Biochemistry in Perspective 101: Advances and Prospects in Biochemical Research

Foreword

Biological organisms, such as humans and their individual cells, are incredibly complex and diverse systems. Nevertheless, certain unifying characteristics exist in all living things, from the simplest bacterium to the human being. The same types of biomolecules are present and they all use energy to function. These molecules are known as proteins, lipids, glycans, and nucleic acids. From the construction, modification, and interaction of these components, our cells develop and carry out specific functions. Biochemistry draws on a wide range of scientific disciplines to explore and study these molecules, cells, and functions. This has sequentially allowed us to gain a better understanding of the human body at the molecular level, which has led to more effective treatments in medicine. We will explore biochemistry in the context of this series, which examines the role it has played and will continue to play in our daily lives. In a nutshell, all life is the embodiment of biochemistry, and everything a living organism does is an expression of a biochemical process.

This 101 series is divided into six articles, including:

1. Biochemistry in Perspective 101: Amplifying Biochemistry Concepts

2. Biochemistry in Perspective 101: Biochemistry of Metabolism in Health and Disease

3. Biochemistry in Perspective 101: Applications of Biochemistry in Daily Life

4. Biochemistry in Perspective 101: Natural Biochemical Cycles

5. Biochemistry in Perspective 101: Biochemistry and its Applications in Drug Development

6. Biochemistry in Perspective 101: Advances and Prospects in Biochemical Research

Biochemistry in Perspective 101: Advances and Prospects in Biochemical Research

Biology and chemistry are interrelated in biochemistry, which delves into the intricate molecular processes that govern life. The field of biochemistry is continually evolving with the progress of technology. Our understanding of cellular mechanisms continues to deepen, revealing new layers of complexity and opening up new research opportunities. In this dynamic landscape, several trends and topics have emerged, shaping the trajectory of biochemistry research.

Genome Editing and CRISPR Technology

The emergence of CRISPR/Cas9 technology [Figure 1] has radically changed functional genomics and genetic engineering. The CRISPR/Cas9 is a gene-editing technology that involves two components: a guide RNA for targeting a specific gene, and Cas9, an endonuclease that breaks double-stranded DNA, allowing modification of the genome. Biochemists are utilizing this powerful tool to manipulate and precisely edit genetic material, enabling the investigation of gene function and regulation. In addition to therapeutic genome editing, CRISPR-based techniques can be used to produce genetically modified organisms for industrial and agricultural use. The use of CRISPR-based technology is exciting due to its potential to be used to treat genetic disorders caused by single gene mutations such as cystic fibrosis (CF), Duchenne’s muscular dystrophy (DMD) and haemoglobinopathies to name a few (Redman et al., 2016).

Figure 1: CRISPR/Cas9 technology depicted (Redman et al., 2016).

This family of DNA sequences, called "clustered regularly interspaced short palindromic repeats (CRISPR)", are found in the genomes of prokaryotes, such as bacteria and archaea. These sequences when combined with CRISPR - associated (Cas) proteins, afford protection against invading viruses, by detecting and destroying DNA from similar bacteriophages. These sequences play crucial roles in developing antiviral defence systems and building up a form of acquired immunity. CRISPR is found in approximately 50% of sequences in bacterial genomes and nearly 90% of sequences in archaeal genomes (Redman et al., 2016). Cas9, one of the associated proteins, is an endonuclease that cuts strands of DNA. In the CRISPR-Cas9 method, specific strands of DNA, that are complementary to CRISPR sequences, are recognized and opened up. The Cas9 protein can be used to find and bind to almost any desired target sequence, simply by adding a piece of RNA to guide it in its search. As soon as the CRISPR-Cas9 sequence finds the desired sequence, it can alter, delete, or change that particular DNA sequence.

The mechanism of CRISPR involves DNA repair. There are two main ways in which DNA repair can occur: through homology-directed repair (HDR) and non-homologous end joining (NHEJ). The HDR procedure uses exogenous DNA, that has a similar sequence to that of the broken DNA, it then functions as a template for repair to take place. If there is no template available, because the Cas9 protein was designed to cut rather than edit, then non-homologous end joining (NHEJ) would take place, where the cell fixes the damage by simply sticking the ends of DNA together. This technique was developed and discovered by Emmanuelle Charpentier and Jennifer Doudna [Figure 2] who received the Nobel Prize in Chemistry in 2020 for their work in gene editing technology (Xue & Greene, 2021).

Figure 2: Emmanuelle Charpentier and Jennifer Doudna (RFJ, 2020).

Gene Therapy

The use of genes to treat, prevent, or cure a disease or medical disorder is known as gene therapy. It uses the manipulation of gene expression to produce a therapeutic effect. Gene therapy often relies on adding new copies of a gene that is broken or replacing a missing or defective gene with a healthy alternative of that gene (Gonçalves & Paiva, 2017). Both sickle cell anaemia and haemophilia, inherited genetic diseases have been treated by means of gene therapy (Herzog & Hagstrom, 2001), as well as some acquired disorders such as leukaemia. The first successful nuclear gene transfer was conducted in 1989 and approved by the National Institute of Health (NIH). The NIH is the primary agency of the United States government, responsible for biomedical and public health. However, the first attempt to modify human DNA was made long before this by Martin Cline in 1980. Since then, over 2900 clinical trials have been conducted using gene therapy, with half of them in phase I. Gendicine, the first regulatory-approved gene therapy medication, is being used to treat cancer patients with cell carcinoma. Since the approval of Gendicine in 2003, several more gene therapy treatments have come to market including Glybera (treatment designed to reverse lipoprotein lipase deficiency), Strimvelis (treatment for people with severe combined immunodeficiency), and more recently Abecma (treatment for multiple myeloma), Adstiladrin (treatment for bladder cancer), Roctavian (treatment for haemophilia A) and Hemgenix (treatment for haemophilia B). These approaches utilize adeno-associated viruses (AAV) and lentiviruses for performing gene insertions. It is important to note that not all gene therapy treatments involve altering a patient's genetic makeup. Some approaches, such as bone marrow transplantation and organ transplantation involve the introduction of foreign DNA (Cross & Burmester, 2006).

As of 2017, most trials involving gene therapy were for cancer treatment. They mostly utilize adeno-associated viruses, because adenovirus vectors are safe and cannot make copies of themselves without outside help. Adeno-associated viruses (AAV) play a crucial role in gene therapy for treating cancer. In this innovative approach, AAV vectors are employed to deliver therapeutic genes into targeted cells with precision [Figure 3 & Figure 4]. The AAV vector serves as a carrier, transporting the therapeutic genetic material to the cancerous cells while minimizing the risk of adverse effects on healthy tissues. The selected therapeutic genes are designed to either replace or supplement the function of mutated or malfunctioning genes within the cancer cells, thereby hindering their ability to grow and divide uncontrollably. This targeted intervention aims to inhibit tumour progression and, in some cases, induce apoptosis, or programmed cell death known as suicide gene therapy, in the cancer cells (Cross & Burmester, 2006). The use of AAV in gene therapy for cancer underscores the potential for a more targeted and personalized treatment approach.

Figure 3: AAV vectors as a platform for gene therapy (Wang et al., 2019).

Another focus for gene therapy has been monogenic diseases (Boudes, 2014). Monogenic diseases are inherited conditions arising from mutations in a single gene. One area where this is being explored is retinal gene therapy. Leber’s congenital amaurosis, a rare genetic retinal disease has been successfully treated with Luxturna, an approved AAV gene therapy, in patients with the disease. Similarly, Zolgensma, another AAV vector gene therapy, has been developed to treat spinal muscular atrophy, a rare neuromuscular disorder that results in the loss of motor neurons and progressive muscle decline. It is often diagnosed in infancy and, if left untreated, can result in death. One other potential focus for the use of AAV vector gene therapy is sickle cell disease, which is caused by autosomal recessive disorders. However, the risks and benefits related to gene therapy being used for sickle cell disease are not yet known (Boudes, 2014).

Targeting infectious diseases such as HIV, hepatitis and malaria has been another focus for gene therapy since 2017 (Strayer et al., 2005). Several trials have already been conducted on these utilizing gene therapy, however many concerns and problems still need to be addressed. Implementing gene therapy for infectious diseases poses notable challenges. One primary concern is the adaptability of pathogens, particularly viruses, which can undergo rapid mutations, potentially leading to the emergence of resistant strains that may evade the therapeutic effects of gene interventions. Precision in targeting infected cells while sparing healthy ones remains a complex task, as off-target effects could have unintended consequences. The immune system's response to viral vectors used in gene therapy is a critical obstacle, as it may hinder the delivery of therapeutic genes or trigger adverse reactions. Balancing the need for a robust immune response against the infectious agent with the potential risks of exacerbating inflammation or unintended immune reactions requires careful consideration. Furthermore, ethical considerations surrounding the alteration of the human genome for infectious disease treatment and concerns about long-term safety and unforeseen consequences add layers of complexity to the development and application of gene therapy in infectious disease scenarios. There are significant economic barriers to widespread accessibility and adoption of certain gene therapies, such as Glybera, alipogene tiparvovec, which amounts to a cost of $1.6 million per patient (Libby & Wang, 2014).

Figure 4: Depiction of how gene therapy works (National Human Genome Research Institute. n.d.).

Structural Biology and Cryo-EM Advancements

Recent years have witnessed remarkable strides in structural biology, driven by cutting-edge techniques such as cryo-electron microscopy (Cryo-EM). Researchers are now able to visualize biomolecules at unprecedented resolutions, unravelling the three-dimensional intricacies of proteins, nucleic acids, and complexes. This capability not only enhances our understanding of fundamental cellular processes but also holds promise for drug discovery and design.

Max Knoll and Ernst Ruska [Figure 5] designed and invented the first transmission electron microscope in 1931. In 1933, they became the first scientists, who were able to break through the resolution limit of the optical microscope and see particles that were even smaller and never before reported. This imaging technique is performed in a high vacuum under radiation conditions, which ultimately causes damage and destruction to biological samples. The following decades saw scientists imaging biological samples through heavy metal staining, and in 1968, De Rosier and Klug reported the first electron microscope structure of the T4 phage tail (Coombs & Eiserling, 1977; Smith et al., 1976). Taylor and Glaeser proposed the use of cryo-electron microscopy imaging in the hope of reducing the damage caused by radiation during the microscopy process. In 1981, Jacques Dubochet and his colleagues introduced the method of rapid freezing, making a breakthrough in electron microscopy technology. This solved the problem of high vacuum and radiation damage by keeping the biological material in its natural state and preventing dehydration.

Figure 5: Max Knoll and Ernst Ruska (Delta Microscopies, n.d.).

During cryo-electron microscopy, the biological material undergoes rapid freezing at temperatures of liquid nitrogen (-180°C), followed by the bombardment of the biological material with electrons, which pass through a lens and create a magnified image on the detector [Figure 6]. The structure is then analyzed and constructed. Cryo-electron microscopy has two main parts. The first is defined as single particle analysis, where 3D structures are created from 2D projections of the same biological sample. The 3D structure is constructed by means of various 2D images and image processing algorithms. The second part is called cryo-electron tomography, which involves capturing multiple images of a single biological sample by tilting the sample at various angles. The electron beam penetrates the sample at these angles which ultimately creates the 3D structure.

Other spectroscopy techniques include X-ray crystallography and nuclear magnetic resonance spectroscopy (NMR). These have limited applications and require large sample sizes. X-ray crystallography also requires the crystallization of samples, whereas cryo-electron microscopy does not require either of these and is suited for imaging at near-atomic resolution. These have all been possible through the advancement of technology and image-processing algorithms. Most viruses, proteins, and ribosomes are now studied utilizing cryo-electron microscopy. One application includes the observation of the structural changes that occur in the p97 protein. The p97 protein is a valosin-containing protein (VCP) or transitional endoplasmic reticulum ATPase (TER ATPase), and its main function is to segregate protein molecules from larger cellular structures such as protein assemblies. It is also thought to be an important target for cancer cell treatment. As a result of the advanced imaging capabilities of cryo-EM, the type of p97 inhibitor binding and contact sites have been observed. With advances in detector technology and sample preparation currently underway, cryo-EM has the potential to further improve resolution and structure elucidation (Davies et al., 2005; Lan et al., 2017).

Figure 6: An example of a cryo-EM image (Medical News, n.d.).

Metabolomics and Systems Biology

The integration of metabolomics and systems biology approaches has become increasingly prevalent in biochemistry research. By comprehensively analyzing the complete set of small molecules in a biological system, researchers gain insights into the metabolic pathways and networks that govern cellular function. This holistic perspective aids in understanding the dynamic interplay of biochemical processes within living organisms. The emerging field of metabolomics has received widespread attention in the past decade. It delves into providing qualitative and quantitative analyses to study various mechanisms within different species.

The metabolite profile of a cell or a tissue is a direct reflection of its molecular phenotype, thus, their profiling can be clinically used in oncology. Tumor initiation, progression, metastasis, and resistance to therapies are all due to the ability of these cells to reprogram their metabolism (Palermo, 2023). Metabolic reprogramming refers to the ability of cancer cells to alter their metabolism in order to support the increased energy request due to continuous growth, rapid proliferation, and other characteristics typical of neoplastic cells. The field of cancer metabolism has seen a broad development of biochemical technologies, which are able to navigate the complexities that are brought forward in cancer research (Danzi et al., 2023).

The concept of targeting metabolism is not a new one; the first antimetabolite therapy dates back to 1947. Dr Sidney Farber [Figure 7], the father of modern chemotherapy, discovered that aminopterin could arrest tumour progression in paediatric acute lymphoblastic leukaemia (ALL) in 1947 (Danzi et al., 2023). The discovery of aminopterin, an antimetabolite, led to the development of an entirely new class of drugs. One of the biggest achievements in the field of cancer metabolism has been the introduction of antimetabolites. Methotrexate (MTX), 5-fluorouracil (5-FU), gemcitabine and cytarabine are all antimetabolite nucleoside analogues that have been widely used in cancer treatment over the past few years (Danzi et al., 2023; Palermo, 2023). A large amount of effort has been committed to understanding the metabolic reprogramming of cancer cells, but its complexity and technical limitations have prevented its full characterization. There is a multidisciplinary approach to cancer metabolism that sits at the interface between diverse technical-scientific fields, including biochemistry, immunology, genetics and microscopy. The ongoing technological advancements in metabolite characterization will bring great success in future endeavours of understanding cancer metabolism and developing effective cancer treatments.

Figure 7: Dr Sidney Farber (Dana Farber, 2013).

Proteomics and Post-Translational Modifications

The proteome of any cell is highly diverse, with proteins performing all the cellular tasks for the cell to stay alive. It is encoded by approximately 20000 genes but the functions are increasingly larger due to the complexities that arise due to genomic recombination, alternative transcript splicing or post-translational modifications. PTMs play a critical role in the regulation of protein functions, structures and cellular processes. Proteomics, the large-scale study of proteins, has evolved to encompass the intricate landscape of post-translational modifications (PTMs). Researchers are exploring how modifications such as phosphorylation, acetylation, and glycosylation influence protein function and cellular signaling. A better understanding of PTMs adds an additional layer of complexity to the regulation of biological processes and may have implications for pathology research (Hermann et al., 2022; Peng et al., 2023).

In biochemical terms, PTM refers to the covalent modification of proteins usually during or after protein synthesis. The modification process involves modifying or adding functional groups to the protein, some of which include: phosphorylation (adding a phosphate group), acetylation (adding an acetate group), glycosylation (the formation of an amide or ether bond), methylations (addition of a methyl group), ubiquitination (controlling substrate degradation), lipidation and proteolysis [Figure 8]. The modification usually increases the functional diversity of the protein, which influences almost all aspects of normal cell biology and pathogenesis. It also has an effect on various cellular processes such as cellular differentiation, protein degradation, signalling, regulatory processes, gene expression and protein-protein interaction. This is why identifying and characterizing PTMs are critical in studying cell biology, disease diagnostics and disease prevention. As might be expected, the identification of PTMs is a tedious process (Peng et al., 2023). Most PTM are present in low quantities and require amplification of the proteins to be performed before identification can take place, which is also limited by mass spectroscopy and the stability of the proteins. In spite of increasing awareness of the impact of post-translational modifications, their reliable detection and quantification remain a major hurdle in translating these novel pathological markers into clinical diagnosis. PTMs have been associated with the onset and progression of various diseases including cardiovascular disease, renal, cancer and metabolic diseases (Peng et al., 2023).

Figure 8: Strategies for post translation modifications (Creative Proteomics, n.d.).

One therapeutic area for targeting PTMs associated with human diseases is through upstream regulators such as kinases, acetyltransferases and methyltransferases. Multiple upstream regulators (inhibitors/agonists) are currently being used as therapeutic treatments and some are in clinical development. However, one major drawback of upstream regulators is that they do not only control the PTMs of disease-related proteins but also of irrelevant substrate proteins. This may introduce unwanted off-target toxicity making the use of these treatments limited. As a result, alternative drugs that solely regulate the specific PTM of the associated disease-related protein have been explored. Chemically induced proximity (CIP) is one such research tool providing such promise. Chemical inducers of proximity have successfully been used to target and regulate protein ubiquitination, phosphorylation, acetylation and glycosylation (Stanton et al., 2018). Several CIP approaches that facilitate targeted protein degradation (TPD), including PROTAC, and molecular glue degraders (MGDs) are currently being used in clinical trial investigations (Duran-Frigola et al., 2023). The potential of CIP research tools opens up a new avenue for treatment methods for diseases that have been related to protein PTMs.

Synthetic Biology and Bioengineering

The idea of synthetic biology was first introduced in 1910 by Stephane Leduc, [Figure 9] (Carrà & Carrà, 2018). Synthetic biology can be thought of as the combination of biochemistry and bioengineering. Researchers are designing novel biological systems, synthetic organisms, and metabolic pathways for applications in medicine, energy, and environmental remediation. This field has witnessed a shift in research approaches, moving away from merely describing and analyzing biological events to actively designing and manipulating desired signal/metabolic pathways, akin to established organic synthesis methods. Presently, synthetic biology has undergone substantial development, evolving into a multidisciplinary field with the goal of creating new biological components, systems, or even organisms based on existing knowledge. Researchers employ an engineering paradigm to craft predictable and robust systems with novel functionalities not found in nature. The integration of synthetic biology spans various subjects, including biotechnology, biomaterials, and molecular biology, offering methodologies and disciplines that contribute to advancements in these fields.

Figure 9: Stephane Leduc (Wikipedia, n.d.).

In recent decades, significant advancements have been made in the field of synthetic biology. These advancements include the construction of delicate biocircuits, the standardization of biological building blocks, and the development of genomic/metabolic engineering tools and approaches. The progress in synthetic biology has been driven by the demands of the medical and pharmaceutical industries. One of the key applications of synthetic biology in these fields is the integration of heterologous pathways into designer cells, enabling the efficient production of medical agents. Additionally, synthetic biology has facilitated the enhanced yields of natural products in cell growth media, rivalling or surpassing those obtained from plants or fungi. Furthermore, novel genetic circuits have been constructed for tumour targeting, allowing for more precise and effective treatments. Synthetic biology has also led to the development of new strategies for treating complex diseases, such as diabetes and cancers, by enabling controllable releases of therapeutic agents in response to specific biomarkers (Yan et al., 2023). Overall, synthetic biology has brought new capabilities to medical and pharmaceutical research, revolutionizing the field and opening up new possibilities for treatment and discovery.

Neurochemistry and Brain Function

The study of neurochemistry is gaining prominence as researchers seek to unravel the molecular underpinnings of brain function and neurological disorders. Advances in imaging techniques and molecular probes allow for a deeper understanding of neurotransmission, neural signalling, and the molecular basis of cognitive processes, offering deeper insights into neurodegenerative diseases. Using neuroimaging is now an essential tool for researchers studying neurological disorders to understand how the brain functions [Figure 10]. Neuroimaging techniques such as functional magnetic resonance imaging (fMRI) and electroencephalography (EEG) are commonly used to observe changes in brain activity (Friston, 2005). In fMRI, magnetic fields and radio waves are used to produce detailed images of the brain. EEG is a noninvasive technique used to record the brain's electrical activity through electrodes that are placed on the scalp. These techniques have contributed significantly to the understanding of brain function and related neurological disorders. However, further noninvasive techniques such as PET (positron emission tomography and computed tomography), MRI (magnetic resonance imaging), and TES (transcranial electrical stimulation) are still necessary to develop new diagnostic and therapeutic strategies for these disorders.

Figure 10: Neuroimaging techniques (The Cochran Firm, n.d.).

There are three main characteristics of autism spectrum disorders (ASDs), namely, impaired communication, impaired reciprocal social interaction, and restricted and repetitive behaviours (Muhle et al., 2018). Research has shown that autism is characterized by abnormalities in brain structure and function, mainly in areas associated with communication, social interaction, and behaviour sensory processing. The prevalence of ASDs in the United States extends to 1 in every 54 children (Maenner et al., 2020). According to the most recent research, there is no single cause or cure for this disease. However, there is growing evidence that neuroimaging studies can provide useful insight into the brain mechanisms underlying neurodevelopmental disorders, such as ASDs. Amygdalas, a brain region primarily associated with emotional processes, have been found in various studies to differ in size and shape between individuals with ASDs. Other studies have found structural and connectivity variations in the prefrontal cortex. The prefrontal cortex is mainly involved in executive functioning and social cognition.

Attention deficit hyperactivity disorder (ADHD) is another neuro-developmental disorder that affects approximately 3-5% of children and 2-4% of adults worldwide (Frank-Briggs, 2011). Neuroimaging has lent insight into the structure and function of certain areas in the brains of people who have ADHD compared to those of people who do not. Areas of the brain of interest include the prefrontal cortex, the basal ganglia, and corpus callosum which is associated with attention, motivation and motor control [Figure 11]. These affect an individual’s ability to pay attention, control impulsive behaviour and regulate activity levels (Frank-Briggs, 2011). The basal ganglia, which play an important role in motor control and reward processing, for example, have been found to be different in size and shape among different individuals. Additionally, other studies have found differences in attentional control and executive functioning in the prefrontal cortex. In general, neuroimaging studies have provided valuable insights into the brain mechanisms of neurodevelopmental disorders. The future of neuroimaging promises significant advancements in technology, enabling a deeper understanding of disorders and facilitating the development of appropriate treatments.

Figure 11: Areas of the brain investigated in ADHD (Get in Flow, n.d.).

Conclusion

These trends and topics demonstrate the endless potential and innovation opportunities in biochemistry research. Scientists continue to be captivated by the complex dance of molecules within living organisms, pushing the discipline into new horizons and transforming applications. Through structural biology, we are able to uncover the three-dimensional architecture of biomolecules, allowing us to design targeted drugs and therapies. We are now able to edit the genome with unprecedented precision thanks to CRISPR-Cas9 technology, enabling us to understand gene function and address genetic disorders more effectively.

The study of metabolomics and systems biology provides a holistic perspective on cellular processes, unravelling metabolic networks in their complexity. We gain a deeper grasp of cellular regulation and signalling through the study of post-translational modifications, enhancing our understanding of protein function. The principles of engineering are converged with the complexities of life through synthetic biology and bioengineering. Custom-designed biological entities hold promise for diverse applications, from medicine to environmental solutions. Neurochemistry, with its focus on brain function at the molecular level, has the potential to shed light on the mysteries of cognition, while addressing neurological disorders that impact millions worldwide. These trends represent an interdisciplinary and collaborative approach to biochemistry research. As chemistry, biology, physics, and engineering are integrated, the field continues to explore uncharted territory, fostering a deeper understanding of life's molecular intricacies.

In reflecting on the present state of biochemistry research, it is evident that the field is driven by curiosity and a desire to understand the molecular world. The world of biochemistry is advancing with every breakthrough, from fundamental biological knowledge to applications that can reshape our healthcare, and agriculture industries. Innovations in biochemistry, thanks to emerging technologies and novel approaches, will lead to a fundamental change in the future of biochemistry. In this growing field, scientists are not only expanding our understanding of life at the molecular level but also laying the foundation for addressing pressing challenges and improving human lives. We walk this scientific journey guided by the trends and topics in biochemistry research toward a future where the molecular intricacies of life are harnessed to improve society.

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