Targeted Anticancer Drug Discovery Using Molecular Diagnostic Proteins

Abstract

Cancer is the uncontrolled and abnormal growth of cells of the body. Every cancer is formed due to abnormalities in the DNA sequence which influences a cell to alter from its normal type to a cancerous cell. Pharmacotherapy of cancer still is a tough process to cure and manage the cancer because of its unwanted effect rather than the intended action. There is growing evidence that intratumoural heterogeneity plays a role in the emergence of anticancer drug resistance. Thus, targeted drug discovery needs a special care for the management of cancer. Tumours can shed certain proteins that enter the bloodstream which can be identified using blood or biopsy. Those proteins exist as biomarker for molecular diagnostics, predicting treatment response and monitoring treatment response and disease recurrence. More research on drug discovery in targeted therapy using genes like BRCA1, BRCA2 genes, P53 and CDKi2A with associated proteins like oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), prostate specific antigen, α-fetoprotein and carcinoembryonic antigen (CEA) will reduce the risk associated with former drugs of chemotherapy and also enhance the efficient treatment for cancer.

1. Introduction

Cancer is a serious worldwide health issue that is characterised by the body’s aberrant cells growing and spreading out of control. It has a significant effect on people, families and societies globally and is a main cause of sickness and mortality. The World Health Organisation (WHO) estimates that 9.6 million people died from cancer in 2018, making it the second highest cause of death worldwide. In order to effectively manage cancer, early identification and therapy are essential. The possibilities for diagnosis and therapy have significantly improved as a result of developments in cancer research and medical technology. These include operations, radiation treatment, chemotherapy, immunotherapy, targeted therapy and precision medical techniques. The prevention, early identification and treatment of cancer still face difficulties. Research efforts are still concentrated on figuring out the molecular processes that lead to the development of cancer, finding fresh biomarkers and creating novel treatments [1].

1.1 Targeted cancer therapies: Importance and impact

By offering more specialised and potent methods of treating the disease, targeted medicines have revolutionised the way cancer is treated. Whilst preserving healthy cells, these treatments directly target critical chemicals, genetic abnormalities or aberrant signalling pathways that promote cancer development and survival. Targeted treatments are significant because they can provide individualised therapy choices based on the unique molecular traits of each patient’s tumour [2]. Targeted therapies have demonstrated considerable therapeutic advantages in a variety of cancer types, enhancing patient outcomes and quality of life. In some circumstances, they can result in higher response rates, longer progression-free survival and even greater overall survival. Tyrosine kinase inhibitors (TKIs), which specifically block certain signalling pathways involved in cancer cell proliferation, are one type of targeted treatment. For instance, by precisely targeting the BCR-ABL fusion protein, medications like imatinib have changed the landscape of CML therapy [3].

Use of monoclonal antibodies, such as trastuzumab, which targets HER2 in breast cancer, that attach to certain proteins on cancer cells is another illustration. These antibodies have the power to obstruct signalling pathways, activate immune responses that attack cancer cells or deliver poisons right to tumour cells. Precision medicine has advanced significantly with the discovery and use of targeted treatments, which have made it possible to customise treatment plans for specific patients. It has also paved the way for combination treatments, which combine a number of targeted drugs or therapies in addition to other forms of care to boost therapeutic effectiveness [4].

1.2 The role of molecular diagnostic proteins in the discovery of targeted drugs

The identification of prospective therapeutic targets and the significant insights into the underlying processes of illness that molecular diagnostic proteins offer are key factors in the development of targeted drugs. These proteins can act as biomarkers by identifying particular molecular changes linked to disease states. Researchers can better understand illness pathways and create tailored medicines that only affect those processes by researching these proteins [5].

Finding oncogenic driver mutations is one illustration of how molecular diagnostic proteins are used in the development of targeted drugs. These mutations cause over-activation of certain proteins or signalling pathways, which aid in the survival and development of tumours.

Researchers can create tailored medications that selectively limit the action of these altered proteins by identifying and characterising these mutations. For example, the use of EGFR inhibitors (such as erlotinib) in lung cancer patients with EGFR mutations has shown to provide significant therapeutic advantages [6].

Furthermore, proteins that are differently expressed or changed in cancer cells as opposed to normal cells can be found using molecular diagnostic proteins. Potential targets for therapeutic intervention might be these proteins. For instance, the creation of targeted medicines like trastuzumab and pertuzumab, which selectively target HER2-positive tumours, was sparked by the overexpression of the human epidermal growth factor receptor 2 (HER2) in breast cancer. Researchers can create and develop medications that selectively target and inhibit these proteins, interrupting the faulty signalling pathways causing the disease, by using the knowledge obtained from researching molecular diagnostic proteins [7].

1.3 Targeted anticancer drug discovery

The identification and development of medications that selectively target molecular processes or proteins essential for the survival and proliferation of cancer cells is known as targeted anticancer drug discovery. Through their invaluable insights into the molecular features of cancer cells and assistance in the identification and development of targeted therapeutics, molecular diagnostic proteins play a critical part in this process [8]. A therapeutic strategy known as targeted therapy focuses on certain biochemical changes or pathways that are essential for the growth and survival of cancer. Targeted treatments, as opposed to conventional chemotherapy, attempt to selectively inhibit or disrupt the activity of certain targets implicated in tumour formation and progression. Traditional chemotherapy frequently affects both malignant and healthy cells. Targeted therapy has the potential to improve treatment effectiveness whilst lowering toxicity [5].

2. Molecular diagnostic proteins in biomarker discovery

In cancer research, biomarkers are quantifiable substances or signs that offer important details regarding the presence, development or features of cancer. They may consist of proteins, genes, mutations, metabolites or other molecular components that are present in cells, tissues or body fluids. Early detection, diagnosis, prognosis, treatment response prediction, illness progression monitoring and therapeutic efficacy evaluation are just a few of the functions that biomarkers may perform.

The use of biomarkers in the study of cancer is complex. They facilitate personalised therapy strategies by enabling the identification and characterisation of certain cancer subtypes. By assisting doctors in deciding which medicines are best for each patient, biomarkers can help with treatment decisions. Additionally, by streamlining clinical trials and permitting focused treatments, they aid in the development and assessment of novel cancer medications and therapies. Biomarkers also play a crucial role in the monitoring and surveillance of cancer patients, providing information on disease recurrence, treatment resistance or the emergence of secondary malignancies. Furthermore, biomarkers can serve as surrogate endpoints in clinical trials, allowing researchers to assess the effectiveness of interventions without having to wait for long-term clinical outcomes [9, 10, 11, 12].

2.1 Methods and approaches for identifying potential biomarkers

The search for possible biomarkers for cancer is conducted using a variety of methods and procedures. The following are a few frequently employed techniques.

2.1.1 Genomic analysis

To find possible biomarkers, genomic analysis examines the DNA, RNA and gene expression levels. Genomic analysis is carried out using methods including microarrays, next-generation sequencing (NGS) and RNA sequencing (RNA-seq) [13].

2.1.2 Proteomics

The full examination of proteins, including their levels of expression, post-translational changes and interactions, is known as proteomics. Proteomic methods based on mass spectrometry are frequently used to find possible protein biomarkers [14].

2.1.3 Metabolomics

The study of tiny molecules involved in cellular metabolism is the main focus of metabolomics. Potential metabolite biomarkers can be found by looking at the metabolite profiles of biological samples [15].

2.1.4 Imaging techniques

In order to help identify imaging biomarkers, imaging methods including positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT) can give useful information on the geographical distribution and functional properties of tumours [16].

2.1.5 Machine learning and bioinformatics

To analyse large-scale datasets, integrate multi-omics data and find possible biomarkers by spotting patterns and correlations, machine learning techniques and bioinformatics tools are used [17].

3. Molecular diagnostic proteins in target identification

It is crucial for the identification of potential targets for therapeutic intervention to use molecular diagnostic proteins. Signalling pathways, molecular alterations or cellular processes can be revealed through these proteins in order to understand how diseases, including cancer, develop and progress. In studying these proteins, researchers can gain insight into the mechanisms underlying a disease and identify specific molecular targets for therapeutic intervention [18].

Analysing the molecular diagnostic protein expression patterns in illness samples in comparison to healthy tissues or cells is one method for target discovery. These proteins’ differential expression can reveal potential targets that are over- or under-expressed in a disease state. For example, it has been possible to develop targeted therapies to inhibit HER2 signalling because of an overexpression of the HER2 protein in breast cancer [19].

The functional role of molecular diagnostic proteins can potentially offer hints for target identification in addition to differential expression. Researchers can pinpoint certain nodes within these signalling networks that could be therapeutically addressed by knowing how these proteins contribute to important signalling pathways or cellular processes involved in the evolution of illness. For instance, proteins like as BRAF or MEK, which are involved in the MAPK/ERK pathway and which support tumour development and survival, have been identified as targets in melanoma [20].

Molecular diagnostic proteins can also be utilised in high-throughput screening methods to find therapeutic drugs or small compounds that interact with these proteins and modify their function. Potential therapeutic candidates that can specifically target proteins linked to diseases may be found thanks to this screening.

Overall, molecular diagnostic proteins are useful tools for identifying targets because they shed light on disease processes and indicate particular molecular targets that may be used to build targeted treatments.

3.1 Molecular pathways and cellular processes involved in cancer development

Dysregulation of several molecular pathways and cellular processes has a role in the development of cancer. Here are several significant pathways and processes frequently linked to cancer (Table 1) [21, 22, 23]:

Pathway/Process	Molecular components involved	Role in cancer development
Cell cycle dysregulation	Tumour suppressor genes (e.g. TP53)	Abnormal control of cell division leading to unchecked growth
	Oncogenes (e.g. CYCLIN D1)
Oncogenic signalling Pathways	MAPK/ERK (RAF, MEK, ERK)	Promotes tumour growth and survival
	PI3K/AKT/mTOR
	Wnt/−catenin
	JAK/STAT
DNA damage response and repair	BRCA1 and BRCA2	Increases genomic instability and cancer risk
Angiogenesis	VEGF	Development of new blood vessels for tumour growth
	Endogenous angiogenesis inhibitors
Apoptosis resistance	Bcl-2 family of proteins	Cancer cells evade programmed cell death
Invasion and metastasis	Epithelial-mesenchymal transition (EMT)	Cancer cells spread to nearby organs and distant sites
	Matrix metalloproteinases (MMPs)
Immune evasion	Major histocompatibility complex (MHC) downregulation	Cancer cells avoid immune system detection and destruction

Table 1.

Dysregulated molecular pathways and cellular processes in cancer development.

3.1.1 Dysregulation of the cell cycle

Cancer is characterised by abnormalities in the control of the cell cycle, which can result in unchecked cell growth. Tumour suppressor genes (like TP53) and oncogenes (like CYCLIN D1) are examples of important genes involved in cell cycle progression that may be altered or mutated to cause dysregulation.

3.1.2 Oncogenic signalling pathway

Oncogenic pathways demonstrate abnormal hyperactivity in cancer, promoting cellular proliferation, survival, and metastasis. Genes in these pathways that are mutated or overexpressed can promote enhanced signalling and the growth of tumours. The MAPK/ERK pathway (for instance, RAF, MEK and ERK), PI3K/AKT/mTOR, Wnt/−catenin and JAK/STAT pathways are a few examples.

3.1.3 DNA damage response and repair

Both internal and extrinsic causes can cause DNA damage. Genomic instability and an increased risk of developing cancer can occur from defects in DNA repair pathways, such as those affecting the BRCA1 and BRCA2 genes.

3.1.4 Angiogenesis

The development of new blood vessels, which is essential for the growth and spread of tumours. Elevated expression of pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), coupled with a reduction in endogenous angiogenesis inhibitors, can precipitate the disruption of physiological angiogenic processes.

3.1.5 Apoptosis resistance

Cancer cells have the ability to avoid apoptosis, or programmed cell death, which allows them to survive and fight treatment. This resistance may be a result of dysregulation of apoptotic pathways, including the Bcl-2 family of proteins.

3.1.6 Invasion and metastasis

Cancer cells develop the capacity to infect close-by organs and migrate to distant areas, resulting in metastases. Epithelial-mesenchymal transition (EMT), extracellular matrix breakdown by matrix metalloproteinases (MMPs) and angiogenesis are processes implicated in invasion and metastasis.

3.1.7 Immune evasion

Cancer cells have the ability to avoid the immune system’s defences against them. Major histocompatibility complex (MHC) molecule downregulation, immunological checkpoint suppression and the production of immunosuppressive substances are a few of the mechanisms involved in immune evasion.

These cellular mechanisms and molecular pathways are interrelated and frequently interact to promote the growth and spread of cancer. For the selection of possible therapeutic targets and the creation of targeted medicines, it is essential to comprehend these pathways and processes.

3.2 Utilising molecular diagnostic proteins to identify potential drug targets

In numerous ways, molecular diagnostic proteins are helpful in finding prospective therapeutic targets. The following strategies were used in this process:

3.2.1 Differential expression analysis

In molecular biology and genomics, differential expression analysis is a popular method for comparing the levels of gene or protein expression amongst various situations or sample groups. By finding genes or proteins that are noticeably elevated or downregulated in disease states, such as cancer, it is especially helpful in discovering possible therapeutic targets.

The process involved in the differential expression analysis typically includes the following steps:

• Biological samples (tissues, cells or body fluids) are collected the individuals.

• Using the proper laboratory methods, RNA or protein is extracted from the gathered materials. Protein extraction is required for proteomic investigations, whereas RNA extraction is conducted for gene expression analyses.

• Quantification: To quantify the amount present in each sample, the extracted RNA or protein is quantified. Numerous techniques, including spectrophotometry and tests based on fluorescence, can be used to do this.

• Gene expression analysis: Methods like microarrays or RNA sequencing (RNA-seq) are used to evaluate the levels of gene expression in RNA-based investigations. Thousands of genes may be simultaneously detected and quantified in a sample using these methods.

• Statistical analysis and validation: To find the genes or proteins that express significantly differently in the experimental groups, statistical tests like t-tests or analysis of variance (ANOVA) are used. Based on the desired degree of confidence, the cutoff for significance is established (for example, p-value 0.05). Additional experimental methods, such as quantitative PCR (qPCR) for gene expression or immunoblotting for protein expression, are used to confirm differentially expressed genes or proteins. This process aids in verifying the accuracy of the preliminary results.

Differential expression analysis enables researchers to pinpoint particular genes or proteins that show notable variations in expression levels between situations, offering possibilities for additional research as possible therapeutic targets. These molecules’ varied expression may be crucial to a disease’s onset, progression or therapeutic response [24, 25, 26].

3.2.2 Proteomic profiling

Proteomic profiling entails a thorough examination of the proteome, which includes the recognition and measurement of proteins in a specific biological sample. Researchers can find proteins that are specifically present or dramatically changed in cancer cells using mass spectrometry-based methods and bioinformatics analysis, offering prospective therapeutic targets [27].

3.2.3 Functional analysis

To comprehend the functional functions and molecular interactions of molecular diagnostic proteins within signalling networks or cellular processes linked to cancer, molecular diagnostic proteins might be researched. Researchers can find key nodes in these pathways that can be drug-targeted by clarifying the functional importance of these proteins.

3.2.4 Pathway analysis

Pathway analysis entails evaluating how certain signalling pathways or cellular processes associated with cancer are impacted by molecular diagnostic proteins. Researchers can find important targets that can be altered for therapeutic intervention by examining the interactions and crosstalk amongst proteins within these pathways (Table 2).

Strategy	Description	Application
Differential expression analysis	Compares gene/protein expression levels between different sample groups	Identifies differentially expressed proteins as potential therapeutic targets
Proteomic profiling	Comprehensive examination of the proteome to identify specific proteins	Discovers proteins specifically present or altered in cancer cells
Functional analysis	Studies the functional roles and interactions of diagnostic proteins	Uncovers key nodes within signalling networks for targeting
Pathway analysis	Evaluates impact on cancer-related pathways by diagnostic proteins	Identifies targets for therapeutic intervention

Table 2.

Molecular diagnostic proteins and their utilisation for target identification.

Researchers can select and rank prospective therapeutic targets for additional study and drug development by using the knowledge gathered by molecular diagnostic proteins [28, 29, 30].

3.3 Examples of successful target identification using molecular diagnostic proteins

In cancer research, there have been several cases of target discovery employing molecular diagnostic proteins. Here are a few noteworthy instances:

3.3.1 HER2 in breast cancer

A protein called Human Epidermal Growth Factor Receptor 2 (HER2) is overexpressed in around 20% of breast cancer patients. Its overexpression is linked to a poor prognosis and aggressive tumour progression. When HER2 was discovered to be a therapeutic target, targeted treatments like trastuzumab (Herceptin) and lapatinib (Tykerb), which selectively block HER2 signalling, were created. Patients with HER2-positive breast cancer now have much better outcomes thanks to these targeted medicines [7].

3.3.2 BCR-ABL in chronic myeloid leukaemia (CML)

In CML, a chromosomal translocation results in the constitutive activation of tyrosine kinase activity and the BCR-ABL fusion protein. The targeted drug imatinib (Gleevec), which was created to limit BCR-ABL activity, has completely changed how CML is treated. Patients with CML have shown significant success with imatinib and other tyrosine kinase inhibitors (TKIs) in producing remission and prolonging life [6].

3.3.3 EGFR in non-small cell lung cancer (NSCLC)

Non-small cell lung cancer (NSCLC) typically exhibits epidermal growth factor receptor (EGFR) mutations, which are linked to accelerated tumour development and a poor prognosis. EGFR inhibitors were created to target the mutant EGFR protein and stop the signalling pathways that promote the proliferation of cancer cells, such as gefitinib (Iressa) and erlotinib (Tarceva). Patients with EGFR-mutated NSCLC have responded well to these targeted treatments [31].

3.3.4 BRAF in melanoma

Melanoma frequently has mutations in the BRAF gene, especially the V600E variant, which causes constitutive activation of the BRAF kinase. As a result, specialised drugs that block the mutant BRAF protein were created, including vemurafenib (Zelboraf) and dabrafenib (Tafinlar). Patients with melanoma that had the BRAF mutation have shown a substantial clinical benefit from these medications [32].

These instances demonstrate how molecular diagnostic protein discoveries have successfully been converted into targeted medicines, which have completely altered the landscape of cancer treatment. Precision and personalised medicine strategies have been established by identifying and focusing on these important proteins, improving patient outcomes.

4. Validation of drug targets

In order to identify and prioritise prospective pharmacological targets for further development, target validation is an important stage in the drug discovery process. Target validation assists in demonstrating the biological significance of a target in the onset, course and therapeutic response of a disease. It shows that the target is directly implicated in the illness pathway and that modulating it might have a significant effect on the phenotype of the disease. The danger of pursuing dangerous or ineffective medication candidates is decreased by validating a target. Researchers can concentrate their efforts and resources on creating medications that have a better chance of success by establishing the target’s importance in illness. Target validation offers insights into the mechanisms of action and prospective therapeutic strategies for a specific target, which may be used to optimise therapy strategies. It aids in determining the most effective modulation techniques for the target, such as monoclonal antibodies, gene treatments or small molecule inhibitors. This information aids in choosing the best therapeutic strategy and creating potent medication candidates. Validated pharmacological targets are important in personalised medicine strategies. Clinicians can customise therapies for specific patients depending on the presence or absence of validated target indicators by identifying targets that are exclusive to particular patient groups or illness subtypes. As a result, medicines can be more focused and successful, which benefits patients’ results. Validation of a target provides a strong rationale for further investment in drug development. It helps attract funding and collaborations, as well as encourages pharmaceutical companies to invest in the development of targeted therapies. Validated targets are more likely to attract interest from the industry and undergo the rigorous process of preclinical and clinical development. Target validation, in short, is crucial for discovering pertinent and interesting drug targets, lowering development risks, enhancing therapeutic approaches, enabling personalised medicine methods and easing the transition of drug candidates from discovery to clinical development [33, 34, 35, 36]. It is a crucial stage in making sure that drug development activities are successful and have an impact.

4.1 Experimental techniques for validating drug targets

Various experimental methods are employed during the drug development process to validate potential therapeutic targets. These methods assist in confirming a target’s involvement in the onset of a disease and determining if a treatment intervention would be appropriate. Several frequently used experimental methods for target validation are listed below [30, 37, 38, 39]:

4.1.1 Gene knockdown or knockout

RNA interference (RNAi): In cellular or animal models, the target gene expression is selectively silenced using small interfering RNA (siRNA) or short hairpin RNA (shRNA) molecules. The effect of target depletion on the phenotypic of the illness is then assessed.

CRISPR-Cas9: The target gene is modified or deleted specifically using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology to determine the functional significance of the gene.

4.1.2 Pharmacological inhibition

Small molecule inhibitors: The development or selection of certain small molecules is done to prevent the target protein from acting. Using in vitro experiments and animal models, the effects of target inhibition on cellular pathways, disease development and treatment response are assessed.

Monoclonal antibodies: The capacity of antibodies created to bind precisely to the target protein to inhibit the activity of the target or to trigger immune-mediated reactions against cells expressing the target is created and assessed.

4.1.3 Biomarker analysis

Protein expression analysis: To evaluate the expression levels and localisation of the target protein in patient samples or animal models, methods including immunohistochemistry (IHC), immunofluorescence (IF) or Western blotting are performed. Investigations are conducted on the relationships between target expression and disease features or clinical outcomes.

Genomic analysis: To find mutations or genetic changes in the target gene that may contribute to the onset or course of illness, genetic sequencing or genotyping procedures are used.

4.1.4 Functional assays

Cell-based assays: To evaluate the effect of target modification on cell proliferation, apoptosis, migration, invasion or other pertinent functional endpoints, several cellular tests are carried out.

Pathway analysis: Target modulation-related changes in gene expression or protein activity are analysed using molecular approaches like proteomics or gene expression profiling. This aids in elucidating the signalling pathways and after-effects of target inhibition.

4.1.5 Animal models

Animal models that have undergone genetic modification, such as transgenic or knockout animals, are utilised to research the effects of target modulation in vivo. These models may be used to assess how target modification affects the onset, progression and treatment response of diseases.

It is vital to remember that a variety of complimentary strategies are frequently used in conjunction to bolster the case for a pharmacological target’s validity.

5. High-throughput screening methods

When developing targeted anticancer drugs, high-throughput screening (HTS) techniques are frequently employed to quickly find promising therapeutic candidates from enormous chemical libraries. HTS enables quick testing of many different drugs against certain targets or cancer cell types. Cell viability tests are used to determine the effects of substances on cell viability and proliferation. Examples of these assays are MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and ATP-based assays. Caspase activation assays and Annexin V staining are two HTS techniques that may be used to find substances that cause cancer cells to undergo apoptosis. Tests for cell migration and invasion gauge a substance’s capacity to prevent the crucial processes of cancer cells migrating and invading. HTS techniques can be used to assess a compound’s inhibitory activity against a particular enzyme implicated in a cancer-related pathway, such a kinase or a protease. Assays for particular protein–protein interactions in cancer signalling pathways determine a substance’s capacity to interfere with a certain protein–protein interaction. High-Content Screening (HCS) evaluates many cell characteristics concurrently, such as protein expression, cellular shape or subcellular localization. HCS combines automated microscopy with image analysis. HCS makes it possible to test substances for their impacts on multiple cellular functions whilst providing thorough information on the method of action. With RNA interference (RNAi) genome-wide screening using large-scale RNAi libraries, it is possible to identify the genes required for cancer cell survival or the activation of certain pathways by silencing the expression of genes in cancer cells. Protein microarrays allow for the screening of substances against a variety of pure proteins, revealing information on the interactions between substances and proteins as well as possible targets. To anticipate the binding affinity of drugs to target proteins, in silico screening techniques use computer algorithms. Virtual screening can assist in reducing the number of substances that need to be evaluated experimentally, saving time and materials. These HTS techniques allow for the fast screening of hundreds to millions of compounds, the identification of lead compounds and the prioritisation of compounds that exhibit the most promising action against the target or cancer phenotype. By quickly providing useful data on compound activity, simplifying hit identification and supporting subsequent hit-to-lead optimisation procedures, HTS approaches considerably speed up the development of targeted anticancer drugs [40, 41, 42, 43].

5.1 High-throughput screening approaches

High-throughput screening (HTS) assays, which are used to quickly evaluate vast chemical libraries for their activity against a target of interest, rely heavily on molecular diagnostic proteins. To improve the effectiveness and efficiency of HTS tests, these proteins are used in a variety of ways. Molecular diagnostic proteins have the following uses in HTS assays [40, 44, 45, 46]:

5.1.1 Target binding assays

To evaluate the interaction between the target protein and possible therapeutic candidates, binding assays can be performed on molecular diagnostic proteins. Surface plasmon resonance (SPR), ELISA and AlphaScreen®/AlphaLISA® assays are a few examples of the assays that can be used. The binding of substances to the target protein may be evaluated, allowing the identification of hits and enabling the use of molecular diagnostic proteins as capture agents or detection probes. Enzyme activity assays: Many drug targets are enzymes, and their activity can be assessed using enzymatic assays. Molecular diagnostic proteins, such as kinase proteins or proteases, can be utilised in enzymatic assays to measure the activity of the target protein. HTS enzymatic assays can be designed to identify compounds that modulate enzyme activity, either as inhibitors or as activators, providing potential leads for drug development [40].

5.1.2 Reporter gene assays

To create reporter constructions, molecular diagnostic proteins can be fused with reporter genes like luciferase or green fluorescent protein (GFP). These constructions can be used in HTS tests to infer information about the target protein’s activity. When a substance interacts with the target, it may modify the reporter gene’s activity, producing a quantifiable signal. High-throughput formats frequently employ reporter gene experiments to find drugs that interfere with certain signalling pathways or biological functions.

5.1.3 Cell-based assays

Molecular diagnostic proteins can be expressed or overexpressed in cell lines to establish reporter cell lines in cell-based HTS tests. These cell lines can be used to assess how different substances affect the target protein’s expression or function in a biological setting. HTS tests can reveal information regarding chemical effectiveness and selectivity in biological systems by integrating molecular diagnostic proteins with fluorescence-based or luminescence-based readouts.

5.1.4 Biomarker-based assays

High-throughput screening (HTS) tests can utilise molecular diagnostic proteins as biomarkers to evaluate a compound’s impact on cellular functions or disease-related signalling pathways. HTS tests can shed light on the possible therapeutic effects of compounds on certain disease states by assessing the levels or activity of these proteins in response to drug treatment.

The quick and effective screening of huge chemical libraries made possible by the incorporation of molecular diagnostic proteins into HTS tests speeds up the discovery of prospective therapeutic candidates. These assays help in the early phases of drug development by offering useful information regarding compound-target interactions, compound potency and compound selectivity.

5.2 Examples of successful drug discovery campaigns using high-throughput screening and molecular diagnostic proteins

There have been a number of effective drug development initiatives that integrated molecular diagnostic proteins with high-throughput screening (HTS). These instances demonstrate the effective application of HTS and molecular diagnostic proteins in the identification and creation of cancer targeted treatments. The rapid screening of chemical libraries against particular molecular targets made possible by the combination of HTS and molecular diagnostic proteins results in the discovery of intriguing therapeutic candidates that can specifically alter disease-related pathways and enhance patient outcomes. Here are a few illustrations:

• Imatinib (Gleevec) is a tyrosine kinase inhibitor that has completely changed how chronic myeloid leukaemia (CML) is treated. Imatinib was discovered by HTS to be a powerful inhibitor of the BCR-ABL fusion protein, which fuels the development of CML cells. The BCR-ABL kinase was the molecular diagnostic protein in this instance, and HTS tests assisted in identifying imatinib as a selective inhibitor, resulting in its development as a very successful targeted treatment for CML [6].

• Erlotinib (Tarceva) is a small molecule inhibitor of the EGFR that is used to treat non-small cell lung cancer (NSCLC). Erlotinib was discovered through HTS, which screened many chemical libraries against EGFR. Erlotinib’s development as a targeted treatment for NSCLC was greatly aided by the molecular diagnostic protein EGFR [47].

• A cyclin-dependent kinase (CDK) 4/6 inhibitor called palbociclib (Ibrance) is used to treat breast cancer that is HER2-negative and hormone receptor positive. Small compounds that preferentially suppressed CDK4/6 activity were found using HTS tests. The development of palbociclib as a targeted treatment for breast cancer and the subsequent target validation both relied heavily on the molecular diagnostic protein CDK4/6 [48].

6. Personalised medicine and molecular diagnostic proteins

Personalised medicine is a method of providing medical care that is tailored to each patient based on their unique traits, such as their genetic make-up, biomarker profiles and other molecular diagnostic data. Molecular diagnostic proteins are essential to personalised medicine because they offer important information about a patient’s disease status, prognosis and response to therapy. Aspects of molecular diagnostic proteins and personalised medicine to consider include the following [49, 50, 51, 52]:

• The precise diagnosis and subtyping of illnesses, including cancer, can be aided by molecular diagnostic proteins. Clinicians can divide their patient populations into subgroups with various illness features by examining the expression patterns or mutations of particular proteins. This subtyping aids in determining the best therapeutic treatments and guiding treatment choices.

• Molecular diagnostic proteins can work as predictive biomarkers by giving data on the propensity for a certain treatment to be effective or for a given therapy to cause harmful effects. For instance, some proteins can predict whether a patient would likely react to a targeted therapy or if a certain medicine may be hazardous to them. This information permits the selection of a personalised course of therapy, improving patient results and minimising unneeded side effects.

• Monitoring of the course of the disease and the effectiveness of the treatment can be done using molecular diagnostic proteins. Clinicians can evaluate a treatment plan’s efficacy and, if required, make modifications by monitoring the levels or activities of particular proteins over time. Real-time monitoring enables prompt interventions and individualised therapy changes.

• Molecular diagnostic proteins can help in the selection and administration of drugs. Protein-based testing, for instance, can detect genetic differences that influence medication metabolism or interactions with therapeutic targets, assisting doctors in determining the ideal dosage or identifying alternative treatment choices. This individualised strategy reduces the possibility of negative medication responses whilst enhancing treatment effectiveness.

• Molecular diagnostic proteins can act as prognostic biomarkers by providing information about a patient’s long-term prognosis and survival. Clinicians can predict the risk of illness development or recurrence by analysing the expression or activity of particular proteins. The creation of specialised follow-up methods and the individual risk classification process are both aided by this knowledge.

• Clinical decisions concerning patient care can be guided by the integration of molecular diagnostic proteins into personalised medicine methodologies, resulting in more specialised and efficient therapies. Personalised medicine strives to enhance patient outcomes, reduce negative effects and maximise healthcare resources by utilising the data offered by these proteins.

6.1 Case studies illustrating the use of molecular diagnostic proteins in guiding personalised therapies

Few case studies that demonstrate the use of molecular diagnostic proteins in guiding personalised therapies are:

• HER2-positive breast cancer and trastuzumab: HER2 (Human Epidermal Growth Factor Receptor 2) is a protein overexpressed in approximately 20% of breast cancers. Trastuzumab (Herceptin) is a targeted therapy that specifically binds to HER2 and inhibits its signalling. Molecular diagnostic proteins, such as HER2 testing, are used to identify patients with HER2-positive breast cancer. These patients are then eligible for targeted therapy with trastuzumab, resulting in improved outcomes and survival rates compared to standard chemotherapy alone [7].

• EGFR-mutated lung cancer and tyrosine kinase inhibitors (TKIs): EGFR mutations are genetic alterations found in a subset of non-small cell lung cancer (NSCLC) patients. These mutations lead to aberrant activation of the EGFR pathway, promoting tumour growth. Molecular diagnostic proteins, such as EGFR mutation testing, are used to identify patients with EGFR-mutated lung cancer. Targeted therapies, such as EGFR tyrosine kinase inhibitors (TKIs) like erlotinib and osimertinib, can then be prescribed to inhibit the aberrant EGFR signalling pathway, resulting in improved response rates and survival outcomes [53].

• BRAF-mutated melanoma and BRAF inhibitors: BRAF mutations are common in melanoma and lead to dysregulated signalling through the MAPK pathway. Molecular diagnostic proteins, such as BRAF mutation testing, are used to identify patients with BRAF-mutated melanoma. Targeted therapies, such as BRAF inhibitors like vemurafenib and dabrafenib, specifically target the activated mutant BRAF protein and inhibit its activity, resulting in improved response rates and overall survival in patients with BRAF-mutated melanoma [54].

• These case studies demonstrate how molecular diagnostic proteins are used to identify specific biomarkers or mutations, guiding the selection of targeted therapies in personalised medicine. By matching the molecular profile of the tumour with the appropriate targeted therapy, these approaches have shown improved treatment outcomes and patient survival rates.

7. Emerging technologies and advancements in molecular diagnostics

The field of medicine is being revolutionised by new technologies and developments in molecular diagnostics, which are allowing for more specialised and precise approaches to illness diagnosis, prognosis and therapy. Here are a few noteworthy developments:

7.1 Next-generation sequencing (NGS)

The quick and affordable sequencing of whole genomes, exomes or targeted gene panels made possible by next-generation sequencing (NGS) technology has revolutionised genomic analysis. Understanding disease causes, patient classification and the creation of targeted therapeutics all depend on the ability to identify genetic variants, including mutations, rearrangements and gene expression patterns [55].

7.2 Liquid biopsies

In liquid biopsies, circulating tumour DNA (ctDNA), circulating tumour cells (CTCs) and extracellular vesicles are examined from peripheral blood samples. Liquid biopsies offer a non-invasive way to identify and track tumour-specific abnormalities, such mutations, changes in gene expression and epigenetic modifications, providing real-time data on the severity of the condition, the effectiveness of treatment and the evolution of resistance [55].

7.3 Single-cell analysis

Through the characterisation and profiling of individual cells within diverse populations, single-cell analytic tools can shed light on cellular diversity, heterogeneity and clonal evolution. The discovery of novel treatment targets, knowledge of cellular connections and identification of unusual cell populations are all made possible by single-cell techniques such as single-cell RNA sequencing (scRNA-seq) and other single-cell methods [56].

7.4 Digital pathology

Histological pictures are digitally transformed and subjected to computer analysis in the field of digital pathology. Artificial intelligence (AI) algorithms are included into digital pathology systems to help with automated image processing, pattern recognition and the discovery of prognostic or predictive indicators. Accuracy, effectiveness and repeatability in pathology diagnosis and research are improved by this technique [57].

7.5 Mass spectrometry-based proteomics

Proteomics using mass spectrometry makes it possible to identify and measure proteins in biological material on a massive scale. Regarding protein expression, post-translational alterations and protein–protein interactions, it offers useful information. Targeted proteomics and multiplexed assays are examples of advanced methods that enable accurate measurement of certain protein targets and enhance the identification and validation of biomarkers.

7.6 CRISPR-Cas9 gene editing

Research in functional genomics and gene editing has been transformed by CRISPR-Cas9 technology. It permits exact manipulation of DNA sequences, making it possible to study gene function, identify therapeutic targets and create new gene treatments. Genes and pathways linked to medication response and resistance can be found using CRISPR-based screening [58].

7.7 Microfluidics and lab-on-a-chip technologies

Lab-on-a-chip and microfluidic systems miniaturise and combine multiple laboratory tasks onto a single device. They provide high-throughput, quick and sensitive examination of tiny sample quantities, including single cells or nucleic acids. These technologies are appropriate for point-of-care diagnostics in resource-constrained situations because they have benefits in terms of speed, mobility and cost-effectiveness [59].

These developments in molecular diagnostics have enormous promise to enhance patient stratification, illness identification and personalised therapy choices. To integrate these technologies into normal clinical practise and maximise their influence on patient care, more research, validation and standardisation are required.

8. Challenges and future perspectives

The development and effective translation of targeted treatments are impacted by a number of obstacles in the targeted anticancer drug discovery process. A significant problem in the treatment of cancer is resistance to targeted therapy. The development of diverse defence mechanisms by tumours against the effects of specific medications might results in treatment failure and the progression of the illness. For better patient outcomes, it is essential to comprehend the underlying molecular processes of resistance and devise methods to combat them. There is a lot of intra-tumour heterogeneity in tumours, and various parts of a tumour could have different molecular profiles. The responsiveness of targeted medicines may be impacted by this heterogeneity, which may also lead to treatment resistance. Designing successful targeted medicines is difficult because it is difficult to identify and target significant molecular changes across many tumour areas [60].

In targeted therapy, biomarkers are crucial for patient classification and treatment choice. Finding trustworthy and prognostic biomarkers that properly predict therapy response, nevertheless, is still difficult. To prove the therapeutic value of a biomarker, substantial study, large-scale clinical trials and the integration of multi-omics data are required. Some targeted medicines could be costly and difficult for some people to receive. Access to these treatments may be hampered by the high cost of targeted medications and the requirement for particular diagnostic procedures. Widespread implementation faces difficulties in ensuring access to targeted therapies that are both affordable and equitable [61].

Preclinical models used today frequently fall short of precisely predicting how patients will respond to targeted medicines. To increase the success rate of targeted drug discovery, it is essential to create more accurate preclinical models, such as patient-derived xenografts (PDX) and organoids, which more accurately reproduce the tumour microenvironment and molecular properties of patient tumours [62].

It is possible to increase the effectiveness of treatment by combining different targeted drugs or targeted treatments with other types of treatment, such immunotherapy or traditional chemotherapy. The issue in creating efficient combination medicines, though, is selecting the best medication combinations and comprehending their synergistic benefits and associated toxicities.

Targeted treatments frequently need regulatory approval, and negotiating challenging regulatory procedures is a necessary part of the development process. The effective use of targeted medicines depends on addressing ethical issues, such as gaining informed permission for molecular profiling and assuring privacy and data security in personalised medicine techniques [63].

Researchers, doctors, regulatory agencies and pharmaceutical corporations must work together to address these difficulties. Targeted anticancer drug development will advance thanks to improvements in technology like high-throughput screening, molecular profiling and computer modelling, as well as a greater comprehension of tumour biology and resistance mechanisms.

8.1 Future prospects and potential breakthroughs in the field

The development of personalised combination therapies, the advancement of non-invasive monitoring techniques like liquid biopsies, the integration of artificial intelligence and data analysis for effective interpretation of results, the emergence of resistance mechanisms, the need for discovery and integration of new biomarkers and the validation and clinical utility of biomarkers are the current challenges in targeted anticancer drug discovery using molecular diagnostic proteins. To turn targeted therapies into efficient and individualised treatments for cancer patients, we must continue our research, make technology improvements, collaborate with interdisciplinary teams and validate our clinical findings [64, 65].

9. Conclusion

By offering invaluable insights into the molecular changes and signalling pathways involved in cancer genesis and progression, molecular diagnostic proteins play a crucial role in the development of targeted drugs. In order to pinpoint specific molecular targets for therapeutic intervention, these proteins act as biomarkers that may be examined. Molecular diagnostic proteins assist researchers in finding potential therapeutic targets that are differently expressed and high-throughput screening. The relevance and viability of these targets for therapeutic development are further supported by target validation studies. Researchers can personalise cancer therapy, choose the best medicines, track treatment response and evaluate therapeutic success by using molecular diagnostic proteins. Ultimately, developing tailored medicines and enhancing patient outcomes in cancer therapy depend on the discovery and confirmation of therapeutic targets supported by molecular diagnostic proteins. The development of molecular diagnostic proteins and associated technologies has enormous potential to transform the way cancer is treated. These technologies have the potential to greatly improve outcomes and quality of life for cancer patients in the future by enabling personalised treatments, accurate target identification and enhanced treatment regimens.

Acknowledgments

I would like to express my deepest gratitude to Prof. Dr. S. Anbazhagan, Principal, and to the management of Surya School of Pharmacy for providing a peaceful environment, which assisted me to write effectively. This endeavour would not have been possible without my mother Mrs. Mayavalli Gunasekaran for her continuous support for creating free time to write this article.

Conflict of interest

“The authors declare no conflict of interest”.