Targeting Caspases 3/6 and Cathepsins L/B May Decrease Laminopathy-Induced Apoptosis in Alzheimer’s Disease

Abstract

Background:

Laminopathy is a pathological manifestation observed in Alzheimer’s disease (AD), leading to neuronal apoptosis.

Objective:

Our objective was to assess inhibitors of enzymes involved in laminopathy.

Methods:

The mRNA expression of the cathepsins L and B, caspases 3 and 6, lamins b1 and b2, granzymes A and B, and lamins A and C were extracted and analyzed from GSE5281 and GSE28146 datasets. A total of 145 ligands were selected for molecular docking. Subsequently, 10 ns and 100 ns atomistic molecular dynamics (MD) and Martini 3 were performed with NAMD for two selected ligands (PubChem id: 608841 and ChEMBL id: 550872).

Results:

The mRNA expression level highlighted caspase 6 and lamin A/C upregulation in the hippocampus of the AD samples, in contrast to cathepsin B, lamin b2, and caspase 3. Moreover, there was a strong correlation between the expression level of cathepsin B, lamin A/C, and caspase 6 in the AD group. The MD results suggested molecule with ChEMBL id of 550872 had higher free binding energy, while in longer simulation the molecule with PubChem id of 608841 was suggested to be more stable in complex with the receptor.

Conclusions:

Our findings suggest that lamins A/C, cathepsins B/L, caspase 6, and lamin B2 are associated with laminopathy as potential factors contributing to apoptosis in AD. We propose that simultaneous inhibition of caspases 6 and cathepsins L may decrease the rate of apoptosis triggered by lamin degradation. Nevertheless, further studies are required to confirm these observations due to the lack of in vivo findings.

Keywords

Alzheimer’s disease laminopathy molecular dynamics transcriptome

INTRODUCTION

Alzheimer’s disease (AD) is a neurodegenerative disorder in which immunological conditions, such as neurotoxic immuno-inflammation concurrent with cytotoxic oligomerization of amyloid-β (Aβ) and tau, play a significant role in the interdependent immunopathic and proteopathic pathogeneses of the disease. 1 It is manifested by a specific pathophysiological condition referred to as laminopathy. Approximately 40 million individuals worldwide are afflicted by dementia, a figure expected to triple by 2050. This surge is attributed to the global population’s aging and the absence of efficacious treatments.2,3, 2,3

Laminopathy is a condition in which the structure of the nucleoskeleton constructed by lamin is disrupted.4,5, 4,5 Multiple enzymes were suggested to be responsible for lamin degradation, among which upregulated cathepsin L was reported as an inducer of lamin b1 cleavage, leading to modification of epigenetic factors in AD. 6 Caspase-6 is another factor involved in laminopathy by acting on lamin b2 with the mediation of multiple enzymes, including Cyclin-dependent kinase 5. 7 In AD, the amyloid precursor protein intracellular domain (ACID) is cleaved from the amyloid-β protein precursor (AβPP) triggering the signaling pathway, resulting in laminopathy in the condition of Aβ aggregation. 8 Correspondingly, ACID bound to the Fe65 is involved in modulating the expression level of the AβPP, glycogen synthase kinase-3 beta (GSK3β), neprilysin (NEP), and Beta-Secretase 1 (BACE1). 8 Caspase 3 is one of the proteases acting on ACID resulting in the production of carboxyterminal 31 residues (C31) and juxtamembrane fragment of AβPP produced by caspase cleavage and gamma-secretase cleavage (Jcasp), leading to apoptosis. 9 Apoptosis, a key pathological feature observed in AD, is characterized by morphological changes in the cell, such as chromatin condensation, DNA fragmentation, and membrane blebbing, ultimately leading to the death of the cell. 10

Likewise, cathepsin B is another protease that cleaves ACID. Accordingly, the upregulated level of cathepsin B correlated with the severity of AD. 11 Eventually, in the study conducted by Zhang et al., it was specified the role of granzyme A and B in the cleavage of the lamin B. In addition, lamins A and C were indicated as the substrates of granzyme A, unlike granzyme B. 12 In the current study, the expression levels of granzymes A and B, cathepsins B and L, caspases 3 and 6, and lamin b2 involved in laminopathy were evaluated. Furthermore, a drug screening was conducted to find the potential small molecules inhibited enzymes that participated in lamincleavage.

METHODS

Transcriptome analysis

The transcriptome data of the AD and normal individuals were acquired from the GSE5281 and GSE28146 datasets from the following web address (https://www.ncbi.nlm.nih.gov/geo/). A total number of 53 samples, including 32 AD and 21 normal samples of the hippocampus were analyzed using the GPL570 (HG-U133_Plus_2) platform using Affymetrix Human Genome U133 Plus 2.0 Array. Data were normalized using the RMA function of the R package “affy”. The preprocessing steps were background correction, standardization, summarization, and log transformation. In this case, the mRNA expression level of granzyme A, granzyme B, cathepsin B, cathepsin L, caspase 3, caspase 6, and lamin b2 was determined. Outlier samples were assessed through hierarchical cluster analysis by calculating Euclidean distance. In this regard, the average linkage method was performed to detect outlier samples, and the correlation coefficient between distance metric and cophenetic distance was determined to assess the quality of the clustering solution. In addition, the optimum number of clusters was determined as 2 and 3 by considering 31 methods including the Elbow, Silhouette, and Gap methods through “parameters” package in R (Supplementary Table 1). Eventually, the samples were checked by k-means outliers detection by setting the number of clusters at 2 and 3. The batch effects of the expression data were removed through fsav function from the “sva” package of R software 13 Subsequently, the expression level of the mRNA was assessed in two groups of AD and normal participants with the aid of the Kruskal-Wallis test through the “tinyarray” package. To this aim, we first assessed the normality of the data distribution using the Shapiro-Wilk test. Moreover, the correlation matrix of the genes was built through “cor” function. This study was approved by the Kerman University of Medical Sciences with the ethical code IR.KMU.AH.REC.1402.014.

Receptor structure information and preparation

The protein PDB files were retrieved from the RCSB data bank (https://www.rcsb.org). Protein selection criteria were 1-encoding by human genome, 2-containing small molecule as an inhibitor bound to protein (small molecule was considered as control positive in this study), and 3-Discovering through X-ray diffraction method with proper resolution (<2.5 angstroms (Å)). In addition, MGL Tools along with AutoDock tool 4.2 was utilized in the preparation of the protein file.14,15, 14,15 In the preparation procedure, the bonds’ order was automatically assigned and systematically adjusted to the AutoDock atom types. In addition, besides removing the water molecules and adding polar hydrogen, Gasteiger-Marsili charges were applied to the molecule.

The value of the Grid box in the specified docking study was determined using AutoDock Tools 4.2. The values were predicted based on the binding sites that the former study suggested for the inhibitory properties of small molecules. Grid box spacing was reported in three dimensions of x, y, and z with spacing = 1Å (Table 1).

Table 1

PDB ID and grid box values of the selected enzymes

Enzymes	PDB id	Grid box
Caspase 3	4JJ8	X = 33.524; Y = 26.515; Z =-4.827
Caspase 6	3OD5	X = 32.461; Y = 7.297; Z = 24.119
Cathepsin B	1CSB	X = 30.288; Y = 37.163; Z = 36.131
Cathepsin L	1GMY	X = 31.182; Y = -25.930; Z =-10.262
Granzyme A	1ORF	X = 15.955; Y = 28.732; Z = 1.666
Granzyme B	1IAU	X = 11.007; Y = 35.877; Z = 74.610

Bioactive library selection and preparation

To explore potential inhibitors, three libraries (PubChem, ChEMBL, and ChemDiv), were selected (sum: 145). The inhibitors were identified based on similarity to the structure of the cathepsin L inhibitor (PubChem Id: 381328630) as the positive control and searched in the mentioned libraries for caspase 6 inhibitors. The search was based on the previously published studies that labeled small molecules as the inhibitors of the caspase family. Similarity to the structure of cathepsin L inhibitor was selected based on having one to three rings and up to three atoms difference in the number of carbon, oxygen, and nitrogen atoms. In addition, ligands suggested by PubChem library as “Related Compounds with Annotation” were included (Supplementary Tables 2 and 3).

The small molecules were modified using AutoDock version 4.2 and OpenBabel (https://openbabel.org/) to be prepared for the protein docking. 16 The modification was briefly mentioned as atom adjustment to the AutoDock atom types, applying Gasteiger-Marsili charges, rotatable bonds assignment, and finally merging non-polar hydrogen bonds.

Molecular docking and hit compounds optimization

Before performing molecular docking using Autodesk Vina, 17 small molecules were evaluated through false positive remover (http://cbligand.org/PAINS/search_struct.php) website to exclude molecules with nonspecific interaction with the receptor leading to false positive responses. These compounds were nominated as promiscuous compounds or PAINS (pan assay interference). Substructures that could interfere in multiple reactions and compounds which are reported to be greater than 85% inhibited 4 or more targets were considered as false positive compounds and excluded from further analysis. Some examples of the substructures that could lead to false positive results are listed as phenolic Mannich bases, rhodanines, hydroxyphenylhydrazones, alkylidene heterocycles, alkylidene barbiturates, 1,2,3-aralkylpyrroles,2-amino-3 carbonylthiophenes, activated benzofurazans, catechols, and quinones. 18

Molecular dynamics

NAMD 2 is a high-performance molecular dynamic (MD) simulator, which was utilized for protein-ligand MD simulation. 19 The PDBQT file of the ligand conformation with the highest binding affinity was obtained from Autodock Vina, considering its drug-likeness score using the Osiris property explorer. In the next step, to determine the topology file of the ligand, CHARMM-GUI was selected. 20 The energy minimization was implemented for 500 ps and simulated for 10 ns. 4 Besides, in the coarse-grained model ligand was converted to the shaped-based coarse-grained through the coarse-grained builder plugin in VMD considering 20– 25 bead number, 4000– 5000 learning steps, initial eps:0.3, initial lambda: 4– 5 and 2 to 3Å bond cutoff.21,22, 21,22 Besides, the enzyme’s topography property was determined through VMD software and subsequently converted to residual-based coarse-grained (Martini 3).23,24, 23,24 The NVT protocol was utilized to calculate the MD simulation lasted for 100 ns, along with 4 ns minimization. The initial solvent box size for the MD simulations was determined based on the protein-ligand complex with 15Å box padding around the solute molecule in all directions. This was done to ensure that the solute was fully solvated and to minimize boundary effects during the simulation. To ensure appropriate solvation and ionic conditions, the solvent box was then neutralized by adding the necessary number of counterions (Na+or Cl-) to balance the net charge of the solute. The ion concentration was set to 0.15 M, which is consistent with physiological salt concentrations and is commonly used in MD simulations of biological systems. This ion concentration helps to mimic the ionic environment within the cell and to screen electrostatic interactions, thereby improving the accuracy of the simulations. 25 In determining the RMSD and RMSF values VMDICE tool was selected. 26 The CaFE tool was utilized to define the free-binding energy. 27 Finally, the molecular mechanics with generalized Born and surface area solvation (MMGBSA) score was calculated through the MolAICal tool. 28

RESULTS

Microarray samples

The hierarchical plots of the expression levels of the proposed enzymes on the laminopathy in this study were built from two datasets of GSE5281 and GSE28146 (Fig. 1). As shown, there were no specific outlier samples to remove. The correlation coefficient between distance metric and cophenetic distance was 0.72 indicating proper fit between the dendrogram and the pairwise distances. The results were checked by k-mean clustering outlier analysis resulting in no potential outlier detection.

Fig. 1

The hierarchical plot of the AD and normal samples obtained from GSE5281 and GSE28146.

mRNA expression levels in AD compared to normal participants

In analyzing the expression level of the proposed enzymes reported in laminopathy, the expression level of caspase 6 is significantly higher than normal individuals in the hippocampus samples. Moreover, lamin b2 depicted a remarkable downregulated in the expression, in contrast to lamin A/C. However, the expression level of other susceptible enzymes involved in the laminopathy in AD (i.e., cathepsin L, granzyme A, and B) did not illustrate any differences between the normal and AD group (Fig. 2).

Fig. 2

The mRNA expression of the enzymes is directly/indirectly involved in the laminopathy along with the expression of the nucleoskeleton proteins in two groups of AD and normal individuals.

Correlation of the expression level of proteases with lamin proteins

The expression level of lamin b2 negatively correlated with cathepsin L expression with a value of 0.017. There is the same association between the expression level of cathepsin B and caspase 6 with a p-value of 0.00047, as well as lamin A/C and cathepsin B (p-value 0.0034). While there is a positive correlation between the expression level of lamin A/C and caspase 6 (p-value 0.0088), as well as lamin b1 and A/C (p-value 0.0032). The correlation of the lamin A/C and lamin b1 is not specified to AD. Other correlation states presented in Fig. 3 did not highlight a p-value of less than 0.05 (Fig. 3A).

Fig. 3

A) Correlation of expression level of proteases with lamin proteins in AD samples. B) Correlation of expression level of proteases with lamin proteins in normal samples.

In addition, in the normal samples, the expression level of caspase 6 negatively correlated with the expression level of caspase 3 and cathepsin L with p-values of 0.016 and 0.0044. Likewise, lamin A/C and caspase 3 depicted a negative correlation (p-value 0.032). Besides, there is a positive correlation between caspase 3 expression level and cathepsin L with a p-value of 0.024, as well as the expression level of lamin A/C and granzyme A (p-value 0.0019). Moreover, same as AD patients in normal individuals there was a positive correlation between lamin b1 and A/C (p-value 0.028). There was no significant correlation in the expression level of cathepsin L and lamin b2. Moreover, the negative correlation between the expression of cathepsin L and caspase 6 suggested an independent activity of caspase 6 and cathepsin L in which one activity inhibited the others. These results suggested that the degradation pattern of lamin A/C altered in AD groups compared to the normal group (Fig. 3B).

Molecular docking

The binding affinity of the confirmed inhibitors of the caspase 3 and 6, and cathepsin B and L were calculated to be defined as the control value. Granzyme A and B were excluded since there was not any significant correlation between granzymes and laminopathy (Table 2). The results in the caspase inhibitor group specified a compound with the PubChem id 608841 nominated as caspase inhibitor x, which had the highest binding affinity in comparison to similar caspase inhibitors. Moreover, in the cathepsin L inhibitors group a compound with the ChEMBL id of 550872 had the highest binding affinity (Table 3). The amino acids involved in the binding site were defined and visualized for both identified inhibitors and positive controls, in which a high percentage of amino acids were common between positive control inhibitors and identified ligands (Fig. 4).

Table 2

The binding affinity values of the ligand and the receptors (kcal/mol) were calculated by Autodock Vina. The first and second values correspond to the two main different postures of the ligand in binding to the active site of the enzyme

	PubChem ID	Caspase 3	Caspase 6	Cathepsin B	Cathepsin L
C₁₄H₁₂O₂	8333	– 5.9	– 5.3	– 6.5	– 5.5
Inhibitor cathepsin B	– 5.8	– 5.2	– 6.1	– 5.3
C₁₇H₂₈ N₂O₇S	125011435	– 5.3	– 5.7	– 6.7	– 4.9
Inhibitor cathepsin B	– 5.3	– 5.7	– 6.1	– 4.8
C₂₅H₂₇Cl₂N₃O₄S₂	49835942	– 7.3	– 6.6	– 5.8	– 6.3
Inhibitor cathepsin L	– 7.1	– 6.5	– 5.2	– 5.7
C₂₃H₂₈N₈O₃	381328630	– 8.2	– 7.4	– 7.8	– 8.3
Inhibitor cathepsin L	– 7.7	– 7.4	– 7.3	– 7.9
C₃₃H₅₀N₆O₁₀	78225326	– 6.8	– 6.0	– 7.8	– 6.5
Inhibitor caspase 3	– 6.7	– 5.8	– 7.8	– 6.4
C₂₂H₃₆N₄O₉	15487887	– 6.9	– 5.8	– 6.9	– 5.6
Inhibitor caspase 6	– 6.6	– 5.6	– 6.8	– 5.6

Table 3

Inhibitors with the highest binding affinity (kcal/mol)

	ID	Caspase 3	Caspase 6	Cathepsin B	Cathepsin L
Searching based on caspase 6 inhibitors
C₁₈H₁₂F₃NO₂S	PubChem id: 608841	7.4	7.2	7.8	7.2
Searching based on cathepsin L inhibitors
C₂₃H₂₇N₇O₃	ChEMBL id: 550872	8.4	7.7	8.1	8.4

Fig. 4

Ligands 2D and 3D docking pose in comparison with positive control inhibitors of the caspase 6 and cathepsin L. A) ChEMBL id of 550872 interacted with cathepsin L B) PubChem id of 608841 interacted with cathepsin L C) Cathepsin L positive control inhibitor (PubChem Id: 381328630) D) ChEMBL id of 550872 interacted with caspase 6 E) Caspase 6 positive control inhibitor (PubChem Id: 15487887) F) PubChem id of 608841 interacted with caspase 6.

Molecular dynamics

Atomistic MD simulation suggested a stable binding status of caspase inhibitor x and cathepsin L inhibitor since the RMSD value was below 2Å. Similarly, the RMSF plot did not specify any difference between the two inhibitors, nor any instability in enzyme residues due to the RMSF value which was lower than 0.5Å (Fig. 5). The MMGBSA results for both inhibitors were the same, except for caspase inhibitor x interacted with cathepsin L, which had approximately half the binding energy of the cathepsin L inhibitor (Table 4).

Table 4

Represents delta MMGBSA values for all-atoms molecular dynamics analysis (kcal/mol)

	ΔE(electrostatic)+ΔG(sol)	ΔE(VDW)	ΔE(G binding)
Cathepsin L
PubChem id: 608841	8.96	– 31.69	– 22.72±0.33
ChEMBL id: 550872	7.54	– 47.31	– 39.76±0.29
Caspase 6
PubChem id: 608841	14.54	– 42.12	– 27.57±0.44
ChEMBL id: 550872	15.38	– 41.13	– 25.75±0.37

E(Vdw), van der Waals energy; G(sol), solvation free energy; E(G binding), free binding energy.

Fig. 5

The RMSD and RMSF plot of the selected inhibitors in complex with cathepsin L and caspase 6 simulated for 10 ns.

The MD simulation extended to 100 ns for the caspase inhibitor x and the cathepsin L inhibitor had high RMSD, RMSF, and free binding energy variation due to choosing Martini coarse-grained which merged each residue of the protein in a single bead. RMSD plot specified the caspase inhibitor x with the higher stability in the stimulation compared to the cathepsin L inhibitor with an overall mean of 14.53 to 26.75 in caspase 6 MD simulation and 13.48 to 21.71 in cathepsin L MD simulation. The result of the RMSF plots suggested more stability in the fluctuation rate of the caspase inhibitor x in comparison to the cathepsin L inhibitor in the first 100 residues, while for the rest of the residues, the interpretation is reversed. Both inhibitors depicted a spick in the residue numbers 145 and 146. The fluctuation rate of the cathepsin L enzyme is almost the same in different residues, in the case of applying caspase inhibitor X or cathepsin L inhibitor. The radius of the gyration (RG) indicated the overall stability of cathepsin L inhibitor in the complex, whereas the multiple spikes in the plot of the cathepsin L inhibitor illustrate the dissection of the ligand and enzyme, in contrast to caspase inhibitor x (Fig. 6). The result of the Molecular mechanics Poisson– Boltzmann surface area (MMPBSA) analysis suggested a detachment of the molecule with ChEMBL id of 550872 in the enzyme complex since the standard deviation value of the MMPBSA is much higher than caspase inhibitor x. Moreover, the total free energy of the caspase inhibitor x is higher than the cathepsin L inhibitor (Table 5).

Fig. 6

The RG, RMSD, and RMSF plot of the inhibitors’ interaction with caspase 6 and cathepsin L simulated for 100 ns.

Table 5

Represents delta MMPBSA values for coarse-grained molecular dynamics analysis with longer simulation periods (kcal/mol)

	ΔEele	ΔVdw	ΔSA	ΔGAS	ΔNPol	ΔTotal
Cathepsin L
PubChem id: 608841	– 1±3	– 354±38	– 4±1	– 355±38	– 357±38	– 359±39
ChEMBL id: 550872	– 9±11	– 260±155	– 3±2	– 268±160	– 263±157	– 271±163
Caspase 6
PubChem id: 608841	1±4	– 360±65	– 3±1	– 360±65	– 363±66	– 362±66
ChEMBL id: 550872	3±1	– 145±35	3±1	– 141±34	– 141±35	– 138±34

Eele, electrostatic contribution; Vdw, van der Waals contribution; SA, solvent area; Npol, nonpolar solvation energy.

DISCUSSION

In the current study, the correlation between the enzymes involved in lamin degradation was evaluated to identify those responsible for laminopathy in AD. Based on microarray analysis, among four lamin-degrading enzymes (caspase 6, cathepsin L, and granzymes A and B), only two enzymes (caspase 6 and cathepsin L) had significant differences in expression of AD patients highlighted laminopathy signaling pathway. Based on the Pearson correlation results, caspase 3 and cathepsin B suggested activating caspase 6, whereas cathepsin L was activated independently through a separate signaling pathway.

Angel et al. demonstrated that the expression of caspase 3 decreased in a caspase 6-knockout 5xFAD model of AD. Meanwhile, the level of caspase 3 correlated with axonal loss in the hippocampus. These results highlighted the correlation of caspase 3 and 6 expression with each other, 29 which was observed in the normal samples in the current study. In addition, it was mentioned that caspase 6 could be activated by the induction of caspase 3. 30 Moreover, substitution mutation of the 73^rd amino acid of caspase 6 reduces the efficacy of caspase 6 in acting on lamin A/C and α-tubulin, which protects against hippocampal atrophy. 31 In our study there was a strong negative correlation between the expression level of lamin A/C and caspase 6, highlighting the role of caspase 6 in lamin A/C degradation.

In addition, the pathology activity of cathepsin B in AD was mentioned in a study by Hook et al. Accordingly, it was reported that cathepsin B is involved in the memory deficit and enhancement of pyroglutamate-Aβ production. Correspondingly, cathepsin B knockout in the hAβPP695 Wt mouse model improves cognitive function. 11 In the current study, cathepsin L inhibitor and caspase inhibitor x had a high binding affinity with cathepsin B (7.8 and 8.1 Kcal/mol, respectively). Caspase inhibitor x was formerly recommended as a potent inhibitor of caspases 8, 3, and 7 in the Binding Database (https://www.bindingdb.org/). Nonetheless, the inhibitory effect of this compound on cathepsin L has not been mentioned.

In the study conducted by Nagakannan et al., it was stated that reactive oxygen species (ROS) activated cathepsin L, leading to cell apoptosis. In addition, lysosomal membrane permeabilization as the result of ROS, activated cathepsin B, subsequently influences caspase 3, leading to cell apoptosis. 32 In another study, ROS was linked to the activation of thioredoxin which influences the activity of caspase 6, causing lamin b1 degradation. 30 In addition, in the Slee and Martin study, it was clearly demonstrated that caspase 3 activated caspase 6, leading to lamin A degradation. 33 These results depicted two main signaling pathways leading to laminopathy; one with the involvement of cathepsin B, caspase 3 and 6, and the other with the participation of cathepsin L.

In the study conducted by Ramasamy et al. (2016), it was declared that Aβ could induce lamin degradation independent from caspase activity. 34 In addition, Hossain et al. study published in 2023 mentioned that Aβ aggregation induced an increase in calcium concentration, resulting in the activation of cathepsin L. Moreover, calcium ions could activate another pathway mediated by caspase 6. However, the result of the study highlighted a difference in the lamin fragment as a result of cathepsin L and caspase 6 activity. 35 The results of these two studies are in line with our findings and highlight the role of cytoplasmic calcium as a mediator of activation of two laminopathy pathways. Besides, there was a direct cell apoptosis pathway mediated by caspase 3 independent of laminopathy. 10 Similarly, besides the caspase 6 role in degrading lamins, it was reported to cleave tau at Asp (D) 421 leading to tau neurofibrillary tangles, highlighting the wide range of caspase 6 pathology in AD. 36 One of our study limitations was the MD simulation which led us to use coarse-grained instead of long-period all-atom simulation due to the servers analyzing time. Moreover, in the current study, the in vitro/in vivo assessments were not implemented, since the inhibitors were not accessible to be purchased for this project. Further studies are needed to perform in vitro/in vivo assessments on the effect of these inhibitors on the proteases involved in AD-related laminopathy.

Conclusion

The transcriptome analysis revealed two main signaling pathways of laminopathy in AD. The first pathway is highlighted by the activation of caspase 6 in the degradation of lamins A/C, while the other pathway is specified by the strong correlation of cathepsin L and lamin b2. There were a few inhibitors that had the potential to inhibit all the key enzymes involved in both signaling pathways of AD laminopathy. Based on the MD results, two molecules (PubChem id: 608841 and ChEMBL id: 550872) had the proper stability in binding with cathepsin L, as well as caspase 6.

AUTHOR CONTRIBUTIONS

Auob Rustamzadeh (Conceptualization; Data curation; Investigation; Methodology; Writing – original draft); Abbas Tafakhori (Conceptualization; Validation); Armin Ariaei (Investigation); Mahdi Heydari (Investigation; Validation); Mehran Ebrahimi Shah-abadi (Conceptualization; Software; Writing – original draft); Farhad Seif (Conceptualization; Methodology; Supervision; Writing – review & editing).

Footnotes

ACKNOWLEDGMENTS

The authors have acknowledgments to report.

FUNDING

The authors have no funding to report.

CONFLICT OF INTEREST

The author acknowledge Kerman University of Medical Sciences for supporting. of this research.

DATA AVAILABILITY

The data of the current study will be available upon reasonable request.

The supplementary material is available in the electronic version of this article: .

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