The Effect of RGD/NGR Peptide Modification of Melanoma Differentiation-Associated Gene-7/Interleukin-24 on Its Receptor Attachment,an In Silico Analysis

Abstract

Melanoma differentiation-associated gene-7 (mda-7/interleukin [IL]-24), a unique tumor suppressor gene, induces selective apoptosis in tumor cells. Secreted IL-24 binds to heterodimeric receptor complexes of IL-20R1/IL-20R2, IL-22R1/IL-20R2, or sigma-1 receptor (Sig1R) that consequently enhances apoptosis. However, this mechanism is not well understood and most likely involves different pathways. Targeting of cytokine by tumor homing peptides (THPs) to the tumor cell surface molecule-like integrin shows to be beneficial in gene immunotherapy approaches. In this study, the in silico targeting of RGD/NGR-modified IL-24 to tumor cells was conducted. In this regard, the sequences of six new synthetic IL-24s that have been modified by RGD (Arg–Gly–Asp) or NGR (CRNGRGPDC) were aligned and their structures were modeled through homology modeling to evaluate their attachment potential to cognate receptor complexes such as IL-20R1/IL-20R2, IL-22R1/IL-20R2, or Sig1R. The results of homology modeling showed that modification of IL-24 with RGD motif in N-terminal and middle of this protein exhibited stronger interaction with cognate receptors. These results also demonstrated that modified IL-24 with RGD motif in the C-terminal has lost native activity. However, the interaction of THP-modified IL-24 with Sig1R would not be affected to that extent, interestingly. Conclusively, in silico analysis showed that modification of IL-24 with THPs needs a more detailed study as these modifications may disrupt native interaction with receptors and reduce apoptosis induction property. This structural analysis gives us a better understanding of mda-7/IL-24 interaction with cognate receptors and helps a more rational design for further cytokine modification.

Introduction

Melanoma differentiation-associated gene-7 (mda-7/interleukin [IL]-24), a multifunctional cytokine, induces apoptosis when expressed in the supraphysiological level. At physiological levels, mda-7/IL-24 acts as a cytokine that plays a role in wound healing, inflammatory response, protection against a number of infectious agents, and somehow exerts a protective impact on cardiovascular disease.¹

This protein belonged to the IL-10 family due to structural similarity and is accepted as an appropriate candidate for cancer therapy to induce apoptosis in cancer cells without any destructive effects in healthy cells.^2,3 IL-10 cytokine family consists of IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, and more distantly related IL-28A, IL-28B, and IL-29.⁴ The IL-20 subfamily comprises IL-19, IL-20, IL-22, IL-24, and IL-26 due to their structural similarity, amino acid sequence, genomic location, and common receptors for signaling pathway. This subfamily induces cellular responses by using the IL-20R1/IL-20R2 (type I) heterodimeric receptor, while IL-20 and IL-24 use an alternative receptor composed of IL-22R1/IL-20R2 chains (type II).⁵

The mda-7 gene has been widely studied as a tumor inhibitor gene and growing evidences support that IL-24 mediated cancer cell killing without damaging normal tissues.⁶ It establishes antitumor activity when expressed endogenously by plasmid/virus constructs.

The apoptosis induction properties of mda-7, mostly depending on its endogenous expression, are related to ER stress and involvement of some molecules, including BiP/GRP78, fas/fasL, p38 MAP kinase (MAPK), PKR, and JNK signaling mediator pathways, as well as Bax/Bak proteins.^1,7,8 However, the secreted mda-7 from expressing cells can also perform the antitumor activity through interaction with different molecules, especially IL-20/IL-22 and sigma-1 receptor (Sig1R or σ₁R), a phenomenon named “bystander effect.”^2,9 As a cytokine, secreted IL-24 performs immunologic activities through two types of heterodimer receptors (IL-22R1/IL-20R2 and IL-20R1/IL-20R2) and the JAK/STAT (I/III) signaling pathway.¹⁰ However, apoptosis induction by secreting mda-7 is mostly independent from the JAK/STAT pathway, but instead is related to MAPK or other undefined pathways. Moreover, recently, sigma 1 receptor has been proposed as another IL-24 binding receptor. The possible role of secreted IL-24-related apoptosis following Sig1R attachment has been proposed.^9,11

Direct targeting of drugs by peptides in tumor cells is a promising approach for improving cancer treatment.¹² Tumor homing peptides (THPs) such as RGD (Arg–Gly–Asp) and NGR (Asn–Gly–Arg) can mediate efficient cellular targeting and entry of soluble protein following their fusion.^13,14 There are small peptide sequences with low toxicity that not only reduce the injected dose but also enhance the therapeutic index.¹³ Application of these peptides to gene therapy would extent and specify the targeting potency of a therapeutic molecule beyond the cognate receptors. The expression of αvβ3 and αvβ5 integrin is significantly upregulated in a wide spectrum of tumor cells and angiogenic endothelial cells. So, these integrins could be an appropriate candidate for tumor targeting by THPs.¹⁵ Due to the high affinity and specificity of RGD peptide to integrins αvβ5 and αvβ3, this peptide may serve as a competitive candidate for targeting drugs to the tumor cells.¹⁶ On the contrary, the tumor-homing NGR peptide can selectively bind to the aminopeptidase N (APN/CD13) that expresses on the specific tumor cell surface.^17
–19 In fact, iRGD binds to the tumor marker integrin αvβ3 directly, while iNGR binds to APN/CD13 at tumor vessels. After binding of iNGR to APN/CD13, cell surface-associated protease cleaved the peptide to expose the cryptic CendR element (RXXK/R) at the C-terminus The interaction of the CendR motif with neuropilin-1 (NRPs) causes the penetration of peptide-binding drugs into the tumor milieu.²⁰

A number of studies have shown that mda-7/IL-24 induces apoptosis in hepatocellular carcinoma-related cell line.^21,22 Since IL-24 partly performs its death induction through attachment to IL-22R1/IL-20R2, IL-20R1/IL-20R2, and Sig1R, the authors hypothesized that fusion of RGD/NGR peptide to mda-7 not only specified its antitumor property by specific adherence and entry but also leaves its native activity intact for receptor attachment, both mechanisms are involved in the bystander effect. For this purpose, the different forms of THPs in fusion with mda-7 were evaluated for apoptosis induction in vitro.^23,24 However, with regard to the previous results, in the present study, the interaction of peptide-modified mda-7/IL-24 with its cognate receptor, IL-22R1/IL-20R2 and Sig1R, was investigated throughout homology modeling. In this study, three-dimensional (3D) structures of native and modified mda-7/IL-24 with RGD or NGR peptides were modeled and evaluated. Then, the interaction of IL-24 with Sig1R, IL-20R1, and IL-20R2 receptors (completely mimicking the structure of type II receptor complex) was investigated. Altogether, these findings provide new insight about the natural attachment property of THP-modified cytokines and can be considered for more precision mda-7/IL-24 targeting.

These THP-modified mda-7/IL-24 have different primary structures as follows: RGD motif in the middle, C or N terminus, 4cRGD, iNGR, and iRGD motifs in the C terminus of IL-24.²⁵ In the pursuit, the binding status of these molecules to cognate receptor as well as Sig1R was evaluated by computational modeling. In another word, the interaction of different peptide-modified mda-7/IL-24 with cognate receptors, IL-20R1/IL-20R2 and Sig1R, was investigated with 3D computational modeling.²⁶

Materials and Methods

Literature overview and analysis

A systematic review of the IL-24 protein/gene literatures in the PubMed revealed that a few studies focused on improving the cytokine efficiency by tumor targeting peptide.^12,24,27,28 In these studies, RGD-4C was coupled to the N-terminus of IL-24 and revealed that both types of IL-24 (wild type and recombinant) have similar behavior for apoptosis induction and adhesion in MCF-7 cancer cells.^16,18,29 Pei et al. individually constructed the RGD-IL-24 by inserting Gly residue into the mda-7/IL-24 between Arg¹⁶⁴ and Asp¹⁶⁵ to form an RGD motif.¹⁶ These studies demonstrated that the RGD motif could be an effective approach to increase the antitumor effect of mda-7/IL-24. Hence, this group designed and constructed two IL-24s, which were modified by RGD4C (full RGD4C and shortened RGD),²³ and evaluated their antitumoral efficiency in vitro. In the other project, a new construct with an RGD motif at the beginning of the gene was provided.²⁴ The aim of these modifications was to improve tumor targeting by specific peptide sequences.

Protein sequence alignment

In the beginning, all of the abovementioned sequences based on article description were reconstructed (Table 1). These six IL-24 models contain the RGD/NGR motif in the primary protein sequences and were designed as follows: (1) the native IL-24 (Fig. 1A) (IL-24-a) and (2) IL-24 with a new signal peptide (shortend residue of 21 instead of 48 residue) plus RGD motif (ACDCRGDCFCG) in the N terminus (IL-24-b), this construct was recently prepared in the laboratory (Fig. 1B), (3) IL-24 with RGD motif (CDGRC) in the C terminus²³ (Fig. 1C) (IL-24-c), (4) IL-24 with iRGD (CRGDKGPDCA) motif in the C terminus (Fig. 1D) (IL-24-d), (5) IL-24 with 4cRGD motif (ACDCRGDCFCG) in the C terminus²³ (Fig. 1E) (IL-24-e), (6) IL-24 with signal peptide at the N terminus and inserting a Gly residue into the IL-24 between Arg¹⁶⁴ and Asp¹⁶⁵ site to form an internal RGD motif^16,18,29 (Fig. 1F) (IL-24-f), and (7) IL-24 with iNGR motif (CRNGRGPDCA) in the C terminus (Fig. 1G) (IL-24-g). These six modified sequences of IL-24 and IL-20 (homologous cytokine) were aligned together by MEGA7 software³⁰ to reveal degree of homology (Fig. 1H). Furthermore, the physicochemical properties of the wild-type and recombinant IL-24 were calculated in ProtParam tool.³¹

FIG. 1.

(A) The schematic representation of sequences of wild-type and six recombinant IL-24s (B–G). The location of signal peptide, IL-24 sequence, and peptide sequences was highlighted. (A) IL-24-a, (B) IL-24-b, (C) IL-24-c, (D) IL-24-d, (E) IL-24-e, (F) IL-24-f, and (G) IL-24-g. (H) Protein alignment of wild-type and recombinant IL-24 and IL-20. Modified sequences at the beginning, middle, and end of protein sequences, and signal peptides at the beginning. IL-20 as homologous cytokine also included to reveal difference and homology between them. The conserved domains of IL-20 and mda-7/IL-24 are extrapolative when surveyed inside the alignment. IL, interleukin; mda-7, melanoma differentiation-associated gene-7.

Table 1.

The Sequences of IL-24 with Modification

	Type of IL24	Amino acid sequences
1	IL24 wild type	MNFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLWSQVSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAALTKALGEVDILLTWMQKFYKLGGSG
2	Signal peptide and RGD motif in the N terminus (IL24-b)	MWWRLWWLLLLLLLLWPMVWA ACDCRGDCFCGGYP AQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAALTKALGEVDILLTWMQKFYKL
3	Signal peptide in the N terminus and RGD motif in the C terminus (IL24-b)	MNFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLWSQVSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAALTKALGEVDILLTWMQKFYKL CDGRC
4	Signal peptide in the N terminus and iRGD motif in the C terminus (IL24-b)	MNFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLWSQVSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAALTKALGEVDILLTWMQKFYKLGGSG CRGDKGPDCA
5	Signal peptide in the N terminus and 4cRGD motif in the C terminus (IL24-b)	MNFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLWSQVSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAALTKALGEVDILLTWMQKFYKL ACDCRGDCFCG
6	Signal peptide in the N terminus and RGD in the middle (IL24-b)	MNFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLWSQVSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSI RGD SAHRRFLLFRRAFKQLDVEAALTKALGEVDILLTWMQKFYKL
7	Signal peptide in the N terminus and iNGR motif in the C terminus (IL24-b)	MNFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLWSQVSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNHHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAALTKALGEVDILLTWMQKFYKLGGSG CRNGRGPDCA

The bold and underlined sequences show the location of RGD modification, the italic sequences show the signal peptide.

IL, interleukin.

mda-7/IL-24 modeling

Up to now, the 3D structure of the IL-24 remained to be determined. To evaluate the effects of RGD/NGR modification on protein structure of IL-24, the 3D structure of these proteins was modeled. For structure prediction, the Iterative Threading ASSEmbly Refinement (I-TASSER) web server, a hierarchical method, was used.³² By retrieving the protein sequence of wild-type IL-24 from NCBI and preparing the protein sequences of recombinant of IL-24 and submission of these sequences in the I-TASSER server, the 3D models of these proteins were constructed.³² On this server, Local Meta-Threading-Server (LOMETS) is used for constructing 3D models, which predicts by collecting multiple-threading alignments from nine locally installed servers that demonstrate a various set of state-of-the-art threading algorithms. I-TASSER web server generated a total of five models based on multiple-threading alignments by LOMETS. The models with the best “Confidence Score” and Z-score are chosen by I-TASSER server. The model with better confidence and Z-score was selected and visualized using Swiss PDB viewer³³ and PyMOL molecular graphics system.³⁴ The server has access to used BioLiP database and PSI-PRED program for study of protein–ligand docking and protein secondary structure prediction, respectively. In the end, the models with the best “Confidence Score” and Z-score were chosen with I-TASSER server.³⁵

Protein–receptor interaction modeling

As remarked, among cytokines related to IL-10, the IL-24, IL-20, and IL-19 are grouped as the IL-20 subfamily. They use a complex of heterodimeric receptor IL-20R1/IL-20R2 (type I), while IL-20 and IL-24 somehow attach to another receptor complex IL-22R1/IL-20R2 (type II).

For interaction prediction of IL-24 with IL-20R1/IL-20R2 complex, the structure of IL20/IL201/IL20R2 ternary complex served as a template in the in silico evaluation of this interaction.⁵ The structural factors of IL20/IL201/IL20R2 ternary complex were deposited in the Protein Data Bank (accession nos. rcsb070584 and 4DOH).⁵ Due to structural homology of IL-24 and IL-20 and based on crystal structure of IL-20/IL-20R1/IL-20R2, the connecting of IL-24 and IL-20/IL-20R1/IL-20R2 complex was applicable as previously described.^5,36 The IL-20 (chain A)/IL-20R1 (chain B)/IL-20R2 (chain R) complex (4DOH.pdb) and IL-24 (model1.pdb) were loaded into the PyMOL program and aligned.³⁴ This ternary complex constructed the final model. For investigating the position of RGD peptides and iNGR in ternary complex, all of the IL-24 models were aligned with PyMOL program and the final models were extracted and evaluated. Moreover, hydrogen bonds of these modifications were also calculated by Swiss PDB viewer program.³³

To investigate mda-7-IL-24 interaction with sigma 1 receptor, its crystal structure was retrieved from the Protein Data Bank. The structure files of the IL-24 and Sig1R were uploaded in the ZDOCK as input.³⁷ ZDOCK can block specific residues from being in the binding site. After specifying PDB files for docking, the 1–36 and 178–220 residues of Sig1R were blocked from the binding site during docking process. Sig1R protein will be stationary in the output predictions, while IL-24 will be moved.

Results

Modeling of wild-type and recombinant IL-24 proteins

Up to now, the crystal structure of the wild-type mda-7/IL-24 was not resolved.³⁸ To predict the structures of wild-type and recombinant IL-24, 3D molecular models of these proteins were constructed in I-TASSER web server based on the structure of IL-20 (PDB ID: 4DOH) and shown in Figure 2A. In this figure, for better observation of modifications, the structures were shown from different angles. The results of modeling showed that these modifications have no undesirable effect on final structure of recombinant proteins. The interesting point in Figure 2A and F is that inserting glycine residue into the mda-7/IL-24 between Arg¹⁶⁴ and Asp¹⁶⁵ left indistinguishable change in the 3D structures of IL-24 protein.^16,18 Table 2 shows the physicochemical properties of the wild-type and recombinant IL-24 that were calculated in ProtParam tool.³¹

FIG. 2.

Representation of IL-24 protein structure and its modifications with RGD and NGR peptides from different angles. (A) IL-24-a, (B) IL-24-b, (C) IL-24-c, (D) IL-24-d, (E) IL-24-e, (F) IL-24-f, and (G) IL-24-g. (B) 3D model of IL-24, which represents the location of RGD and iNGR modification and formation of hydrogen bonds. (A) IL-24-b, (B) IL-24-c, (C) IL-24-d, (D) IL-24-e, (E) IL-24-f, and (F) IL-24-g.

Table 2.

In silico Physico-Chemical Properties of IL-24 Obtained from ProtParam Tools

No	Parameters	Wild type	IL24-b	IL24-c	IL24-d	IL24-e	IL24-f	IL24-g
1	Theoretical pI	8.96	8.22	8.41	8.40	7.81	8.61	8.73
2	Molecular weight	18,651.4	22,663.4	18,927.7	19,654.5	19,524.4	18,450.2	19,681.5
3	Sequence length	162	193	163	172	169	159	172
4	Extinction coefficients (M⁻¹ cm⁻¹ at 260 nm)^*	21,095–20,970	55,835–55,460	21,220–20,970	21,220–20,970	21,345–20,970	66,140–65,890	21,220–20,970
5	Asp + Glu	16	18	17	18	62	16	17
6	Arg + Lys	18	20	19	20	57	18	20
7	Instability index	40.63	41.67	40.87	36.93	41.87	39.90	37.76
8	Grand average of hydropathicity	−0.187	−0.017	−0.194	−0.240	−0.154	−0.181	−0.244
9	Aliphatic index	90.25	96.48	89.69	85.58	87.10	91.95	85.58

First value is based on the assumption both cysteine residues from cystine and the second assumes that cysteine residues are reduced.

The second aim of this study was to predict the suitable modification of IL-24 that selectivity attached and upregulated the integrin receptor on the surface of cancer cells. In Figure 2B, the spatial situation of the RGD motif was delineated after 3D modeling of recombinant IL-24. In the recombinant models of IL-24 with RGD modification in N- and C-terminal loop, some amino acids have interaction with RGD sequence and stabilize this loop (Fig. 2B). According to these figures, different amino acids such as Ser, Glu, Cys, and Asp are involved in the interaction of RGD with IL-24. These results show that in the original design of recombinant IL-24, the RGD motif should not have any unsuitable interaction with other amino acids. Furthermore, the side chains of these amino acids (RGD) should be solvent exposed to provide the favorable function of this motif.³⁹

Receptor complex modeling

Common receptors of IL-20, the IL-20/IL-20R1/IL-20R2 ternary complex, served for modeling and binding of different IL-24 models. The 3D structures of IL-20R1 and IL-20R2 subunits are shown in Figure 3. Two important sites involved in binding to the receptors were displayed by site 1 and site 2. At first, the 3D structure of wild-type IL-24 was put in the receptor place and showed a similar situation of the IL20-IL20R1/IL20R2 receptor. This means that both IL-24 and IL-20 occupy similar sites in the receptor. In the following, the recombinant models of IL-24 were superimposed onto the IL-24 and their orientations were evaluated on the IL20R1/IL20R2 receptor, a complex that similarly mimics the behavior of IL-22R1/IL-20R2.

FIG. 3.

Superimposition of wild-type and recombinant IL-24 with IL-20 in IL20-IL20R1/IL20R2 receptor. (A) IL-24-a, (B) IL-24-b, (C) IL-24-c, (D) IL-24-d, (E) IL-24-e, (F) IL-24-f, and (G) IL-24-g.

Modeling studies show that modification of IL-24 with RGD/INGR in some positions can interfere with binding with the receptor complex. On the other hand, the recombination of IL-24 with RGD in the N-terminal or middle portion can have a little or any interfered with receptor connection. As shown in Figure 4F, in recombinant IL-24 with RGD motif in the middle of IL-24 (IL-24f), the RGD motif was located outside of the IL-24-receptor complex and was solvent exposed, which could have a positive effect on its performance. In the recombinant models of IL-24 with RGD motif in the N terminus, the RGD made an inside loop (shown in yellow) that when located in the IL-20R1/IL-20R2 receptor, very slightly impressed site 2. This site has an important role in stabilizing the IL-24- IL-20R1/IL-20R2 complex.⁴⁰ However, this motif was available to the solvents making positive effects on their expected function. In other words, the yield of the solvent accessibility and effectiveness of IL-24 on site 2 determine the ultimate function of this IL-24 recombinant.

FIG. 4.

Focusing on interaction of RGD motif in recombinant IL-24 proteins with IL-20 receptors. (A) IL-24-b, (B) IL-24-c, (C) IL-24-d, (D) IL-24-e, (E) IL-24-f, and (F) IL-24-g.

While in these models, the RGD motifs are solvent accessible and slightly influence site 2, in the other recombinat models of IL-24 with RGD motif in the C terminus, the RGD made an inside loop (shown in yellow) that, when located in IL-20R1/IL-20R2 receptor, impressed and interfered in site 1. This site has a very important role in stabilizing the IL-24-IL-20R1/IL-20R2 complex and disruption of this binding site, effected on the connection of IL-24 to site 1 and reduced the affinity of recombinant IL-24 and the receptor. Finally, in the recombinant IL-24, this motif was not available to solvents that have a negative effect on the expected function of this motif (Fig. 4).

Interestingly, modeling of THP-modified mda-7/IL-24 revealed that all of them exhibited a similar pattern of interaction with Sig1R. These modeling results showed that all of the modified mode-7/IL-24s are tolerable and have no detrimental effect on Sig1R attachment. The structure of Sig1R is folded in such a way that interaction motifs expose perfectly to natural as well as modified mda-7/IL-24 (Fig. 5). ZDOCK will filter results such that only those with specified residues in the binding site are returned. Results from the top 2000 ZDOCK predictions are filtered using the user-defined residues and 6 Å distance cutoff. The PDB file of the results was evaluated by Swiss PDB viewer (Fig. 5).

FIG. 5.

Stereo presentation of docking situation of IL24 into the sigma 1 receptor created by PyMOL. Predictions are only kept if all residues selected are within 6 Å of the partner protein.

Discussion

Directing therapeutic agents to the tumor site is among the encouraging strategies to enhance anticancer agents, efficacy on their side-effect decrement.^12,13 mda-7/IL-24 induces apoptosis in tumor cells without any side-effect on normal cells. This is a major reason that made it an appropriate candidate for cancer therapy. To improve the apoptotic impact of IL-24, some studies tried to modify secreted IL-24 protein by THPs, targeting peptide, that would consequently enhance its bystander effect.^12,16,24 Different kinds of peptides especially RGD- and NGR-related ones have been used for mda-7 targeting on cancer cell surface.

mda-7/IL-24 induces its extracellular effect through attachment to IL-20R1/IL-20R2 or IL-22R1/IL-20R2 heterodimer receptor complex as well as Sig1R. As a cytokine, to perform apoptosis induction, secreted IL-24 is mostly independent from the JAK/STAT pathway, but instead is related to MAPK or other undefined pathways such as Sig1R.^9,11,41 To evaluate the impact of THP fusion on the natural properties of secreted mda-7/IL-24, bystander effect, preliminary studies have been performed in vitro previously.^23,24 However, regarding those disappointed findings, here the interaction of THP-modified mda-7/IL-24 with its cognate receptor, IL-22R1/IL-20R2 as well as Sig1R, was investigated by in silico analysis. Even though modification of mda-7/IL-24 by THPs retarget it toward receptors such as integrins on the surface of majority of tumor cells, attachment to cognate receptor remained partly responsible for further apoptosis induction. So, the authors focused on a question if THP modification may leave a drastic effect on native attachment of mda-7/IL-24 with cognate receptors.

The functional IL-22 receptor complex is expressed on the nonhematopoietic cells such as the liver, lung, skin, pancreas, intestine, and kidney.⁴² Since this computational modeling and other literatures showed the similar 3D structure of both receptors, here the authors assessed the proposed subjects with the IL-20R1/IL-20R2 complex.

For improving the cytokine efficacy by peptide targeting, in a pioneer effort, RGD4C was fused to N-terminus of hTNF through a glycine/serine linker.²⁵ The results showed that RGD4C-TNF in the integrin-positive tumor cells has a more antitumor effect in comparison with native form of IL-24. In 2008, Xiao et al., coupled RGD-4C to the N-terminus of IL-24 and revealed that both types of IL-24 exhibit similar behavior for apoptosis induction and adhesion in MCF-7 cancer cell.¹² In separate studies, the RGD motif was created inside the IL-24 body by inserting a glycine residue between Arg¹⁶⁴ and Asp1⁶⁵.^16,18,29 These works showed that adding an RGD motif could be an efficient approach to increase the antitumor effect of IL-24. In this concept, to improve cell targeting by mda-7, this group modified mda-7/IL-24 with different kinds of peptides such as iRGD, NGR, and RGD4C (full RGD4C and shortened RGD) and evaluated their antitumoral efficiency in vitro.^{16,18,23

–42}

In these studies, contrary to expectations, the addition of an RGD motif to IL-24 not only does not improve the apoptotic effect of IL-24 but also may reduce the rate of apoptosis effect.³⁶ On the contrary, some previous studies mentioned the in vitro beneficial impact of tethering RGD peptide in the IL-24 protein. Two versions of modified IL-24 were constructed throughout the preceding RGD4C in the 5′ end and the other with the following RGD4C at the 3′ end of mda-7/IL-24 CDNA, named RGD/IL-24 and IL-24/RGD, respectively. The experimental results showed that expression of mda-7 with RGD in 5-terminus induces more apoptosis than native form, a difference hard to be considered.²⁴ In contrast, adding RGD to 3-end left a destructive effect on mda-7 as apoptosis properties reduced significantly. This phenomenon was repeatedly achieved when iNGR was added to C-terminus of mda-7 in similar experiments.⁴³

Given that the crystal structure of IL-24 was not determined and due to the structural similarity with IL-20 and its receptor, 3D modeling and protein–protein interaction were stepwise performed by the help of deposit data from 3D structure of IL-20 complex.⁵ However, the authors have previously modeled the structure of cytosine deaminase, firefly luciferase, HBV and HCV proteins and have a good experience in homology modeling technique.⁴⁴ Therefore, to investigate whether the position of the RGD and iNGR sequences in the recombinant IL-24 affects the natural structure of IL-24, 3D structure models of wild-type and recombinant IL-24 and binding of these IL-24s to the receptor were modeled and evaluated for comparing all previous and ongoing structures.

The results of homology modeling showed that these modifications have no effects on the 3D structure of native IL-24. These findings highlight these changes and clearly indicated that most disturbance of natural structure related to adding the peptide to C-terminus of protein. Hence, in these cases, the integrated peptide will be hidden among the structures of proteins and consequently will keep far away from accessibility of integrins. In two cases, including the peptide insertion in the corresponding position of the polypeptide backbone^16,18,29,45 and at the N-terminal of protein, the availability of peptide for binding to integrin was much better than others. In these forms, the natural structure of proteins remained nearly similar to unmodified IL-24 proteins and indicated the lower impact of modification on the final structure of proteins.¹⁶ Binding of IL-22 to its receptor (IL-22R) utilizes Jak/STAT and Tyk2 cascade of signaling transduction.⁴⁶ A similar mechanism of action could be supposed by RGD-IL-24 to improve apoptosis of tumor cells. However, these assumptions demand further investigation.

Really with the aim of improving the peptide targeting of mda-7, some studies obtain results with a successful design. Previous studies demonstrate that RGD- IL-24 has more potent antitumor effects than unmodified mda-7/IL-24.^16,18 Their works profoundly support this in silico analysis of IL-24 modifications. The in silico assessment revealed that those kind of designs completely left mda-7 structure accessible to receptor attachment. In contrast, this study indicated that adding RGD in C-terminus of IL-24 disrupts receptor attachment and reduces apoptosis property. This achievement was completely in agreement with in silico survey.²³

Also, the results of modeling show that in some models, the conformational changes of IL-24 interfere in its bindings to receptor. The first exception was IL-24 with RGD in the N-terminus that will affect site 2, but since this motif is accessible to solvent, the normal function of this recombinant IL-24 was not changed in this study. In fact, this achievement was also supportive of this idea as the result of apoptosis induction by modifying IL-24 in N-terminus was encouraging compared to the native format of IL-24. In this regard, adding RGD peptide to protein induces more apoptosis when compared to other previous constructs.²⁴ The second exception was to the IL-24 with RGD in the middle of polypeptide backbone that was completely solvent accessible and has a positive effect on the function of this recombinant IL-24. From the experimental studies^16,18,29 and the previous results, it was strongly proved that these finding were in agreement in both experimental and in silico analysis. The interesting similarity in both kinds of results showed the advantages of computational modeling in the saving of cost and time in comparison with laboratory test.

Hence, all THP-modified mda-7/IL-24s exhibited a similar pattern of interaction with Sig1R, a structure that was investigated for the first time for this purpose. The results of modeling showed a tolerable and nondetrimental structural impact of THP modification of the native structure of mda-7/IL-24, a finding that was not achieved before. This finding clarified that any kind of peptide fusion in mda-7/IL-24 is acceptable in the case of Sig1R importance. The structure of Sig1R showed extended features that would accept all forms of modified cytokine.

From all the abovementioned results, it can be concluded that THP modification of mda-7/IL-24 may afford a destructive effect on its natural attachment to cognate receptors, IL-22R, while leaving it unaffected for Sig1R attachment. Moreover, fusion of the RGD motif within the N-terminus or insertion in the middle structure of cytokine would render it a better conformational change for further receptor attachment. The results also are applicable for further development of other cytokine immunotherapies even with a different structure.

Footnotes

Disclosure Statement

No competing financial interests exist.

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