Review for QSAR studies and drug design of selected heterocyclic nucleus of antitubercular drugs

This review article explores the pressing issue of drug-resistant strains of Mycobacterium tuberculosis, which have emerged due to the widespread and uncontrolled use of antibiotics in clinical settings over several decades. In response to this challenge, various methods have been developed for synthesizing new antitubercular compounds. Among these, the fragment-based drug discovery (FBDD) approach has shown promise as an effective strategy. One class of compounds that has exhibited significant potential in combating tuberculosis is 1,2-diazoles. The article discusses the importance of these compounds and their potential as future antitubercular drugs. Additionally, it delves into the various strategies employed in drug development, emphasizing the relevance and efficacy of FBDD. Analytical methods play a crucial role in characterizing antitubercular antibiotics, and the article highlights liquid chromatography and voltammetry as preferred techniques for determining these compounds. The redox (oxidation/reduction) properties of antituberculars make them amenable to analysis using electrochemical methods, with voltammetry being particularly suitable. Furthermore, the article underscores


Introduction
Tuberculosis is a chronic infection characterized by caseous necrosis and granuloma formation.It is caused by the bacteria called Mycobacterium tuberculosis.Most cases of tuberculosis involve the lungs, where the disease produces hemoptysis, or bloody sputum, along with the typical cough 1 .However, it can also affect other sites of the body known as Extrapulmonary TB.In 1882, Dr. Robert Koch discovered the organism responsible ie., Bacillus Mycobacterium tuberculosis.This widely-known disease is one of the diseases of stigmatization and affects one-third of the world population 2 .World Health Organization (WHO) reports that a relatively small proportion (5-10%) of the estimated 1.7 billion people infected with M. tuberculosis will develop TB disease during their lifetime.However, the probability of developing TB disease is much higher among people infected with HIV; it is also higher among people affected by risk factors such as malnutrition, diabetes, smoking, and alcohol consumption.Mycobacterium tuberculosis is a small, aerobic, nonmotile, rod-shaped bacillus, 2-4 μm in length and 0.2-0.5μm in width.The human host serves as the only natural reservoir for this bacterium, but it can be cultured in the laboratory.It divides every 16 to 20 hours, which is an extremely slow rate compared with other bacteria 5 .The cell wall of this bacterium is composed of complex lipids, such as mycolic acids that make up over half the cell envelope of the mycobacteria.Their hydrophobicity prevents the diffusion of many chemicals including drugs into the bacterium.Mycolic acids also play a major role in virulence of M. tuberculosis, by protecting the organism from complement fixation and damage from lysozymes and free radicals in the phagolysosomes of neutrophils.The cell wall also contains acyl glycolipids and other substances such as free lipids and sulfolipids.The high concentration of lipids in the cell wall of this bacterium has been associated with unique clinical characteristics such as impermeability to stains and dyes, resistance to many antibiotics, resistance to killing by acidic and alkaline compounds, resistance to osmotic lysis via complement deposition, resistance to lethal oxidations and survival inside macrophages.

Clinical classification of mycobacteria
According to their growth rate, the Mycobacterium genus is usually separated into two major groups:  Slow-growing species including M. tuberculosis, M. bovis, and M. leprae. Fast-growing species such as M. smegmatis. 6ong the pathogenic species, the most relevant for human health are M. tuberculosis and M. leprae, are the causative agents of two of the world's oldest diseases, tuberculosis and leprosy, respectively.M. canettii and M. africanum, which also can cause human TB, are most commonly isolated from African patients.M. bovis demonstrates the broadest spectrum of host infection, affecting humans, domestic or wild bovines, and goats.M. microtia can also cause disease in immunocompromised human patients.M. kansasii, M. malmoense, and M. xenopi represent pulmonary opportunists, while M. marinum is the skin pathogen infecting organisms by entering through damaged skin.

Pulmonary Tuberculosis:
TB disease most commonly affects the lungs; this is referred to as Pulmonary TB.Patients with Pulmonary TB usually have a cough and an abnormal chest radiograph and may be infectious.Although the majority of TB cases are Pulmonary, TB can occur in almost any anatomical site.

Extrapulmonary Tuberculosis 7 :
Extrapulmonary TB isease occurs in places other than the lungs, including the larynx, the lymph nodes, the pleura, the brain, the kidneys, or the bones and joints.In HIV-infected persons, Extrapulmonary TB disease is often accompanied by Pulmonary TB.

Miliary Tuberculosis 8 :
Miliary TB occurs when tubercle bacilli enter the bloodstream and disseminate to all parts of the body, where they grow and cause disease in multiple sites.This condition is rare but serious."Military" refers to the radiograph appearance of millet seeds scattered throughout the lungs.It is most common in infants and children younger than 5 years of age and severely immune-compromised persons.

Pathogenisis 9,10 :
Infection occurs when a person inhales droplet nuclei containing tubercle bacilli that reach terminal alveoli in the lungs.The first step is the recognition of mycobacteria as invading pathogens, followed by activation of innate host defense responses.Cell-mediated immunity is usually developed within approximately two to eight weeks from the initial infection, followed by the initiation of adaptive immune responses.The activated T lymphocytes, macrophages, and other immune cells form granulomas that limit further replication and spread of the tubercle bacilli.

Latent tuberculosis 12
Latent tuberculosis (LTB), also called latent tuberculosis infection (LTBI) Latent TB infection occurs when Mycobacterium tuberculosis runs away from the immune system.Latent Tuberculosis infection is generally not contagious and produces no symptoms, but the bacilli may be present in the body.Individuals who have a latent infection cannot transmit infection to others.In this infection, most of the individuals do not develop active disease, but the problem occurs when latent infection becomes active.Approximately 10% of the individuals develop active Tuberculosis disease.Some of them develop active disease soon after the infection, whereas, other people develop later when their immune system becomes weak for one or the other reason.The global prevalence of LTBI broadly reflects the local prevalence of TB.Those with documented contact with a case of transmissible TB are at the highest risk of infection (up to 50% chance).In low-incidence countries, the prevalence of infection in the general population is approximately 1% among young adults but can increase to 8% in elderly adults, 5and up to 25% in adult migrants from high-incidence countries.In some cases, can develop active tuberculosis if they do not receive treatment for latent tuberculosis.The Mantoux test is the standard method of determining whether a person is infected with Mycobacterium tuberculosis.The local skin reaction to Tuberculin Purified Protein Derivative (PPD) injected into the skin is used to assess the individual's sensitivity to tuberculin protein.

TB disease burden 17 :
Worldwide, tuberculosis (TB) is one of the top 10 causes of death, and the leading cause from a single infectious agent (above HIV/AIDS); millions of people continue to fall sick with the disease each year.
In 2017, TB caused an estimated 1.3 million deaths among HIV-negative people, and there were an additional 300,000 deaths from TB among HIV-positive people.

DRUG-RESISTANT TB (MDR and XDR) 18,19 :
Drug-resistant TB is caused by M. tuberculosis organisms that are resistant to the drugs normally used to treat the disease.
Drug-resistant TB is transmitted in the same way as drug-susceptible TB and is no more infectious than drugsusceptible TB.However, delay in the recognition of drug resistance or prolonged periods of infectiousness may facilitate increased transmission and further development of drug resistance.
Multidrug-resistant TB (MDR TB) is caused by organisms resistant to the most effective anti-TB drugs, Isoniazid and Rifampin.These drugs are considered first-line drugs and are used to treat most persons with TB disease.
Extensively drug-resistant TB (XDR TB) is a relatively rare type of drug-resistant TB.XDR TB is resistant to Isoniazid and Rifampin, plus any Fluoroquinolone and at least one of three injectable second-line drugs (i.e., Amikacin, Kanamycin, or Capreomycin).Because XDR TB is resistant to first-line and second-line drugs, patients are left with treatment options that are more toxic, more expensive, and much less effective.

Need for novel anti-tuberculosis agents
Several once-active Anti-tuberculosis drugs have now become inactive due to the ever-increasing rise in drug-resistant strains of tuberculosis.
Drug-resistant TB is a major public health concern.
A growing awareness of the increasing drug resistance and a great need for therapy shortening together with killing also latent forms of M.tb led to the discovery of more efficient and less toxic treatment regimens.
The emergence of Multi-resistant (MDR) strains and high susceptibility of human immunodeficiency virus (HIV) infected persons to the disease forced scientists to develop novel anti-tuberculosis agents.
It is evident from these facts; that there is an ever-growing need to develop novel agents for the treatment of tuberculosis.These new agents should be potent, fast acting, have an excellent Pharmacodynamics / Pharmacokinetics profile have a high therapeutic index, and preferably have a novel mechanism of action to avoid cross-resistance with other agents.

Current tb research and development:
A small number of technologies emerged in 2017-2018 and several have not demonstrated adequate performance in field evaluation studies.
There are 20 drugs in Phase I, II, or III trials for the treatment of drug-susceptible TB, multidrug-resistant TB, or latent TB infection.
Various combination regimens with new or repurposed drugs are in Phase II or Phase III trials 21 .
Twelve vaccine candidates are in clinical trials: Four in Phase I, Six in Phase II, and two in Phase III.
They include candidates to prevent the development of TB infection and disease, and candidates to help improve the outcomes of treatment for TB disease.

Drug discovery 22 :
Drug discovery is a process, which aims at identifying a compound therapeutically useful in treating and curing a disease.The process of drug discovery involves the identification of ligands, synthesis, characterization, screening, and assays for therapeutic efficacy.The phase between hit identification and lead selection is called the hit-to-lead phase.The dominant and the most widely applicable technique for the identification of lead compounds is HTS an experimental screening technique based on robotingwhere large numbers of different compounds are screened in a time as short as possible and at reasonable costs.

Computer-aided drug design in the drug discovery pipeline 24 :
CADD is capable of increasing the hit rate of novel drug compounds because it uses a much more targeted search than traditional HTS and combinatorial chemistry.It not only aims to explain the molecular basis of therapeutic activity but also to predict possible derivatives that would improve activity.CADD can be classified into two general categories:

Structure-based drug design 25 :
It relies on the knowledge of the target protein structure to calculate interaction energies for all compounds tested.The core hypothesis of this approach is that a molecule's ability to interact with a specific protein and exert a desired biological effect depends on its ability to favorably interact with a particular binding site on that protein.
Steps Involved in Structure-based Drug Design  Identification of drug target  Determination of target structure  Identification of binding site  Computational drug design methods  Evaluation of potential lead candidate

Ligand-based drug design
It exploits the knowledge of known active and inactive molecules through chemical similarity searches or the construction of predictive Quantitative Structure-Activity Relation (QSAR) models.The overall goal is to represent these compounds in such a way that the physicochemical properties most important for their desired interactions are retained, whereas extraneous information not relevant to the interactions is discarded.

Molеcular docking 26 :
Molecular Docking is the technique which еnvisagеs thе "prеfеrrеd orientation of one molеculе to a sеcond whеn bound to еach other to form a stable complеx in thrее dimensional spacеs".The success of a docking program depends on two components: the Search algorithm and the Scoring function.

search algorithm
The search algorithm finds different conformations for the ligand.Systemic searches explore all possible binding modes between the ligands and receptors.However, this takes a huge amount of computational time, especially for large flexible ligands.The amount of conformational space explored and the computational time required for the search must be balanced.

Scoring functions:
Scoring aims to quantify the free energy associated with protein and ligand in the formation of the protein-ligand interactions.They are used to rank the different conformations obtained by the search algorithm.The score in the empirical scoring function is derived from the individual energy contributions of each component involved in intermolecular interactions.ΔGbind = ΔG0+ΔGhb∑h-bonds+ΔGionic∑ionic-int+ΔGlipophilic│A │+ ΔGrotNROT Where: ΔG0-empirically derived offset that in part corresponds to the overall loss of translational and rotational entropy of the ligand upon binding.
|Alipo| is the surface area of lipophilic contact between the ligand and receptor.

Biological target 27
Various biosynthetic enzymes are essential for the survival of the Mycobacterium and are considered potential drug targets.Some of the target enzymes are,

Review of study about Tuberculosis:
Manishakotadiya et al., (2018)  37 , reported that the Advances in TB drug development over the past decade are leading to the development of enhanced MDR-TB treatments with simple and short regimens.Additionally, efforts must be made to reduce the development of resistance to these valuable new TB drugs during treatment 38 , reported that almost no resistance to the tested second-line Antituberculosis drugs among non-MDR-Mtbs.Anti-tuberculosis regimen with Pyrazinamide, Ethambutol, Fluoroquinolone, Kanamycin, Cycloserine, and p-Aminosalicylic acid can be empirically used for newly diagnosed MDR-TB cases. 39, confirmed that drug resistance, including MDR, observed against all first-line TB drugs was a real threat in the management of TB infection in Indonesia.The resistance pattern identified in this study could assist clinicians in providing appropriate treatment regimen to TB patients and improve their clinical outcome.

Cucunawangsih et al., (2015)
Padmanesan Narasimhan et al., (2013)  40 , summarized that the risk of progression from exposure to the tuberculosis bacilli to the development of active disease.Exogenous factors play a key role in accentuating the progression from exposure to infection among which the bacillary load in the sputum and the proximity of an individual to an infectious TB case are key factors.Similarly endogenous factors lead in progression from infection to active TB disease. 41, described the mechanisms by which the interactions among the antituberculosis drugs used in the basic regimen can cause drug induced hepatitis, and discussed about the alternatives in this situation.

Review of study about Enoyl acyl carrier protein reductase (InhA) enzyme:
Denise A. Rozwarski et al., (1999)  42 , reported the Crystal Structure of the Mycobacterium tuberculosis Enoyl-ACP Reductase, InhA, in Complex with NAD + and a C16 Fatty Acyl Substrate.(1994)  43 , reported that the EnvM protein was purified from an overproducing Escherichia coli strain.It showed NADH-dependent enoyl-acyl carrier protein (ACP) reductase activity using both crotonyl-ACP and crotonyl-CoA as substrates.It wasconcluded that EnvM is the NADH-dependent enoyl-ACP reductase of E. coli and we propose to rename the corresponding gene fabI.

Review of study about 1, 3, 4-Thiadiazole derivatives:
Sevgi et al., (2010) 44 were synthesized 5-[-4-(4fluorobezoylolamino) phenyl]-2-subsitutedamino-1,3,4-thiadiazole 27 and evaluate the cytotoxic activity. 45have been introduced the synthesis of various thiadiazolederivatives compounds and evaluated for anti diabetic activity. 46synthesized a series of 3-aryl amino/amino-4-aryl-5-imino-D2-1,2,4-thiadiazoline .The Anticonvulsant activity of all the synthesized compounds was evaluated against maximal electroshock induced seizures (MES) and subcutaneous Pentylenetetrazole (ScPTZ) induced seizure models in mice.Melby Mendoza-Aguilar et al., (2012)  62 concluded that the MABA appears to be a useful model for the selection of drugs that are effective for the treatment of murine leprosy.To further validate our results, a broader study involving the use of novel anti-mycobacterial agents and other agents, including particular anti-tuberculosis drugs, should be performed. 63applied the Alamar blue assay to determine the susceptibility to antituberculosis pharmaceuticals.It is a reliable method todetermining the drug susceptibility to pharmaceuticals.(1997)  64 reported the high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium using Microplate AlamarBlue Assay (MABA) and compared with BACTEC 460 Assay System.

Review of study about Spectroscopy:
reported an in silico evaluation of the ADMET profile of the Streptome DB database.An assessment of the "drug-likeness" and pharmacokinetic profile of >2,400 compounds of natural origin, currently available in the recently published Streptome DB database was also reported.

Review of study about
Salla Virtanen et al., (2013)  70 developed a novel method for virtual screening that employs the negative image of the binding site.They were shown that the VS results by this method are often better compared to docking and ligandbased VS.Drug design, sometimes referred to as rational drug design or simply rational design, is the inventive process of finding new medications based on the knowledge of a biological target. (72)The drug is most commonly an organic small molecule that activates or inhibits the function of a biomolecule such as a protein, which in turn results in a therapeutic benefit to the patient.In the most basic sense, drug design involves the design of molecules that are complementary in shape and charge to the biomolecular target with which they interact and therefore will bind to it.Drug design frequently but not necessarily relies oncomputer modeling techniques. (73)This type of modeling is often referred to as computer-aided drug design.Finally, drug design that relies on the knowledge of the threedimensional structure of the biomolecular target is known as structure -based drug design. (39)In addition to small molecules, biopharmaceuticals and especially therapeutic antibodies are an increasingly important class of drugs and computational methods for improving the affinity, selectivity, and stability of these protein-based therapeutics have also been developed. (74)ug design with the help of computers may be used at any of the following stages of drug discovery:  Hit identification using virtual screening (structure-or ligand-based design)  Hit-to-lead optimization of affinity and selectivity (structure-based design, QSAR, etc.)  Lead optimization optimization of other pharmaceutical properties while maintaining affinity

TYPES
There are two major types of drug design.The first is referred to as ligand-based drug design and the second, structure-based drug design. (75)

Ligand-based
Ligand-based drug design (or indirect drug design) relies on knowledge of other molecules that bind to the biological target of interest.These other molecules may be used to derive a pharmacophore model that defines the minimum necessary structural characteristics a molecule must possess in order to bind to the target. (76)

Structure-based
Structure-based drug design (or direct drug design) relies on knowledge of the three dimensional structureof the biological target obtained through methods such as x-ray crystallography or NMR spectroscopy. (77)If an experimental structure of a target is not available, it may be possible to create a homology model of the target based on the experimental structure of a related protein.Using the structure of the biological target, candidate drugs that are predicted to bind with high affinity and selectivity to the target may be designed using interactive graphics and the intuition of a medicinal chemist.Alternatively various automated computational procedures may be used to suggest new drug candidates. (78)

Binding Site Identification
If the structure of the target or a sufficiently similar homolog is determined in the presence of a bound ligand, then the ligand should be observable in the structure in which case location of the binding site is trivial.However, there may be unoccupied allosteric binding sites that may be of interest.Furthermore, it may be that only apoprotein (protein without ligand) structures are available and the reliable identification of unoccupied sit es that have the potential to bind ligands with high affinity is non-trivial.

Scoring functions
Structure-based drug design attempts to use the structure of proteins as a basis for designing new ligands by applying the principles of molecular recognition.Selective high affinity binding to the target is generally desirable since it leads to more efficacious drugs with fewer side effects.Thus, one of the most important principles for designing or obtaining potential new ligands is to predict the binding affinity of a certain ligand to its target (and known antitargets) and use the predicted affinity as a criterion for selection. (81)e early general-purposed empirical scoring function to describe the binding energy of ligands to receptors was developed by Bohm. (82),(83)This empirical scoring function took the form: Where: ΔG0 -empirically derived offset that in part corresponds to the overall loss of translational and rotational entropy of the ligand upon binding.

ΔGhb -contribution from hydrogen bonding
ΔGionic -contribution from ionic interactions ΔGlip -contribution from lipophilic interactions where |Alipo| is surface area of lipophilic contact between the ligand and receptor ΔGrot -entropy penalty due to freezing a rotatable in the ligand bond upon binding  85), ( 86), (87)  Docking is done by using ARGUS LAB Software  Protein preparation.

 Open pdb
Open 'residues' Open 'misc'  From 'Misc' delete the inhibitor and hetero residues [Note: Do not delete Co-factor]  Then I Opened water press shift, selected all water molecules and deleted. Added hydrogen atoms. Go to Calculation on the toolbar energy by UFF method start. Saved the prepared protein as *.agl file format in the desktop.

Q-SITE FINDER o
Step: 1  Open Q-Site finder through online. Upload / Import the PDB format of the Protein  Find all the active site and make a list out of the common amino acid residues.o Step: 2  Open residues open Amino acids. Press control and select the amino acid Which were listed from the Q-Site finder. Make sure that all amino acid residues listed are selected. Right click on the mouse make a group from the selected residues give name Binding site Ok.

Toxicity prediction
All the data set molecules were subjected to the toxicity risk assessment by using Osiris program, which is available online.The OSIRIS property Explorer shown in this page is an integral part of Actelion's in house substance registration system.It allows drawing chemical structures and also calculates various drug relevant properties whenever a structure is valid.
Prediction results are color coded in which the red color shows high risks with undesired effects like mutagenicity or a poor intestinal absorption and green color indicates drug-conform behavior. [88]lecular property prediction includes  Toxicity risk assessment  Clog P prediction  Solubility prediction  Molecular weight  Drug likeness prediction  Drug likeness score 4.1.11.Lipinski's rule of five (89), (90)   Lipinski's rule of five also known as the Pfizer's rule of five or simply the Rule of five (RO5) is to evaluate drug likeness or determine if a chemical compound with a certain pharmacological or biological activity has properties that would make it a likely orally active drug in humans.
The rule was formulated by Christopher A.Lipinski in 1997.The rule describes molecular properties important for a drug's pharmacokinetics in the human body, including their absorption, distribution, metabolism, and excretion ("ADME").However, the rule does not predict if a compound is pharmacologically active.
Lipinski's rule states that, in general, an orally active drug has no more than one violation of the following criteria:

conclusion
Decades of widespread and uncontrolled application of antibiotics in clinical has resulted in the emergence of drug resistant strains of M. tuberculosis.To face drug resistance, several methods are developed for the synthesis of new antitubercular compounds.Among many strategies that are actually used in the drug development, fragment-based drug discovery (FBDD) approach has emerged as a promising strategy.1,2-diazoles are more promising antitubercular drugs in future.Many analytical methods are used to characterize antitubercular antibiotics.Liquid chromatography and voltammetry are mostly preferred for the determination of antitubercular compounds.The redox (oxidation/reduction) properties of antituberculars make them analyzable by electrochemical methods.The quantification of the categories of antibiotics by voltammetry in both dosages forms and human body fluids seems to be the cheapest for developing countries.

Figure 2
Figure 2 Pathogenesis of Mycobacterium tuberculosis 11
Drug Design: Surabhi et al., (2018) 67 overviewed about the Computer aided drug design and development of a new drug.Ram BabuTripathi et al., (2016) 68 reviewed about the In-silico expectations of pharmaceutical industry to design of new drug molecules.Ntie Kang F et al.,(2015)


To study the docking studies drug design and docking software. To study the series of 1,2 diazole derivatives3.4.2.Plan of work Study of docking software  Prepare the series of 1,2-diazole derivatives 4. Docking studies drug design:(71)


Selection of active site (Q-Site finder). Ligand Preparation. Docking Procedure. Visualization / Interpretation of Docking.4.1.6.Protein preparation o Step: 1  Protein (pdb) ID is entered in the protein data bank.(1KPI)  I clicked the download files and select pdb as text file. Saved the downloaded pdb (text file) to the desktop.o Step: 2  After I Opened Argus lab file Open Imported pdb file from the desktop. 3D Structure of the protein will appeared in the workspace of Argus lab. Left side of the screen shows molecular tree view.

Table 1
Structural activity relationship for various compound


Draw the structure from Chem sketch and save as MDL Mol format. Imported the ligand into workspace of Argus lab. Cleaned Geometry, Cleaned Hybridisation. I Selected the ligand, Right click on the mouse Make a group from the residues give name ligand Ok.  DOCKING PROCEDURE  Selected the set up a Dock Ligand calculation from the toolbar. Argus Dock as the Docking Engine. Dock was selected as calculation type. Flexible for the scoring function.Molegro Molecular viewer will help in analysing the energies and interaction of the binding. View Secondary Structure view. View Hydrogen bond interaction. Ligand map Interaction overlay.