesenchymal transition. The key pathways involved in increased expression of Epithelial-to-mesenchymal transition include Integrin/FAK/PI3K/AKT, Wnt/β-catenin, and TGF-β/SMAD. PROTACs can target proteins involved in the regulation of these pathways, such as FAK, Smad3, p38, IGF-IR, TCF, etc. (3)

Conclusion:

In the bottom line, PROTAC technology has extensively progressed from its start in 2001, with significant efficacy in improving cancer treatments. However, there is still a need to have more clinical trials and evidence-based research on its efficacy. The effects of PROTACs in controlling cancer progression and its counterattacking mechanisms during different steps of cancer cell growth, proliferation, and spread have been studied. However, developing different types of PROTACs with specific advantages and overcoming disadvantages and therapeutic efficacy and safety need more study.

References:

1. Xie H, Liu J, Alem Glison DM, Fleming JB. The clinical advances of proteolysis targeting chimeras in oncology. Explor Target Antitumor Ther. 2021;2(6):511–21.
2. Qi SM, Dong J, Xu ZY, Cheng XD, Zhang WD, Qin JJ. PROTAC: An Effective Targeted Protein Degradation Strategy for Cancer Therapy. Front Pharmacol. 2021 May 7;12.
3. Wang C, Zhang Y, Zhang T, Shi L, Geng Z, Xing D. Proteolysis-targeting chimaeras (PROTACs) as pharmacological tools and therapeutic agents: advances and future challenges. J Enzyme Inhib Med Chem. 2022 Dec;37(1):1667–93.
4. Li X, Song Y. Proteolysis-targeting chimera (PROTAC) for targeted protein degradation and cancer therapy. J Hematol Oncol. 2020;13(1):50.
5. Li X, Pu W, Zheng Q, Ai M, Chen S, Peng Y. Proteolysis-targeting chimeras (PROTACs) in cancer therapy. Mol Cancer. 2022;21(1):99.
6. Ocaña A, Pandiella A. Proteolysis targeting chimeras (PROTACs) in cancer therapy. J Exp Clin Cancer Res. 2020 Sep 15;39(1):189.

Most Preclinical CROs provide studies that aim at providing information about safety and efficacy of a drug candidate before testing it in humans. Furthermore, they can provide evidence for the compound’s biological effect and usually include both in vitro and in vivo studies. Preclinical studies have to comply with the guidelines dictated by Good Laboratory Practice to ensure reliable results and are required by authorities such as the FDA before filing for approval as IND. Insights into the compound’s dosing and toxicity levels are essential to determine whether it is justified and reasonably safe to proceed with clinical studies and are provided by studies on pharmacokinetics, pharmacodynamics, and toxicology. (Honek, 2017)

Pharmocodynamics (what does the drug do to the body?). Pharmacodynamics describes the relationship between the concentration of a drug in the body and its biological effect (dose response). This includes addressing the question, how potent and efficacious the drug is with regard to its desired pharmacological effect, including safety aspects and AEs (adverse events). Thus, pharmacodynamics establishes the therapeutic index of a drug, describing the ratio of the dose causing toxicity and the dose eliciting a therapeutic effect. Ideally, the therapeutic index is large to indicate a wide therapeutic window.

Pharmacokinetics (what does the body do to the drug?). The effect of a drug is determined by the amount of active drug present in the body particularly at the target site. This, in turn, depends on absorption, distribution, metabolism, and excretion (ADME) of the compound. Pharmacokinetics describes changes in plasma concentrations over time as a consequence of ADME. ADME profiling is critical for establishing dose range and administration schedule for subsequent phases of the clinical trial.

Most drugs are administered orally and need to be absorbed in the gastrointestinal tract to enter the bloodstream, allowing them to be transported to their site of action. On its way to the target site, the drug reaches the liver, where first-pass metabolism takes place. Consequently, the drug concentration – and thus its bioavailability – is reduced before entering systemic circulation. Intravenous drug administration bypasses the first-pass effect, resulting in greater bioavailability. Once in the circulation, the drug is transported to different tissues. Distribution of the compound throughout the body is determined by (i) the drug’s affinity for plasma proteins, (ii) the drug’s molecular properties and polarity, and (iii) tissue vascularisation. After entering the body, drugs are metabolised to facilitate elimination. Metabolism refers to the chemical alteration of the parental drug into pharmacologically active or inert metabolites. To ensure adequate long term dosing and appropriate steady-state concentrations of the drug, it is critical to obtain information on drug elimination from the body (clearance). Clearance is mainly achieved via the renal and hepatic routes; however, pulmonary clearance plays a major role for volatile drugs such as anaesthetics. Concomitant disease, lifestyle factors, and patient’s age can affect clearance and these are frequently studied in later stages of the clinical trial. When the rate of clearance equals the rate of absorption, a so-called steady state is reached. Typically, maintaining a stable steady state level is desirable and can be achieved through repeated dosing. Eventually, the drug and its metabolites are excreted from the body mainly through urine or feces.

Toxicology (it is efficacious, but is it safe?). To determine whether a drug is safe for testing in human subjects, preclinical toxicology studies are performed to identify the treatment regimen associated with the least degree of toxicity and thus determine a suitable and safe starting dose for clinical trials. Additionally, they can be used to establish biomarkers for monitoring potential AEs later. Starting with single-dose studies to identify organs that might be subject to drug toxicity, preclinical in vivo studies continue with repeated-dose approaches. The treatment regimen ideally mimics the intended clinical

design with respect to treatment duration, schedule, and route of administration. Other studies evaluate carcinogenicity, genotoxicity, and reproductive toxicity. While the drug’s genotoxic effect is usually studied based on its potential to induce mutations in yeast-based in vitro systems, carcinogenicity and reproductive toxicity studies typically involve rats. As the tumorigenic effect of a drug may only become evident after prolonged exposure, carcinogenicity studies comprise continuous drug administration for a minimum of six months.

In vitro models (studying the drug in a petri dish). In vitro studies are a relatively fast, simple, and cost-efficient way of preclinical testing. Those studies utilize cell, tissue, and organ cultures, or focus on particular cell components such as proteins or other biological macromolecules. In vitro studies permit tight control and monitoring of experimental settings and often provide mechanistic evidence for the investigational compound’s mode of action. While having the potential to provide mechanistic insights, in vitro models are constrained by the fact that isolated cells may not behave in a petri dish as they would within the body where they partake in crosstalk and interaction with millions of other cells. Consequently, more sophisticated preclinical models are required to establish the investigational compound’s safety profile before transitioning to a clinical setting.

In vivo models (is the mouse the best experimental animal?). In vivo studies consider the complete organism based on various animal models. The choice of appropriate animal models depends on myriad criteria and requires understanding of species-specific physiology and similarity with regard to the target organ, metabolic pathways as well as financial, regulatory, and ethical considerations. Typically, in vivo studies are performed in a rodent (e.g, mouse, guinea pig, hamster) and non-rodent model to comply with FDA requirements. Mice, rats, and dogs are among the most frequently used animal models while testing in primates (e.g., monkeys, apes, etc.) is performed occasionally and typically for larger molecules.

Discovering and developing a new therapeutic can take 10-15 years, on average, with costs often exceeding $1 billion. For every 10,000-15,000 compounds initially evaluated, only five advance to human testing, and only one is ultimately approved for commercialization. The increasingly complex drug development process requires therapeutic expertise, advanced technological capabilities, and familiarity with the increasingly complex regulatory process. As the time and resources needed to develop new compounds rises, jobs that used to be performed by biopharmaceuticals in-house laboratories are increasingly being outsourced to CROs that can complete them 30% more quickly. With the enormous costs at hand and risks involved throughout the process, biopharmaceutical companies, understandably, seek outsourcing partners that possess the necessary expertise and scale to maximize the chances of ultimate approval, navigate the regulatory hurdles, compress the development timeline where possible, and produce a quality end-product with a successful clinical trial. This generally positions larger CROs more favorably than smaller providers, given their broader therapeutic expertise and global footprints, regulatory expertise in numerous geographies, and advanced technological capabilities. In addition to discovering and developing new compounds, biopharmaceuticals also often task CROs with improving existing drugs. The advantages of CROs in this process can save biopharmaceutical companies three to five months’ time and generate $120-150 million more revenue. (Wilson, Willoughby, & Wallach, 2016)

Pre-Discovery. At this early stage, researchers attempt to understand the causes of a disease at a molecular level and identify diseases that new therapeutics could potentially target. Recent advances in molecular medicine and powerful technological tools that enhance computational capacity greatly improve the efficiency of this process and enable researchers to better understand human diseases at the molecular level. Biopharmaceutical companies often perform basic research independently, as well as in partnership with external researchers and academic institutions.

Drug Discovery. The ultimate goal of the drug discovery phase is to find a promising molecule, or lead compound, that has the potential to become a new medicine. Researchers assess the underlying disease pathway and identify potential target compounds, narrowing the field of compounds to one lead compound that shows potential to influence the target. Researchers can create a molecule from living or synthetic materials and using high-throughput screening techniques, select a few promising molecules from an initial pool of as many as 10,000-15,000 compounds. Researchers can also identify compounds found in nature or genetically engineer living systems to produce disease-fighting molecules.

Preclinical Research (Pre-human). Relevant compounds are tested in-vitro (test tubes) and in-vivo (animals) over a wide range of doses to establish relative toxicity of the compound and detect any potential adverse reactions to the therapeutic. If results of preclinical research indicate that the compound is safe and potentially effective, the sponsor submits initial study results to the FDA along with a complete Investigational New Drug Application (IND). An IND includes, among other things, preclinical study data, Chemistry, Manufacturing and Controls (CMC) information, and an investigational plan for clinical trials, and it must become effective before proceeding to clinical trials. An IND automatically becomes effective 30 days after receipt by the FDA, unless the FDA raises concerns relating to proposed clinical trials within the 30-day time period, in which case the FDA’s concerns must be addressed before clinical trials can commence. Before clinical trials can begin at a study site, the site’s Institutional Review Board (IRB), an independent expert body charged with protecting patient safety and privacy, must give their approval, separately from the IND submission.

Clinical Trials. Of the 250 compounds that advance to preclinical testing for a particular project, only five, on average, progress to clinical (human) testing. Clinical trials are completed to determine the safety and efficacy of a drug. Clinical trials can last six to seven years and comprise Phases I-III, with Phase IV or post-commercial marketing studies often required by the FDA as well. These trials often involve the use of placebos, where some subjects receive the new drug candidate and others receive an alternative treatment (placebo), with randomization (patients are randomly selected to receive either the actual compound or a placebo) and double-blinded protocols (where neither the researcher or subject know which patients are given the actual drug candidate or a placebo) in order to minimize biases. We describe the primary clinical testing phases in more detail below.

Phase I: During the earliest phase of clinical trials, testing is focused on basic safety and pharmacology, typically completed using 20 to 100 healthy human volunteers, though sometimes stable patients that exhibit the targeted disease are also included. Inpatient studies often take place at specialized research centers known as a Clinical Pharmacology or Clinical Research Units (CPU or CRU). These studies evaluate human metabolic and pharmacologic reactions to the compounds, the duration of effectiveness and activity, how it is affected by other drugs, how it is tolerated and absorbed, and how it is broken down and excreted from the body. Multiple dosage ranges and methods are analyzed, with side effects also carefully monitored. Once these studies are completed with satisfactory results, testing of efficacy is commenced.

Phase II: Sometimes referred to as proof-of-concept studies (POC), this stage of clinical testing focuses on basic efficacy, with dose-range testing completed in 100 to 500 patients afflicted with the targeted disease or condition. During this stage, though the primary focus is efficacy evaluation, further safety testing is also completed, along with determination of optimal dosage levels, dosage schedules, and administration routes. If Phase II studies yield satisfactory results, Phase III can commence, provided that no hold is placed on further studies by the FDA. It is during Phase II that the majority of drugs under development fail.

Phase III: Trials at this stage are completed at a larger scale, across multiple testing centers, in populations of 1,000-5,000 patients afflicted by the target disease. During this stage, advanced efficacy and safety testing is completed in order to provide enough data for valid statistical conclusions required by the FDA and other relevant regulatory bodies, as well as to provide an adequate basis for product labeling, optimal dosage, formulations, and administration methods. This stage is typically the longest and most expensive phase, and two successful Phase III trials demonstrating a drug’s safety and efficacy are often required to obtain FDA approval. Roughly 50% of drugs that enter Phase III testing fail. Once Phase III test results are approved by the FDA, the drug sponsor can submit a new drug application (NDA) or biologics license application (BLA), depending on the nature of the compound and disease.

FDA Review. After determining that the results of the clinical trials indicate that the compound is safe and effective, the sponsor submits an NDA or BLA to the FDA requesting approval to market the drug. Included with the NDA or BLA submission are the comprehensive testing results and supporting data and analysis from both preclinical and clinical testing, along with proposals for manufacturing plans and labeling. These applications are often over 100,000 pages. There are strict protocols that govern the submission process, and failure to abide by them can be grounds for rejection.

Post-Marketing Surveillance and Phase IV Studies. Once a drug is approved by the FDA, the agency often requires the sponsor to collect and periodically report additional safety and efficacy data to the FDA. At times, this can occur throughout the entire marketed lifespan of the product. If the product is marketed internationally, surveillance reports must include data from all countries in which the drug is sold. The FDA may require additional studies (Phase IV) even following approval, to test the compound for other potential indications, or new dosage formulations. The FDA and other regulatory agencies may also require license holders to prepare risk management plans that assess areas of product risk and plans to actively manage such risks.