Established Drug Modalities Part 3: Protein and peptide modalities
Article summary
Peptide and protein therapeutics have revolutionized medical treatment, from life-saving insulin to modern blockbuster weight-loss drugs like Ozempic and Wegovy.
These biopharmaceuticals offer unique therapeutic advantages such as high specificity and complex and diverse mechanisms of action, ranging from enzyme replacement therapies to immunomodulatory drugs and hormones, contributing to their therapeutic success.
Despite challenges like limited oral bioavailability and immunogenicity, advancements in peptide chemistry and protein engineering are continually expanding their therapeutic scope and range of medical indications.
The application of peptide and protein drugs extends to personalized medicine and targeted therapies, underscoring the versatility of these established drug modalities.
Peptide Therapeutics: From Life-Saving Insulin to Blockbuster Weight-Loss Drugs
Peptide therapeutics have a rich history, initially finding success in treating hormone deficiencies. The discovery of insulin, a 51 amino acid peptide, and the subsequent development of strategies to make it available to patients, is one of the monumental achievements in drug discovery. In 1923, insulin became the first commercially available peptide drug (developed by Eli Lilly), and its discovery was awarded with a Nobel Prize in Physiology or Medicine, paving the way for the successful development and FDA approval of dozens of peptide drugs for treatment of various disease conditions, including diabetes, cancer, infectious, and cardiovascular diseases. Notable examples of peptide therapeutics include:
Human glucagon-like peptide 1 (GLP-1) analogs Trulicity, Victoza, and Ozempic for diabetes
Human glucagon-like peptide 2 (GLP-2) analog Gattex for short bowel syndrome.
It is noteworthy that GLP-1 analog peptide drugs are also used for weight loss and Ozempic and Wegovy (developed by Novo Nordisk) have both reached a blockbuster drug status.
What is a blockbuster drug? A blockbuster drug refers to a drug that generates substantial and sustained commercial success by achieving exceptionally high sales revenue. Specifically, a blockbuster drug is characterized by annual sales exceeding the billion-dollar threshold, often propelled by broad demand and effectiveness in treating prevalent medical conditions. The term is commonly used in the pharmaceutical industry to describe drugs that significantly contribute to the financial success of the pharmaceutical company that develops and markets them.
Diverse Origins of Peptide Therapeutics: From Natural Sources to Innovative Designed Peptides
Peptide therapeutics derive from various sources. Hormones serve as foundational contributors, exemplified by insulin. Additionally, other endogenous peptides, originating within the human body, have also been used as a basis for the design and development of peptide therapeutics. Natural products, including peptides derived from plants, microbes, and animals, present a broad range of bioactive compounds that have fueled peptide drug discovery and development. Advances in protein engineering have enabled the de novo design of peptides tailored to specific targets. The multifaceted origins of peptide therapeutics underscore the interdisciplinary nature of peptide drug discovery, drawing inspiration from hormones, nature, and innovative engineering approaches.
Navigating the Advantages and Limitations of Peptide Therapeutics
Peptide therapeutics, as a distinct drug modality, have unique therapeutic advantages. They have a typical molecular size in the range of 500 – 5000 Da and are typically made of 5 to 50 amino acid residues linked in linear chains. Because of their structural properties, peptides present interaction surfaces that can be highly complementary to relatively shallow binding sites on targets that usually cannot be engaged by small molecule drugs. As a consequence, peptides can effectively block protein-protein interactions, a property that distinguishes them from a typical small molecule drug.
Structure is essential for activity and various approaches have been developed to stabilize the functional structure of peptides. For example, stapled peptides include a covalent linkage of side chains to stabilize helical peptide structures, disulfide bonds can be engineered into the peptide sequence to form a stabilized beta-sheet structure, and peptide cyclization, via the formation of covalent bonds between the peptide termini or between side chains, can stabilize non-canonical peptide structures.
Despite their advantages, peptides have limitations similar to the limitations of mAbs and, more generally, of protein therapeutics. Peptides face challenges such as limited oral bioavailability and cell membrane crossing. Limited oral bioavailability is due to the susceptibility of natural peptides to enzymatic degradation when administered orally. Because peptides are typically highly polar, they are also generally unable to effectively cross the cell membrane and enter cells, limiting their mechanism of action to cell surface targets. Nevertheless, recent advances in peptide engineering–modifications such as hydrocarbon stapling, cyclization, and introduction of disulfide bonds–not only increase the stability of peptides, enabling the development of oral peptide drugs, but can also enhance their cell membrane permeability which allows peptide drugs to engage intracellular targets.
What is oral bioavailability? Oral bioavailability of a drug refers to the proportion of a drug that reaches systemic circulation when administered orally. It is an important factor in determining the effectiveness of a drug, as it influences the dosage required to achieve therapeutic levels in the bloodstream. Limited bioavailability, caused for example by the drug being metabolized quickly, means that less of the drug reaches systemic circulation, reducing its effectiveness.
Another challenge that peptide therapeutics face is renal clearance. Peptides, being relatively small molecules, can be filtered by the kidneys and excreted from the body, potentially decreasing their time in systemic circulation. Various strategies, including modifications and special formulations, can be used to mitigate the challenges associated with renal clearance in order to enhance the effectiveness of peptide therapeutics.
What is renal clearance? Renal clearance is the process by which the kidneys remove molecules from systemic circulation in the body. Renal clearance plays a central role in determining the efficacy of a drug since rapid clearance limits the exposure of target tissues and organs to a drug. Decreasing renal clearance allows a drug to circulate in the body for longer, increasing the likelihood of reaching targets and exerting therapeutic effects.
From Chemical Synthesis to Recombinant Expression for the Production of Peptide Therapeutics
Therapeutic peptides can be produced via various approaches, with the peptide characteristics largely defining the approach of choice. Chemical synthesis is widely adopted for the production of relatively short to medium-sized peptides. This approach allows for unnatural amino acids to be incorporated, facilitating the production of chemically modified peptides, and the process can be automated, enabling the effective production of large peptide quantities. Peptides can also be produced recombinantly in expression hosts, and this is typically required for relatively long peptides or for peptides with multiple disulfide bonds, which cannot be easily synthesized via chemistry. Chemical ligation can be used to combine shorter peptides, obtained via either chemical synthesis and/or recombinantly, into longer and more complex peptides carrying unnatural amino acids and other modifications.
What are unnatural amino acids? Unnatural amino acids are synthetic amino acids not found in the standard 20 amino acids encoded by the genetic code. They can be incorporated into peptides and protein to enable additional chemical modifications that enhance therapeutic properties like stability, specificity, and efficacy.
Exploring the Unique Properties of Protein Therapeutics
The first recombinant protein drug, human insulin, received approval from the FDA in 1982. Genentech, in collaboration with Eli Lilly, developed recombinant insulin, which was a groundbreaking achievement in biotechnology because it showcased the successful application of recombinant technology for the production of therapeutic proteins. The development and commercialization of recombinant insulin laid the foundation for the subsequent development of recombinant protein therapeutics.
What is a recombinant protein? Proteins obtained via recombinant technology are referred to as recombinant proteins. Recombinant technology involves genetically modifying organisms like bacteria or yeast to produce specific proteins. This method starts with inserting a gene, which codes for the desired protein, into a host organism using a vector. The host then uses its own cellular machinery to produce the protein encoded by the inserted gene. This technique allows for large-scale production of therapeutic proteins like human insulin and various therapeutic antibodies.
Because proteins perform a vast range of functions in cells, their use as biologic therapy covers a broad range of medical indications. The following examples illustrate the versatility of protein therapeutics.
Antibodies are perhaps the largest class of protein drugs. Their predominant mechanism of action is to bind cell surface proteins and thus block signaling pathways or induce cell death. Antibodies can also be used to deliver cytotoxic molecules to cancer cells. Since the first therapeutic antibody was approved in 1986, antibodies have been a highly successful therapeutic modality, with over 100 distinct entities available to patients. (Established Drug Modalities Part 2: Monoclonal Antibodies provides a historical perspective on monoclonal antibodies).
Enzymes catalyze the reactions required for the functioning of cells. Enzyme therapeutics are typically used in replacement therapies to substitute a deficient or absent enzyme activity. Dozens of enzyme replacement therapies have been approved since 1991 when the FDA approved this drug modality for the first time. Replacement therapies continue to be developed for the treatment of various disease conditions, for example, the recent FDA approval of Lamzede, brining hope for patients with rare genetic conditions.
Hormones are signaling molecules that regulate physiology. Because many hormones are proteins, recombinant hormones can be used to correct hormone deficiencies, for example insulin for diabetes (see sections above for a historical perspective and overview of insulin as a drug).
Cytokines are a class of small proteins involved in signaling and immune response. Recombinant cytokines can be used in a variety of indications, with their most frequent use being as immunomodulatory drugs. The first cytokine therapy approved by FDA was interferon alfa-2b. It received approval in 1986 for the treatment of leukemia and, over time, received approvals for additional indications, including certain cancer types and virus infections.
What is an immunomodulatory drug? Immunomodulatory drugs are therapeutics designed to modify the immune system's response. They typically work either by enhancing (immunostimulants) or suppressing (immunosuppressants) the body's immune activity. These drugs are commonly used to treat autoimmune diseases, where they reduce harmful immune responses, and in cancer therapy, where they stimulate the immune system to target cancer cells more effectively. Their application extends to a wide range of conditions with an immunological basis.
Coagulation factors are proteins involved in blood clotting. As therapeutics, recombinant coagulation factors typically correct clotting-related deficiencies in patients. Currently there are a handful of FDA-approved coagulation factor therapeutics.
Protein Therapeutic Modalities: Large Molecules with Complex Structure and Diverse Mechanisms of Action
Protein therapeutics, a cornerstone of biologic therapy, are typically significantly larger than small molecule drugs and peptide therapeutics, with molecular weights of up to 150,000 Da in the case of monoclonal antibodies (mAbs). Because of their large size, protein therapeutics have complex structures and can be composed of multiple domains with various functions and modes of action. The large interaction surfaces of protein therapeutics are made of numerous protein groups that typically make many interactions with their targets, bestowing high target specificity on them. The ultimate example is the tremendous specificity with which mAbs recognize their targets. Because of their large interaction surfaces, proteins can bind to relatively shallow binding sites with high affinity, which enables them to effectively block protein-protein interactions, a property that differentiates them from a typical small molecule drug.
The complex structure of proteins enables them to exert more diverse and complex functions than small molecule drugs and peptides. For example, in addition to recognizing their targets with high affinity and specificity, enzyme modalities can be used to restore catalytic activities that are deficient or lacking in patients while hormone modalities can correct hormone deficiencies.
Advancements in our understanding of how protein sequences relate to protein function and recent progress in computational modeling approaches have enabled the de novo design of proteins with desired and novel functions, paving the way for development of designer protein drugs and their application in personalized medicine.
Navigating and Overcoming the Limitations of Protein Therapeutics
While protein therapeutics have a number of advantages over other drug modalities, they also have unique challenges and limitations. Proteins are inherently susceptible to denaturation, degradation, aggregation, and rapid clearance from the body, all of which typically lead to loss of activity. Further, because proteins are typically large molecules with heterogeneous and polar surfaces, most proteins cannot effectively penetrate the cell membrane, limiting the range of therapeutic targets to cell surface proteins.
Proteins are composed of linear chains of amino acid residues the folding of which produces the complex protein structure required for activity. The protein structure is held together by a large number of relatively weak interactions, making the majority of proteins modestly stable. Changes in temperature or in environmental physicochemical properties can cause changes in protein structure and denaturation, resulting in loss of activity.
The peptide bonds that link protein amino acid residues together in linear chains are susceptible to cleavage by enzymes in the physiological context of the human body. The cleavage of protein peptide bonds eventually leads to protein degradation and loss of activity.
Proteins have complex surfaces that confer them with exquisite specificity for their targets. However, the complexity of their surfaces can also lead to non-specific protein-protein interactions and, in certain cases, aggregation. Partially and fully denatured proteins are exceptionally susceptible to aggregation. Regardless if in their native state or denatured, aggregated proteins cannot carry on their function, resulting in loss of activity.
Kidneys filter molecules based on their size. Because most proteins are not large enough to be retained by the kidneys, and because proteins do not typically penetrate cells, protein drugs are quickly removed from systemic circulation via renal clearance.
The large size of protein therapeutics also contributes to their immunogenicity: proteins are susceptible to triggering the body’s immune response. A typical immune response is the production of antibodies against the protein therapeutic. Antibodies bind to the protein and reduce or eliminate their activity.
Advancements in protein engineering and chemical modifications enable the ability to circumvent some of these limitations and improve the properties of protein drug modalities. For example, the attachment of polyethylene glycol chains to the protein surface, or fusion of the therapeutic protein to other proteins, for example to human serum albumin, can reduce kidney clearance rates and make proteins less susceptible to degradation and aggregation, while glycosylation–the attachment of sugar chains to the protein surface–can also reduce immunogenicity.
Advancements in computation tools allow the properties of therapeutic proteins to be optimized. For example, protein stability and solubility can be increased and immunogenicity can be decreased, by specific changes in the protein amino acid sequence that leads to changes in the protein surface. Not only can protein function be enhanced via computational approaches, but proteins with desired functions can be designed de novo. Notably, computational design tools now allow for the design of new proteins that can bind specific targets with high affinities. Applying de novo computational protein design approaches to generate protein therapeutics that specifically recognize disease-causing mutated protein variants also paves the way for the use of protein therapeutics in personalized medicine. Importantly, protein engineering has also enabled the design of proteins that can more effectively cross the cell membrane, laying the foundations for the design and development of protein therapeutics that can engage intracellular targets.
Therapeutic Proteins are Typically Recombinant
Therapeutic proteins are most often produced recombinantly in expression hosts. While certain proteins, such as insulin, do not require specific modifications (e.g., glycosylation) for their function and can be produced in prokaryotic expression hosts, many therapeutic proteins require such modifications for activity and to reduce immunogenicity and need to be produced in eukaryotic hosts. For example, while mAbs were first produced using the hybridoma technology, they are now predominantly produced recombinantly in mammalian cells.
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