The Current Perspectives in Clinical Research: Computer-Assisted Drug Designing, Ethics, and Good Clinical Practice

In the era of emerging microbial and non-communicable diseases and re-emerging microbial infections, the medical fraternity and the public are plagued by under-preparedness. It is evident by the severity of the Coronavirus disease (COVID-19) pandemic that novel microbial diseases are a challenge and are challenging to control. This is mainly attributed to the lack of complete knowledge of the nov el microbe’s biology and pathogenesis and the unavailability of therapeutic drugs and vaccines to treat and control the disease. Clinical research is the only answer utilizing which can handle most of these circumstances. In this review, we highlight the importance of computer-assisted drug designing (CADD) and the aspects of molecular docking, molecular superimposition, 3D-pharmacophore technology, ethics, and good clinical practice (GCP) for the development of therapeutic drugs, devices, and vaccines. ,


INTRODUCTION
The world is still fighting the Coronavirus Disease (COVID- 19) pandemic, which practically challenged humankind on every front. It has become necessary for pharmaceutical companies to constantly develop new drugs targeting prevalent diseases and emerging and re-emerging ones 1-3 . Drug designing can be broadly classified into two categories: structurebased and ligand-based drug designing. The structure-based method considers the structures of both target and the ligand.
At the same time, the ligand-based approach utilizes only the structure and target of the ligands 4,5 . Once the designing method is finalized, the new drug undergoes four phases before entering the consumer usage market. The drug development involves four phases, including phase 1, 2, 3, and 4 6 . Phase 1, also termed the drug development phase, evaluates humans' drug dosage and toxicity. A minimal amount of the drug is given to healthy and physiologically sound male volunteers. In this phase, the dosage with the first sign of toxicity is noted 7 . Phase 2 is considered a pre-clinical phase where the trial drug is assessed for its efficacy against a specific disease.
In this phase, a small amount of the new drug is given to the patient volunteers, who are followed up on a timely basis. This phase decides the optimum dosage for patient use 8 . Phase 3 is called the clinical development phase, and wherein many patients are recruited to evaluate and confirm the results obtained in the previous two phases. The  the current treatments or uses a placebo, and its efficiency is identified. The complete data on the efficacy and safety of the drug is collected and placed before the international and national regulatory agencies like the Food and Drugs Administration (FDA), the United States of America (USA), and the Central Drugs Standard Control Organization (CDSCO), India for final approval of marketing 9 . Phase 4 involves post-marketing studies, which are also called pharmacovigilance. During this phase, the long-term safety and efficacy of the drug are assessed in a larger population group 10 .
Various techniques to discover drugs have evolved from finding a natural substance to treat diseases and using computerassisted drug designing (CADD) for manufacturing the drugs (Figure 1). The latest addition to this array of technology is molecular docking and artificial intelligence 11, 12 . The molecular docking process consists of two main stages: ligand conformation and positioning of the ligand within the target sites 13 . In the current review, we comprehensively discuss the nuances of clinical research, which include CADD, discovery, molecular docking, molecular superimposition, 3D pharmacophore technology, ethics, and good clinical practice (GCP).

THE NUANCES OF DRUG DESIGN AND DISCOVERY
Drug design and discovery involve a complex process. Given the improved scientific and technological advances, the drug discovery process has shifted from the traditional processes to the more synthetic approaches. Drug design has transformed from when the drugs were discovered from the purification and alteration of a known natural substance to the novel technique of producing the drugs from chemicals. Improved knowledge of the disease, from physiological to molecular and atomic levels, and the availability of advanced technologies have significantly influenced the drug design and the discovery process 14 . The drug design and discovery process can be depicted in stages that include identifying the problem/disease, finalizing the compound, and conducting the phase-wise trials (phase 0, phase 1, phase 2, phase 3, and phase 4). After clearly understanding the process involved in drug design and discovery, we move towards developing and manufacturing the drug. An increased understanding of the disease/problem and the genetic basis of the disease enables the identification of the target protein that cures the disease 15 (Figure 2).
Since several diseases like Alzheimer's, Parkinson's, and malignancies have different contributing factors, identification of those factors and finding/discovering the modulating compounds using molecular and computer-assisted approaches are considered multidimensional approaches to drug discovery 16 . Although technological advancement proves to be a boon to drug design and discovery, there will still be issues identifying the appropriate drug target for a particular disease and the rational approaches to its discovery 17, 18 . The essential components of drug design and discovery include the identification of a problem/disease/target. The case here could be when a satisfactory treatment is unavailable, or there is not yet any therapeutic drug available to treat. Once a target is identified, a search for any natural substance with known therapeutic value is searched and further analyzed for the hit compound, which is further purified and evaluated through clinical trials 19 . The hit molecule is purified using medicinal chemistry studies. The pre-clinical studies are performed to assess the biological activities and toxicological characteristics of the cell cultures and experimental animals before being evaluated in humans in different phases of clinical research. The compound can be synthesized synthetically from chemicals or by modifying a known compound 20 . The CADD, also called the 'in silico method,' has been instrumental in studying and analyzing the compound in recent times. Even with the increased technological advances, the process of drug design and discovery is a lengthy (time-consuming), costly, complex, and highly unpredictable process 21

MOLECULAR DOCKING: A CLUE TO DRUG DESIGN AND DISCOVERY
After identifying the target and finding the desired compound/hit, the most critical drug design and discovery process is to validate the compounds' complementarity with the molecular docking technique. Molecular docking studies enable researchers to find the best confirmation between the protein target and the ligand 13 . Molecular docking identifies the configuration where the protein-ligand complex shows maximum interaction with the least energy. It also finds different protein targets and inhibitors of the target proteins and designs appropriate molecules or ligands to bind to them. This process is influenced by several factors, including intramolecular (bond length, bond angle) and intermolecular forces (electrostatic, van der Waals forces, and others). The docking type includes protein-protein, protein-ligand, lock-key, and fitting and flexible docking 29, 30 . Molecular docking is a computational methodology where the target protein and ligand interactions are carefully studied regarding their best sites of attachments/interactions. The molecular docking studies use computer programs to analyze various ligand-protein binding confirmations and rank these confirmations, which forms an essential aspect of pharmaceutical research 31 . The discovery of whole human genome sequencing has improved the understanding of various disease processes and has been instrumental in identifying better drug targets and binding sites.
Molecular docking also helps study the small molecule binding affinities to the target protein and the biochemical processes involved in the ligand-protein bindings 32 .
Of all the newer in silico techniques available for drug discovery, molecular docking is considered a key concept for successful drug discovery using structure-based drug design (SBDD) 33 . Identification of newer molecular entities/blockbuster drugs is a tedious and costly affair that the newer molecular docking technology can overcome 34 . Using molecular docking, the novel binding site for the drug (HIV-1 integrase) for combating human immunodeficiency virus (HIV) infection was discovered 35 . In recent times, the molecular docking mechanism has been used to study the molecular and quantum mechanics of the proteins, using these studies to discover newer antimicrobial therapeutic agents and assess the role of larger protein-protein complex interactions in developing drugs 36 . In the SBDD, the ligand/protein binding capacity with the receptor is analyzed for the strengths of the bond, stability, and affinities using various scoring

SYNTHESIS OF PHARMACOPHORE ELEMENTS USING CADD
The most significant part of a drug design and discovery is the synthesis of three-dimensional ligands, also called 3D- X-ray crystallographic studies are used to study the molecular structure/confirmations of the ligand (spatial arrangements and electrochemical properties) and the receptor. High-affinity ligands are more suitable for attachment to the receptors and show no steric repulsions with receptors. The pharmacophore technology assists in studying the ligand's binding sites (high affinity/low affinity), modifying the binding site/molecular structures to improve the binding capabilities of the ligand with the receptor/protein (Figure 4) 43 . Pharmacophore-based ligand synthesis methods will help identify the suitable biological target, as noted from a recent study that found hepatocyte growth factor receptor (c-Met) as a suitable target for new compounds 44 . The quantitative structure-activity relationship (QSAR) and three-dimensional ligand-based pharmacophore models are frequently used to identify the target binding sites on the ligand, as noted from the research studies on Alzheimer's disease 45 .
In CADD, synthesizing pharmacophore elements is crucial for designing and discovering a new drug. Recent research elaborated on using a pharmacophore model to synthesize new quinolone derivatives for their antioxidant activities 46 .
Pharmacophore modeling was used to synthesize a ligand-based pharmacophore model to synthesize the serotonin receptor antagonist, which has a therapeutic application in managing various clinical conditions, including anxiety and others 47 . Because CXCR2 is an essential receptor in the development and metastasis of cancerous conditions, the ligandbased pharmacophore model was prepared using the computational method (virtual screening) to synthesize the CXCR2 antagonists 48 .

THERMODYNAMICS OF DRUG DESIGN AND DISCOVERY
The success of the drug design and the discovery depends on the thermodynamics of the ligand-receptor complexes. This concept discusses the conformational modes of the ligand and its multiple binding sites to the protein/receptor. It also elaborates on the two important mechanisms of assessing the binding affinities of the ligands to the protein/receptor molecules: free energy perturbation (FEP) and thermodynamic integration (TI) 49 . The nature of the receptors includes those without the endogenous ligand (enzymes, ion channels, proteins, and nucleic acids) and those with the endogenous Ligand based screening Library preparation Pharmacophore / 3D Elements Structure based screening

In Vitro Studies
In Vivo Studies regulatory ligands (hormones, auto acids, neurotransmitters, growth factors, and cytokines). By using the CADD, the conformational properties of the ligand-receptor/protein complex may be studied/understood using the quantum chemical methods that include the Schrödinger equation. In this equation, the molecule is considered a collection of positively charged nuclei and negatively charged electrons moving under the influence of Coulombic potentials 50 .
The ligand and the receptor interactions will decide the complex's stability and the drug delivery potential. The proteinligand of the ligand-receptor interactions depends on the complexes' enthalpy and entropy 51 . The bioactive conformational energies of the ligand-receptor/protein complexes assume greater significance because the higher the affinity, the greater the complexes' stability. The affinities depend on the free energy difference (ΔG) between the bound ligand-protein complex and the unbound protein and the ligand 52 . The water affinities and the hydrophobicity associated with the stable ligandreceptor complexes depend on the protein's polar, non-polar, and topographical complex concavities, as noted in a previous study 53 .
Drug design and discovery is a complex process involving several versatile research areas. The ligand-binding ability of the receptor (drug-target complex) is checked using thermodynamic studies, and those ligands which are faulty can be eliminated, and those with improved binding capacity can be selected for further research. The thermodynamic studies include the assessment of the free energies (ΔG) of the ligands, their enthalpies (ΔH), and the entropies (ΔS) 54 .
Thermodynamics is the study of the heat change that occurs when two molecules interact. It is used to identify inhibitors and antagonists to minimize antimicrobial drug resistance due to mutations, reduce side-effects caused by non-specific attachments, and water solubility to increase bioactivity, as noted from the available research findings 55 . The lead optimization studies apply thermodynamics considering three essential aspects that include the presence of appropriate enthalpy in the hydrogen bonds, there is favorable entropy in hydrophobic interactions, and conformational changes that are entropically unfavorable 56 .

DESIGN
Among the most significant advantages of CADD, the technique of molecular superimposition assumes great significance.
Understanding the process of molecular superimposition and molecular mechanisms involved in drug design and discovery is essential. After preparing a 3D-pharmacophore element, the molecular superimposition helps to compare different molecules for their conformational properties and ability to bind or fit into the model. The molecular superimposition may be done using either the atoms/fragments or the molecules. Molecular superimposition can be rigid or flexible 57 . The computer method QUASIMODI is used to perform superimposition and the Patterson-density-based similarity index, and the electron-density derived similarity is applied to optimize the confirmations. The FLEXS, FLASHFLOOD, SUPERFLEX-SIM, and the FLASH methods are applied to perform a flexible alignment. The semiflexible approach can be applied using the computer program, the SUPERPOSE, and the CATALYST 58 . However, molecular superimposition ensures that various atoms and molecules are checked for their confirmations, and binding abilities, the stability of a 3D-pharmacophore element also depends on the molecular mechanics of the molecule that is assessed. The molecules are a combination of atoms, and the stability of the complex depends on the bond lengths, bond angles, torsional angles, and the non-bonded distances between atoms of the molecule 59 .
Clathrin is a protein present on the cell membranes of eukaryotes with various functionalities that include the uptake of bacteria, membrane-bound proteins, and others. A recent study reported using a flexible docking mechanism to identify the confirmations on the clathrin for its binding ability to the Bolinaquinone to inhibit its activities 53 . Most synthetic drugs are synthesized by using organic molecules containing carbon atoms. Therefore, medicinal chemists play an active role in drug design and discovery. Molecular mechanics involve synthesis, alteration, and representation of 3D structures of the molecules. Molecular mechanics include applying computational technologies to study the molecular and biological properties of various protein/receptors/targets using theoretical and experimental data. The molecular mechanics involve X-ray crystallographic studies to understand the 3D conformation of the molecule and the ability of the molecule to bind to the target/receptor. Molecular mechanics are inexpensive and easy to manage and are used to reproduce molecular confirmations matching and adjusting the bond lengths, bond angles, and torsion angles to equilibrium values to the one it has been designed to bind/attach 60 . The QSAR study is a technique that quantifies the anatomical and biological properties of the molecules/ligands/proteins. The physicochemical properties include hydrophobicity, structural, ion-ion interactions, and steric effects. A recent study attempted to combine the molecular docking technique with the QSAR method to find the binding sites on the transforming growth factor-β (TGF-β) necessary to stop invasion and tumor metastasis 61 .

3D-PHARMACOPHORE ELEMENTS IN DRUG DESIGN AND DISCOVERY
The 3D-pharmacophore and the typical feature of the pharmacophore include hydrogen-bond donors and acceptors, positively and negatively charged ions, and hydrophobicity. The pharmacophore elements form the basis/core of medicinal chemistry. The pharmacophores are synthesized by using the active molecules in such a way that they retain the biological activity, and a slight change in the configuration of the molecules may influence the biological activities. The pharmacophore technology is to synthesize the ligand and receptor antagonists, as noted in the case of dopamine antagonist receptors and the serotonin (5-hydroxy tryptophan) receptors. The 3D-pharmacophore elements are prepared using the atoms and the molecules bound by various bonds/forces like the hydrogen bonds, electrostatic forces, and the van der Waals forces. Also, the pharmacophore elements may contain the heteroatoms such as oxygen, nitrogen, and polar functional groups such as carboxylic acids, amides, and hydroxy groups 62 .
There are two types of pharmacophore elements, structure-based (X-ray) and ligand-based (derived from active compounds) pharmacophore elements 63 . Since not all protein structures have been elucidated, the ligand-based pharmacophore synthesis is most opted by the researchers. The software used in the molecular modeling pharmacophores includes the MOE and Phase 64 . Pharmacophore technology is essential in drug design when the structural data on a target receptor is unavailable. The pharmacophore method is used to perform lead discovery, lead optimization, and to assess the similarity and variations in the structural confirmations of the ligand and the receptor 65 . According to the international union of pure and applied chemistry (IUPAC), the pharmacophore is defined as the interactions of molecular structures to their molecular target by the steric and electric features and defining a specific biological property. The pharmacophore technique uses molecular interaction to define a ligand's binding ability to the receptor, including features such as hydrogen bond donors, hydrogen bond acceptors, positive and negative charged ion groups, and hydrophobic regions 66 .

HUMAN PARTICIPANTS IN CLINICAL TRIALS
Clinical research is usually undertaken to solve a current medical/public health problem. The problem in most instances would be the patients suffering from various diseases that include both infectious (microbial infections) and non-infectious conditions. The solution looked for is to find a treatment for a disease that has neither a therapeutic intervention available nor a vaccine present, and when the current treatment is plagued with complications/severe adverse effects. Although the pharmaceutical substances are designed based on CADD and other in silico methodologies, they are tested on healthy and diseased people to assess their safety and efficacy before being approved by the regulatory authorities for prescription purposes. The regulatory bodies stress the need for human subjects' protection, informed consent, and support for the  With the international guidelines as a parameter, national guidelines for protecting human rights were implemented by the

respective countries, including India's Indian Council for Medical Research's (ICMR) Ethical Guidelines for Biomedical
Research on Human Subjects in the later years 78 . The ethical code of conduct during clinical research involving human subjects has gained significance due to the infamous human experiments during World War I and II. Also, the Tuskegee Syphilis human research that led to unethical practices involving a particular group of humans, including the prisoners and mentally ill people, was instrumental in framing ethical code in clinical research involving humans 79 . Clinical research becomes ethical by satisfying seven requirements that include research to enhance further understanding of a disease/condition, scientific methodology used while conducting the research, including appropriate participants after following scientific procedures, and favorable risk-benefit ratios. An independent review board approves the study protocol, informed consent is taken without any influence, and respect for privacy protects the well-being 80 .
Since clinical research is conducted for a good social cause and the improvement of human health, such research must satisfy ethical concerns and justify the research concerning its social value requirement (SVR). The SVR is justified in all cases of clinical research, which satisfy eight ethical concerns that include safeguarding the rights of participants who cannot give consent, respect for autonomy, investigator integrity (not exposing subjects to undue risks), deceiving participants (promising undue advantages/incentives), not exploiting the participants, stewardship of public resources (spending for a social cause), imparting public trust (benefiting public), compensating any deviations for the above rules (make sure the competent adults are recruited for research on the non-social cause, only expose to no more than moderate risks, compensate the undue risk with benefits, preserving privacy, and not use public funds) 81 . The most significant part of clinical research is the implementation of good clinical practice (GCP) guidelines. Once the lead compound is identified and optimized, the next step toward drug discovery is the application for a new drug testing (investigational new drug application-INDA). The potential drug is approved for animal testing (pre-clinical phase to assess for safety and toxicity) and later in humans (clinical research phase 1-4) 88 . While the clinical research is being conducted, the GCP guidelines must be followed at various stages. The GCP guidelines state that regulatory authorities must satisfactorily evaluate the clinical trials like the FDA. The FDA must evaluate each phase of a clinical trial. The GCP guidelines ensure that the clinical trials are approved by the regulatory authorities (IRB), ensuring the trial processes, designing the case report form (CRF), analyzing research planning, and assessing the study reports at regular intervals after completion of the study 89 . The sponsor and investigators are responsible for updating the modifications/amendments in the study protocol both to the institutional review boards and to the FDA. Also, they are entitled to notify any information amendments (increase/decrease in drug exposure), safety reports (reporting adverse events), and annual reports detailing the status of the study. Protection of human rights, the safety of the participating subjects, and the reliability of the data being generated imply the quality of the clinical research. A rigorous review of the study protocol by the respective institutional review boards and stringent informed consent practices will demonstrate the high scientific standards of a clinical trial study.

ETHICS IN CLINICAL RESEARCH: INFORMED CONSENT
Continuous monitoring of the trial and regular audits will ensure the quality of a trial 91 .
Most clinical trials evaluate the efficacy, safety, and adverse events associated with medical products, including drugs. The clinical trial involves a large group of qualified medical professionals, including the principal investigator, co-investigators, clinical research associates, and the sponsors who fund the trial (Figure 7). The clinical trial must follow a protocol (background and purpose of study, trial design, infrastructure required, procedural details, and statistical methods to analyze results), standard operating procedures (SOP), study manuals, and other guidelines, including a well-structured plan of action document. All the deviations in the protocol must be so as not to harm the study participants, and any harm must be addressed and informed to the regulatory authorities 92