CANDIDATURE OF THE SYNTHETIC CASPASE INHIBITORS AS NEW ANTI-SARS-COV-2 DRUG DISCOVERY, IN-SILICO MOLECULAR DOCKING

CANDIDATURE OF THE SYNTHETIC CASPASE INHIBITORS AS NEW ANTI-SARS-COV-2 DRUG DISCOVERY, IN-SILICO MOLECULAR DOCKING

Adham M. M. Zaki ^{* 1}, Yasmin Moustafa Ahmed ² and El-Shimaa M. N. Abdelhafez ³

Department of Medicinal Chemistry ¹, Faculty of Pharmacy ³, Minia University - 61519, Minia, Egypt.

Department of Pharmacology and Toxicology ², Nahda University, Beni Suef Governorate, Egypt.

ABSTRACT: The new pandemic virus identified as severe acute respiratory syndrome Coronavirus 2 is the etiological agent responsible for the pneumonia outbreak that ended with respiratory failure. The main protease (M^PRO) is a key enzyme for SARS-CoV-2, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus. Here, we determine the mechanism-based for binding and interactions between the reported caspase inhibitors and M^PRO, by computer-aided drug design molecular docking. In addition to the prediction of physicochemical properties, pharmacokinetics, and drug-likeness profile in-silico as well as reviewing the previously reported antiviral activity of the caspase inhibitors and how could the viruses get an advantage from the apoptosis process. Molecular operating environment is used for docking between thirteen reported caspase inhibitors compounds and SARS-COV-2 main protease using HWH (~ {N}- [2- (5- fluoranyl-1~ {H}-indol-3-yl) ethyl] ethanamide) as a reference. The results have shown that almost all of the reported anti-caspase compounds revealed acceptable binding with good docking scores and favorable in-silico pharmacokinetic profile that might promise a rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.

3C-like protease, SARS coronavirus, Drug Discovery, Caspases, Molecular docking

INTRODUCTION: In November 2002, in Guangdong Province, China, a virus known as SARS-CoV was identified and involved in the extreme acute respiratory syndrome. By December 2019, after seventeen years coronavirus was identified again from Wuhan, china which able to transmit from human to human as an aerosol infection then causes severe acute respiratory syndrome corona virus (SARS-CoV-2) ¹.

In December 2020, SARS-CoV-2 had infected more than 65 million cases and 1, 501, 350 deaths across 213 countries ². Coronaviruses are envelo-pedpositive - sense single-stranded RNA viruses of 30 kb ³. It subdivided into four genera; α, β, γ and δ based on their genomic structure, α and β genera are a wide variety of host species infected mammals ^4-5.

Lymphocyte such as cytotoxic T cells and nature killer cells are playing an important role in viral infection management ⁶. From what’s been happening lately, it seems like lymphopenia is the major diagnostic inductor of SARS-COV-2 infection ⁷, where lymphocytes are depleted as a direct result of virus-induced apoptosis which causes suppression of immune response leading to combat viral infection along with a wide infection spread rate inside the body, multiple organ failures, and increase of mortality rate ^8-9.

Mechanism of Human Immune Response against SARS-COV-2 and SARS-COV-2 Patho-genesis:

Inflammatory Cytokines Role: The innate immune system can recognize the molecular structures that are produced by the invasion of the virus, which is called pathogen‐associated molecular patterns (PAMPs) ¹⁰. Coronavirus is considered RNA virus where PAMPs are founded in the shape of viral genomic double‐stranded RNA which are recognized by several events lead to activation of specific signaling pathways such as nuclear factor (NF‐κB), activator protein 1 (AP‐1), interferon response factor 3 (IRF3), IRF7 lead to stimulation of many molecules which required for inflammatory responses, including inflammatory cytokines (e.g., [TNF] tumor necrosis factor and IL‐1). Interferon type I (IFN‐α and IFN‐β) production is promoted by IRF3 and IRF7, which are important for the antiviral activity of the innate immunity and ultimately able to suppress viral replication at an early stage. This process can cause complications such as cough, exhaustion, and weakness in patients ¹¹.

In the case of SARS-COV-2 infection, low-grade fever and cough are moderate signs although approximately 15% of cases are related to respiratory compromise; on the opposite hand, severe cases lead to acute respiratory distress syndrome (ARDS) with systemic inflammation within which lung injury is related to the discharge of inflammatory cytokines which plays a very important role in immunopathology during infection ¹².

There is significant literature implicating the delayed release of cytokines and chemokines occurs in respiratory epithelial cells, dendritic cells (DCs), and macrophages at SARS-COV-2 early stages of infection ¹³. However, within the late stages, the cells secrete low levels of the antiviral factors interferon’s (IFNs) and high levels of (IL)-1β, IL-6, tumor necrosis factor (TNF), CCL-2, CCL-3, and CCL-5 ^14-15. Recent literature implicating that the Nod-like receptor familypyrin domain containing 3 (NLRP3) inflammasome and cytokine storm together plays an important role in SARS-COV-2activation of innate immunity to recognize pathogens in viral infections pathogenesis ^16-17. However, we SARS-COV 3aprotein activates the NLRP3 in lipopoly-saccharide-primed macrophages with 3a-mediated IL-1β secretion associated with K+ efflux and mitochondrial reactive oxygen species (ROS) ¹⁸. By contrast, SARS-COV-2 patients with mild symptoms were experienced lower levels of IL-6, together with activated T lymphocytes and IgM SARS-CoV-2- binding antibodies ¹⁵. The recent studies reported an increase in inflammatory cytokine response mediates severe disease; however, the low responses are also related to an adaptive response that favors disease resolution ¹⁹. The response of immunity to production IFN-I or IFN-α/β reflects the key natural immune defense response against viral infection, as IFN-I is that the fundamental molecule that has an important role mechanism as an antiviral within the early stages of viral infection ²⁰. In the early SARS-COV-2 infection, delayed IFNs release delays the body antiviral response. Later, the rapid increase in both cytokines and chemokines appeal to several inflammatory cells leading to excessive infiltration of the inflammatory cells resulting in tissue injury ²¹.

These studies indicate that the unorganized and overproduction of both cytokine and chemokine and IFN-I delayed responses by SARS-CoV-infection that would play an important role in the pathogenesis of SARS-2, looks as If lymphopenia is that the major diagnostic inductor of SARS-COV-2 infection ⁷, where lymphocytes are depleted as a direct result of virus-induced apoptosis ²² which causes suppression on immunologic response resulting in combat viral infection together with a large infection spread rate inside the body, multiple organ failures and increase of mortality rate ⁹.

Apoptosis between Immunity and SARS-COV-2 Infection Strategies: Apoptosis or programmed cell death is one of the processes that regulate many cellular functions such as homeostasis between multicellular organisms and limit viral spreading across human cells ²³. It could be a common host cell response against viral infection as it limits viral spreading by removing infected cells ²⁴. A family of cytokines called interferons plays a significant role within the regulation of apoptosis by stimulating cytotoxic T cells (CTLs) and many intracellular genes that directly induce apoptosis and prevent virus replication ²⁵. Viruses that encode anti-apoptotic protease are likely to inhibit the apoptosis process to facilities its replication and spreading ²⁶. Several similar viruses encode Bcl-2 homologs, which inhibit activation of apoptotic caspases and block the death of the host cell ²⁷ as well as interfering with other pro-apoptotic molecules, for example papillomavirus E7 and HHV-8 that encodes several versions of the IRFs, which interferes with normal IRF function ²⁸. Many viruses take advantage of apoptosis to facilitate its replication ^27; the major protein involved in the apoptosis process is a family of cysteine-dependent aspartate-directed proteinases called caspases ²⁹. Caspases have a vital role in diverting non-apoptotic processes like migration and differ-rentiation ²⁷, the lytic infection by virus-induced apoptosis leads to the formation of apoptotic bodies that are absorbed by neighboring cells, which help in dissemination progeny as well as limiting immune responses ³⁰.

Structural Proteins Implicate Caspase Induced Apoptosis by SARS-COV-2: SARS‐CoV‐2 has four main structural proteins, Spike (S), Envelope (E), Nucleocapsid (N), and M membrane (M) ³¹. SAR-COV-N protein cleavage is essential for viral replication, which depends on host cell caspase cleavage ^32-33.

The more surprising correlation is with SARS-COV-2 infected cells upon treatment with pan-caspase inhibitor Z-VAD-FMK N-protein cleavage were also inhibited because of the reduction in viral replication ³³.

Membrane Protein: SARS-CoV-2 membrane protein is considered the most concentrated protein in viral envelope ³⁴. The M protein facilitates membrane fusion and controls viral replication; furthermore, viral packaging RNA in matured virions ³⁵. It has been demonstrated that M is abundant within the Golgi body of viral-infected cells ³⁶ inductions of apoptosis by overexpression of M protein in HPF cells are reported ³⁷. M protein induces apoptosis in host cells by activates caspase 3-8 and -9.

Moreover, over-expression of the M protein results in down-regulation of Akt phosphorylation as shown in Fig. 1, which reducing cell survival signal and induced apoptosis; additionally, misloca-lization of mitochondrial cytochrome C and nuclear condensation was observed in M-expressing cells ^38-39.

FIG. 1: THE DIAGRAM IS SHOWING THE DYNAMIC INTERACTION NETWORK BETWEEN SARS-COV-2 AND HOST CELL WHICH INVOLVING S-GLYCOPROTEIN BINDING TO THE ACE2 RECEPTOR, WHICH ENHANCING VIRAL ENTRY VIA ENDOCYTOSIS AND SUBSEQUENT UN-COATING AND RELEASING VIRAL GENOME. CELLULAR FUNCTIONS IN VARIOUS COMPARTMENTS (CYTOPLASM, PLASMA MEMBRANE, ER, GOLGI, ERGIC NUCLEUS, AND MITOCHONDRIA) MAY BE INTERFERED WITH SARS-COV-2 PROTEINS EVENTUALLY LEAD TO ACTIVATION OF CASPASE CASCADE, ER STRESS, MITOCHONDRIA DYSFUNCTION, RELEASING OF CYTOCHROME. THE ABBREVIATION: ANGIOTENSIN-CONVERTING ENZYME 2 (ACE2), THE ENDOPLASMIC-RETICULUM–GOLGI INTERMEDIATE COMPARTMENT (ERGIC), THE OPEN READING FRAME (ORF) 7A, C-JUN N-TERMINAL KINASES (JNKS), B-CELL LYMPHOMA-EXTRA-LARGE (BCL-XL), NUCLEAR FACTOR KAPPA-LIGHT-CHAIN-ENHANCER OF ACTIVATED B CELLS NF-ΚB, FOCAL ADHESION KINASE (FAK), A MITOGEN-ACTIVATED PROTEIN KINASE (MAPK)

Nucleocapsid Protein: SARS coronavirus N protein is a pro-apoptotic protein; it regulates activation of many pathways slike virus life cycles, cell proliferation, host cell apoptosis ⁶, AP-1 signal transduction pathway, caspase-3, -7 and 9 ^42-40. The N protein cleavage is essential for viral replication ³². The mechanism of activation of caspase cascade by N protein is not clear, but it was reported that N protein activates apoptosis through activation of mitochondria pathway by increasing the release of cytochrome C and ROS into cytosol ⁴³. Interestingly, the Caspase-3,-6 are involved in N protein cleavage, which explains why are viral replication reduced upon the addition of pan-caspase inhibitor Z-VAD-FMK ³³. However, the mechanism is not fully known, but there is two possibilities either N is translocated into the nucleus, leading to the activation of caspase-6 or N can be imported into the nucleus only if casapse-6 is involved during apoptotic processes ⁴⁴. Also, down-regulation of FAK (focal adhesion kinase) and fibronectin expression by SARS-CoV have been reported ⁴⁰.

SARS-CoV-2 Envelope Protein: SARS-CoV-2E protein shares high sequence similarities to SARS-CoV-1 E protein, which is strongly conserved in N-term regions and it is the smallest of all 8–12 kDa structural proteins ^45-46. SARS-CoV-2 E proteins share a conserved Bcl-2 Homology 3 (BH3)-like motif in their C-terminal region; a well-studied motif shown to be necessary for SARS-CoV E binding to Bcl-xL47; it has been found that apoptosis is promoted by Ectopic expression of SARS-CoV E ³¹.

Accessory Proteins Implicate Caspase Induced Apoptosis by SARS-COV-2: Eight putative accessory proteins are encoded on the SARS-Co V genome (ORFs 3a, 7a, 3b, 7b, 6, 8a, 9b and 8b), which does not have any homologs ⁴⁸. In other words, there are three main SARS-CoV-2 accessory proteins involved in the induction of apoptosis ⁴⁹.

First, ORF-3a (also called U2 74, SARS X1, or ORF-3) is suggested to be located in several sub-cellular compartments as its multifunctional protein 50 causes caspase 8 cleavages to facilitate nuclear translocation of viral component ⁵¹ and ultimate maturation of viral particles ^49-52.

On the other hand, ORF-3b, also called ORF-4, which is a predominantly localized protein in the nucleolus and partially in the mitochondria, plays a role in G0/G1 arrest and apoptosis indication in transfected cells ⁵³. ORF-7a (also known as X4 or ORF-8) that encodes a type I transmembrane protein located in the cell surface and Golgi apparatus ^54-55, consisting of a 15 residue N-terminal signal peptide, an 81 residue luminal domain, a 21 residue transmembrane segment, 122 amino acid and a 5 residue cytoplasmic tail ⁵⁵. ORF-7a not only induce apoptosis but also inhibit cellular protein synthesis through activating p38 mitogen-activated protein kinase (MAPK) signaling pathway, activate NF-κB56, block cell cycle progression, and interact with the cellular transcription factor small glutamine-rich tetratricopeptide repeat-containing protein (SGT); furthermore the anti-apoptotic regulator Bcl-XLthis could imply that the induction of apoptosis by these different proteins occurs at different stages of the infection cycle ^57-52. The encoding of these pro-apoptotic proteins on the SARS-COV-2 genome suggests that apoptosis is useful for the cleavage of viral protease, which facilitates SARS-COV-2 replication ⁵⁸.

Main Protease (MPRO): SARS-COV-2 Main protease (MPRO) or chymotrypsin-like cysteine protease named 3C-like protease (3CLpro) enzymes are required for translation of polyprotein from viral RNA ⁵⁹ and viral replication ⁶⁰. The auto-cleavage of polyprotein to 1a and 1ab by main protease and papain-like protease is essential for SARS-COV maturation and replication; also, caspase-3 cleavage is considered as the most important step on polyprotein cleavage ⁶¹. Therefore, it would be suggested that inhibiting the caspase-3 enzyme would block viral replication ⁶². The MPRO Inhibitors are unlikely to be toxic as no reported human protease with the identical cleavage specificity is known ⁶³. A significant increase in ROS level and inhibition of AP1-dependent transcription, likewise as activation of NF-κB-dependent reporter  caspase-3 and caspase-9 on the cell expressing SARS-CoV 3CLpro was reported ⁶⁴ which induct that 3CLpro activate a series of signaling events ultimately leading to programmed cell death or apoptosis induced by SARS-COV-2 ⁶⁵.

The Antiviral Activity of Caspases Inhibitors:

TABLE 1: CASPASE INHIBITORS AND THEIR ANTIVIRAL ACTIVITY WITH THEIR 2D STRUCTURE IN SEVERAL RECORDED STUDIES DEMONSTRATING THE ROLE OF CASPASE INHIBITORS AS ANTIVIRAL ACTIVITY

Compound Name	Stracture/  Iupac Name	Antiviral Effect
Z-FA-FMK	compound  1benzyl N-[(2S)-1-[(4-fluoro-3-oxobutan-2-yl)amino]-1-oxo-3-phenylpropan-2-yl]carbamate	It selectively inhibits caspases 2, 3, 6 and irreversible inhibitor of cysteine proteases like cathepsins B, L, and S Which caused a reduction in reovirus orthoreovirus (PRV) replication as well as aquareovirus (CSRV), and rhabdovirus (IHNV) replication 66, 67-68
Z-VAD-FMK	compound  2 methyl (3S)-5-fluoro-3-[[(2S)-2-[[(2S)-3-methyl-2-(phenylmethoxycarbonylamino)butanoyl]amino]propanoyl]amino]-4-oxopentanoate	A pan-caspase inhibitors, which inhibit the proteolytic activity of the CVB3 proteases 2A and 3C which cause a reduction on both viral RNA and viral proteins  69
Q-VD-OPH	compound  3(3S)-5-(2,6-difluorophenoxy)-3-[[(2S)-3-methyl-2-(quinoline-2-carbonylamino)butanoyl]amino]-4-oxopentanoic acid	A pan-caspase inhibitors, which suppress the release of newly produced CVB3 from infected cells which suppressed the production of viral progeny 70
Z-LEHD-FMK	compound  4methyl (4S)-5-[[(2S)-1-[[(3S)-5-fluoro-1-methoxy-1,4-dioxopentan-3-yl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-4-[[(2S)-4-methyl-2-(phenylmethoxycarbonylamino)pentanoyl]amino]-5-oxopentanoate	A caspase 9 inhibitor, inhibits the pro-apoptotic activity of SADS-CoV-271 and reduce the replication of HIV-1,and SADS-CoV-2 71, 72-73
IETD-FMK	compound  5(4S)-4-[[(2S,3S)-2-amino-3-methylpentanoyl]amino]-5-[[(2S,3R)-1-[[(2S)-1-carboxy-4-fluoro-3-oxobutan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-5-oxopentanoic acid	A caspase 8 inhibitor produced modest inhibition of apoptosis and subsequent HIV-1 replication as well as it preserves and maintains HIV-1-specific immune responses 72-74
Z-ASP-CH2-DCB	compound  1(3S)-5-(2,6-dichlorobenzoyl)oxy-4-oxo-3 (phenylmethoxycarbonylamino) pentanoic acid	A caspase-3 inhibitor that significantly reduced the replication of highly pathogenic avian influenza virus A/FPV/Bratislava/79 (H7N7) and calicivirus 75-76
Ac-DEVD-CHO	compound  2(4S)-4-[[(2S)-2-acetamido-3-carboxypropanoyl]amino]-5-[[(2S)-1-[[(2S)-1-carboxy-3-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-oxopentanoic acid	A caspase-3 inhibitor responsible for a pronounced inhibition of ADV viralgenomic replication, gene expression on and viral titer 51 A caspase-3 inhibitor
Z-YVAD-fmk	compound  8 methyl (3S)-5-fluoro-3-[[(2S)-2-[[(2S)-2-[[(2S)-3-(4-hydroxyphenyl)-2-(phenylmethoxycarbonylamino)propanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-4-oxopentanoate	A Caspase-1 inhibitor that reduced oncolytic herpes simplex virus replication and oncolytic herpes simplex virus 77
Z-VEID-FMK	compound  9 methyl (4S)-5-[[(2S,3S)-1-[[(3S)-5-fluoro-1-methoxy-1,4-dioxopentan-3-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-4-[[(2S)-3-methyl-2-(phenylmethoxycarbonylamino)butanoyl]amino]-5-oxopentanoate	A Caspase-6 inhibitor which preventing an Arbovirus from acquiring a replicative advantage by delayed mosquito bites as well as it inducedcutaneous innate immune responses 78. Also, it reduced the classic swine fever virus replication 51-78
Z-WEHD-FMK	compound  10methyl (4S)-5-[[(2S)-1-[[(3S)-5-fluoro-1-methoxy-1,4-dioxopentan-3-yl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-4-[[(2S)-3-(1H-indol-3-yl)-2-(phenylmethoxycarbonylamino)propanoyl]amino]-5-oxopentanoate	A caspase-1 inhibitor which rescued more than half of the cells undergoing HCV- PCD and inhibits HCV replication 79
Z-DEVD-FMK	compound  11 methyl (4S)-5-[[(2S)-1-[[(3S)-5-fluoro-1-methoxy-1,4-dioxopentan-3-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-[[(2S)-4-methoxy-4-oxo-2-(phenylmethoxycarbonylamino)butanoyl]amino]-5-oxopentanoate	It is a caspase-3 inhibitor thatwas used for reducing SARS-COV dissemination by inhibiting caspase-3 activation caused by the ORF-6 induced apoptosis pathway 80. Furthermore, Z-DEVD-FMK inhibits Fowl Adenovirus Serotype 4 (FAdV4) replication 81

METHODS AND RESULTS:

Molecular Docking Results: Ligand-protein docking was carried out for all reported compound 1-13 and N-[2-(5-fluorenyl-1H-indol-3-yl)ethyl] ethanamide (HWH) 14 as a reference for the purpose of prediction the binding interactions between all reported caspase inhibitor (anti-apoptotic) compounds and SARS-CoV-2 protease enzyme that obtained from protein data bank (PDB:5R7Z) using version 2014.09 of Molecular Operating Environment (MOE®) software and for validation of the method we used HWH as reference. The theoretical prediction results obtained from the molecular docking study were found to be a good illustration of observed SARS-CoV-2protease inhibition activity.

FIG. 2: A REFERENCE COMPOUND FOR PREDI-CTION THE BINDING INTERACTIONS BETWEEN ALL REPORTED CASPASE INHIBITOR (ANTI-APOPTOTIC) COMPOUNDS AND SARS-COV PRO-TEASE ENZYME

All studied compounds were successfully docked into the active site of the SARS-CoV protease enzyme. The binding free energies from the major docked poses are listed in Table 1, and the most favorable poses of the tested compounds are shown in Fig. 2-15. Most of the tested compounds have a high binding of the reference compound HWH is completely consistent with the mode of action of tested anti-apoptotic compounds. The 2D & 3D diagrams showed a crucial binding ARG298 with phenyl ring functionality. However, compounds 2-5, 7, 8, 10-13 showed greater binding as well as docking scores than the reference. Docking Results with SARS-CoV protease revealed that all of the tested compounds show a good binding with ARG298 amino acid residue in addition to several interactions comparable to that of the reference HWH. Compounds 9 Fig. 10 exhibited the same interaction with the conserved hydrophobic interaction (Pi-H) with ARG298 as HWH, both compounds 10 and 13 possess the greatest interaction amongst the tested compounds to interact with the same amino acid with additional hydrogen bonding with MET6 amino acid residue and hydrogen bonding with GLY302 and THR304 Fig. 11 and 14.

Although compounds 2, 4, 5, 7, and 11 showed hydrogen bonding with MET6 amino acid residue, besides, compounds 4, 11, and 12 exhibited hydrogen bonding interaction with CYS300, while compounds 1 and 3 only showed interaction with TYR154 Fig. 2-15. On the other hand, compound 6 Fig. 9 dismissed hydrogen binding interactions with the amino acid residue ARG 298, explaining its weak activity, and compounds 5 and 8 kept only hydrogen bond interaction with SER1 Fig. 9.

TABLE 2: MOLECULAR DOCKING DATA FOR COMPOUNDS 1-13 AND HWH IN SARS-COV-2 ACTIVE SITE (PDB ID 5R7Z

ΔG (Kcal/mole) a; the binding free energies

FIG. 3: THE BINDING MODE OF COMPOUND 1 INTERACTED WITH THE SARS-COV-2 ACTIVE SITE (PDB ID 5R7Z) IS SHOWN AS 2D AND 3D DIAGRAMS

FIG. 4: THE BINDING MODE OF COMPOUND 2 INTERACTED WITH THE SARS-COV-2 ACTIVE SITE (PDB ID 5R7Z) IS SHOWN AS 2D AND 3D DIAGRAMS

FIG. 5: THE BINDING MODE OF COMPOUND 3 INTERACTED WITH THE SARS-COV-2 ACTIVE SITE (PDB ID 5R7Z) IS SHOWN AS 2D AND 3D DIAGRAMS

FIG. 6: THE BINDING MODE OF COMPOUND 4 INTERACTED WITH THE SARS-COV-2 ACTIVE SITE (PDB ID 5R7Z) IS SHOWN AS 2D AND 3D DIAGRAMS

FIG. 7: THE BINDING MODE OF COMPOUND 5 INTERACTED WITH THE SARS-COV-2 ACTIVE SITE (PDB ID 5R7Z) IS SHOWN AS 2D AND 3D DIAGRAMS

FIG. 8: THE BINDING MODE OF COMPOUND 6 INTERACTED WITH THE SARS-COV-2 ACTIVE SITE (PDB ID 5R7Z) IS SHOWN AS 2D AND 3D DIAGRAMS

FIG. 9: THE BINDING MODE OF COMPOUND 7 INTERACTED WITH THE SARS-COV-2 ACTIVE SITE (PDB ID 5R7Z) IS SHOWN AS 2D AND 3D DIAGRAMS

FIG. 10: THE BINDING MODE OF COMPOUND 8 INTERACTED WITH THE SARS-COV-2 ACTIVE SITE (PDB ID 5R7Z) IS SHOWN AS 2D AND 3D DIAGRAMS

FIG. 11: THE BINDING MODE OF COMPOUND 9 INTERACTED WITH THE SARS-COV-2 ACTIVE SITE (PDB ID 5R7Z) IS SHOWN AS 2D AND 3D DIAGRAMS

FIG. 12: THE BINDING MODE OF COMPOUND 10 INTERACTED WITH THE SARS-COV-2 ACTIVE SITE (PDB ID 5R7Z) IS SHOWN AS 2D AND 3D DIAGRAMS

FIG. 13: THE BINDING MODE OF COMPOUND 11 INTERACTED WITH THE SARS-COV-2 ACTIVE SITE (PDB ID 5R7Z) IS SHOWN AS 2D AND 3D DIAGRAMS

FIG. 14: THE BINDING MODE OF COMPOUND 12 INTERACTED WITH THE SARS-COV-2 ACTIVE SITE (PDB ID 5R7Z) IS SHOWN AS 2D AND 3D DIAGRAMS

FIG. 15: THE BINDING MODE OF COMPOUND 13 INTERACTED WITH THE SARS-COV-2 ACTIVE SITE (PDB ID 5R7Z) IS SHOWN AS 2D AND 3D DIAGRAMS

FIG. 16: THE BINDING MODE OF COMPOUND 14 INTERACTED WITH THE SARS-COV-2 ACTIVE SITE (PDB ID 5R7Z) IS SHOWN AS 2D AND 3D DIAGRAMS

Computational Analysis:

Prediction of Physicochemical Properties, Pharmacokinetics and Drug-Likeness Profile in-Silico: Because of the costs required for the production of a new drug, in addition to inappropriate ADME parameters (distribution, excretion, absorption, and metabolism), design and applied new drugs is considered to be complicated. Therefore, evaluating the pharmacokinetic properties of a new drug is a critical step in the process of drug development and may lead directly to optimizing efforts to recover analogs ⁸².

Currently, the most promising compounds can be picked in-silico ADMET screens, reducing the chance of degradation of drugs in late stages ⁸³. To achieve a desired in-vivo response, it should be balance between pharmacodynamics and pharmacokinetic properties. Further, information about regimen and drug dose is given by the prediction of brain penetration, volume of distribution, oral bioavailability, and clearance ⁸⁴. Many parameters such as drug solubility S, partition coefficients, polar surface PSA, cell permeability, human intestinal absorption HIA and drug-likeness score have been studied during virtual screening methods. An available orally drug elected in agreement with Lipinski's rule if the molecular weight is less than 500 and LogP is not higher than 5, the number of hydrogen bond acceptors is less than 10  and the number of donor hydrogen bond donors is less than 5 ⁸⁵.

The number of rotatable bonds reflects molecular flexibility that plays an important role in oral bioavailability and means less orally active in a flexible molecule. The number of hydrogen bonding groups has also been suggested as a consideration to substitute for the polar surface area (PSA) and also to measure the percentage absorption (% ABS) as it is in inversely proportional to tPSA

% ABS = 109 − 0.345 tPSA.

The higher oral bioavailability was exhibited by Compounds with tPSA of less than 140 A2 and 10 or fewer rotatable bonds ⁸⁴. Herein, we used Pre-ADMET ⁸⁵, Molinspiration ⁸⁶, Molsoft ^87, and Swiss ADME software ⁸² for predicting the pharmacokinetic parameters of the reported compounds. The results are shown in Table 2, Fig. 17 Aexhibit that compounds 1, 2 and 5 obey the Lipinski's rule with LogP values from 1.76 to 3.1 7 (<5), aMW range from 386.42 to 467.49 (<500) and HBD from 2 to 5 (≤ 5) and HBA from 5 to 7 (<10). They would theoretically show a strong oral absorption and this property cannot be attributed to variations in their bioactivity. Besides, the topological PSA values of the compounds ring between ⁸⁴. 50 and 119.00A2 (< 140A2) and the corresponding percentage oral absorption was between ⁸⁰, 54 and 60, 73% exhibit strong permeability, absorption and transport across biological membrane.

Furthermore, the drug-likeness model score and solubility for compounds was confirmed by Mol soft software Table 3, Fig. 17B. Aqueous solubility can change the absorption and distribution characteristics. The more positive drug-likeness model scores, the more likely it is to be a drug molecule; these compounds have fulfilled their solution ability specifications at values between 1.80 and 5.43 mg / l (above 0.0001 mg / L). Positive model-scores (0.07 and 0.29, respectively) were anticipated for compounds 3 and 11, while that for other compounds was negative (0.38 to -1.74). Additionally, the following pharmacokinetic parameters were experimented in-silico using Pre-ADMET software; blood-brain barrier partition coefficient (BBB), cytochrome inhibition of cytochrome P4502D6 (CYP2D6), Caco2, coefficient (human colon adenocarcinoma), MDCK (Madin-Darby canine kidney cells) permeability coefficient, human intestinal absorption (HIA) and human plasma-protein binding  (PPB).

The results of the ADME parameters are shown in Table 4 Fig. 17C, findings from compounds with moderate CNS absorption ranges between 0.0324858 and 0.165928 (≤ 0.1); investigated compounds exhibited medium to low cell permeability in Caco-2 the MDCK models range from 13.105, 20.908 nm/s, and 0.043 to 1.14061 nm/s, respectively.

This is aligned with non-inhibitors of the CYP2D6 enzyme and thus may pretend no interactions with CYP2D6 inhibitors and/or inducers. Furthermore, they showed high human intestinal absorption values, 31.582 to 80.537% (≤80%), indicating very well-absorbed compounds. The examined compounds were found to be highly-bound to human plasma proteins from 22.99 to 91.41%.

FIG. 17: ANALYSIS OF THE PHYSICOCHEMICAL PROPERTIES OF ANTI-APOPTOTIC COMPOUNDS 1-13 DISPLAYED IN TERMS OF (A) LIPOPHILICITY PARAMETERS, (B) LIPINSKI DRUG-LIK` ENESS

TABLE 3: PHYSICOCHEMICAL AND LIPOPHILICITY OF THE TARGET COMPOUNDS USINGSWISSADME & MOLINSPIRATION SOFTWARE PHYSICOCHEMICAL PROPERTIES, (C) ADME DATA

TABLE 4: LIPINSKI DRUG-LIKENESS OF THE TARGET COMPOUNDS USING MOLSOFT & SWISSADME SOFTWARE

^aS: solubility ^aMW, molecular weight; ^bMR, molar refractivity; ^cTPSA, topological polar surface area; ^d% ABS: percentage of absorption.

TABLE 5: ADME DATA OF TESTED COMPOUNDS CALCULATED USING PREADMET SOFTWARE

^aBBB: blood-brain barrier penetration; ^bCACO-2: permeability through cells derived from human colon adenocarcinoma; ^cHIA: percentage human intestinal absorption; ^dMDCK: permeability through Madin-Darby canine kidney cells; ^ePPB: plasma protein binding; ^fCYP2D6: cytochrome P450 2D6

CONCLUSION: The theoretical predictions results obtained from the molecular docking study were found to be a good illustration of observed SARS-CoV protease inhibition activity that plays a key role in apoptosis. The 2D & 3D diagrams showed a crucial binding ARG298 with phenyl ring functionality; however, compounds 2-5, 7, 8, 10-13 showed greater binding as well as docking scores than the reference. On the other hand, SARS-CoV-2 protease revealed that all the tested compounds show a good binding with ARG298 amino acid residue in addition to several interactions comparable to that of the reference HWH.

ACKNOWLEDGEMENT: The authors are thankful to Ahmed M. R. Eisa, Minia, Egypt for his efforts in reviewing the linguistic errors of Manuscript and DR Mahmoud said, faculty of pharmacy tanta university for check the plagiarism for our manuscript.

CONFLICTS OF INTEREST: We declare that we have no conflicts of interest.

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    Zaki MMA, Ahmed YM and Abdelhafez MNE: Candidature of the synthetic caspase inhibitors as new anti-sars-cov-2 drug discovery, in-silico molecular docking. Int J Pharm Sci & Res 2021; 12(1): 104-19. doi: 10.13040/IJPSR.0975-8232.12(1).104-19.

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