**4. Drugs capable of stabilizing the KRas4B/PDE6δ protein complex**

The search for new targeted therapies aimed at trying to improve the quality of life of patients with pancreatic cancer has taken about 30 years; along this time, researchers have looked for compounds that can inhibit the signaling pathway of the KRas4B oncoprotein. One of the most important mechanisms for the activation of KRas4B is the transport from the cytosol toward the plasma membrane by the PDE6δ, which recognize the farnesyl group of KRas4B present at carboxyl terminal (**Figure 1a**). It was believed that KRas4B/PDE6δ was transported as a dimer, and it is now known that it forms a cluster of 6–12 proteins or 3–6 dimers (**Figure 1b**). Because of this, our work group looked for a plate of the heterodimer using the crystal of the heterodimeric complex of the cluster of 6 proteins (**Figure 1c**) in order to identify small organic molecules capable of stabilizing the interaction of the molecular complex KRas4B/PDE6δ with the purpose of avoiding the activation of KRas4B as well as its signaling pathway dependent of this oncoprotein. An exhaustive search was carried out in public chemical libraries of organic compounds

#### **Figure 1.**

*Types of interactions between KRas4B/PDE6δ heterodimeric complex crystallized. (A) Interaction between K-Ras4B (pink) and PDE6δ (aqua) proteins. (B) Cluster formation among K-Ras4B/PDE6δ in multiheterodimeric molecular complexes crystallized. (C) Template of K-Ras4B/PDE6δ heterodimeric complex in a cluster used to docking and drug identification. (D) Molecular docking of D14 (N-[(2H-1,3-benzodioxol-5-yl)methyl]-2-[4-(5-chloro-6-oxo-1-phenyl-1,6-dihydropyridazin-4-yl)piperazin-1-yl]acetamide) and C22 (3-(2-{[1-(4 chlorophenyl)ethyl]amino}acetamido)-N-cyclopropylbenzamide) compounds and using a cluster of the heterodimeric K-Ras4B/PDE6δ molecular complexes.*

#### *KRas4BG12C/D/PDE6δ Heterodimeric Molecular Complex: A Target Molecular Multicomplex… DOI: http://dx.doi.org/10.5772/intechopen.93402*

with pharmacological potential, which could stabilize the KRas4B/PDE6δ complex. The identification of the compounds that had an *in silico* interaction with the complex, in addition these compounds was selected considering that they complied with the Lipinsky rule, which states that (1) the compounds should not have more than five hydrogen bridge donors; (2) they must not contain more than 10 hydrogen bridge acceptors; (3) they must have a molecular weight of less than 500 g/mol; (4) the compounds must have an octanol/water partition coefficient of less than five (log P < 5). Compounds identified as D14 and C22 showed different *in silico* interaction energies on the KRas4B/PDE6δ and K-Ras4BG12C/PDE6δ heterocomplex crystals; these interaction energies ranged from −143 to −162 ΔG [28].

An *in silico* analysis on the prediction of absorption using the ADME software made it possible to identify that compounds D14 and C22 have good absorption at the intestinal level and have low uptake by the permeability glycoprotein proteins that belong to the ABC transporter family. Their values are very low compared with the absorption of Gemcitabine and Deltarasin, which indicates that compounds D14 and C22 have a low chemoresistance when they are used as a treatment for pancreatic cancer cells (**Figure 2a**). Additionally, it was possible to observe a metabolism of compounds D14 and C22 by cytochromes P450 (CYP450), which indicated rapid liver metabolism and low toxicity since these compounds are coated by these enzymes (**Figure 2b**). Furthermore, it was observed that these compounds may have low toxicity compared to that obtained with the treatment of choice for pancreatic cancer such as Gemcitabine and with the PDE6δ Deltarasin inhibitor (**Figure 2c**).

Once identified that compounds D14 and C22 do not have toxic effects, we assessed the presence of KRas4B and PDE6d in pancreatic cancer cell lines by immunofluorescence using different cell lines with KRas4B (BxPC3), KRas4BG12D (PANC-1), and KRas4BG12C (MIA PaCa-2); we observed a greater presence of these proteins in the KRas PANC-1 and MIA PaCa-2-dependent cell lines (**Figure 3**). Having identified the cell lines with the highest presence of KRas4B and PDE6d, we treated them with a concentration of 200 μM of compounds D14 and C22 comparing their effect with hTERT-HPNE, which is a noncancerous cell line, and with 5 μM of Deltarasin (**Figure 4**). The results showed that compound D14 had a greater cytotoxic effect on the PANC-1 and MIA PaCa-2 cell lines, while compound C22 had a greater cytotoxic effect on the MIA PaCa-2 cell line. The comparison of these results with the effect obtained from Deltarasin, where the normal hTERT-HPNE cell line of pancreas was affected, suggested that our compounds do not have cytotoxicity in noncancerous cell lines.

Taking into account the results described above, we carried out Ras activation assays using the MIA PaCa-2 cell line. We obtained a dose response curve during 60 min, measuring Ras-GTP uptake by means of G-Lisa assays. Compounds D14 and C22 significantly decreased Ras activation over time; we obtained a 50% decrease in Ras activation at 60 min after treatment with the compounds (**Figure 5a**). As mentioned earlier during this chapter, the constitutive activation of KRas4B is essential for the development, progression, and maintenance of pancreatic cancer, and therefore, we performed subcutaneous xenograft tests by grafting 5 million cells of the MIA PaCa-2 cell line and administered via intraperitoneal 10 and 20 mg/kg of weight of compounds D14 and C22 for 15 days. The result was a 50% decrease in tumor growth in tumors treated with 20 mg/kg of weight of the two compounds, compared to the vehicle used as a control (**Figure 5b** and **c**).

Pancreatic ductal adenocarcinoma (PDAC) remains one of the leading causes of death by cancer, in addition to being one of the most aggressive types of cancer. The pancreatic cancer stem cell population (PCSCs) has been linked to this aggressiveness and poor prognosis. The cancer stem cell model proposes that tumor initiation,

#### **Figure 2.**

*Prediction of ADME processes of compounds D14 and C22. (A) Absorption of compounds D14 and C22 in epithelial barriers and their uptake by permeability glycoprotein proteins. (B) Metabolization of compounds D14 and C22 by means of cytochrome P450. (C) Toxicity of compounds D14 and C22.*

maintenance, and growth are directed by the population of stem cancer cells (CSC) [34, 35], which have been identified in several types of cancer, *e.g.* breast, brain, head and neck, colon, and pancreas [36, 37]. CSCs are defined as those tumor cells

*KRas4BG12C/D/PDE6δ Heterodimeric Molecular Complex: A Target Molecular Multicomplex… DOI: http://dx.doi.org/10.5772/intechopen.93402*

capable of self-renewal and production of heterogeneous lineages that comprise tumor volume [38]. In addition, several studies have reported evidence of the contribution of CSCs in resistance to conventional therapy, which causes metastasis and tumor recurrence [36, 39]. Different immunphenotypes have been reported for the identification of pancreatic cancer stem cells (PCSCs) [36, 37]. Due to the high fatality of PDAC, the importance of CSCs, and the participation of oncogenic KRas4B, we decided to evaluate the effect on the tumorigenicity of compounds D14 and C22 in CSC of PDAC; in this sense, cancer steam cells from BxPC3, PANC-1, and MIA PaCa-2 pancreatic cancer cell lines, as well as in the hTERT-HPNE noncancerous cell line, were growing in nonadherent conditions, forming spheroids or pancreatospheres and selected with the immunophenotypes positive to CD44, CD24, and ESA markers, which indicates an enrichment of PCSC (**Figure 6**). These

**Figure 4.**

*Morphological visualization of hTERT-HPNE, PANC-1, and MIA PaCa-2 cell lines treated with compounds D14 and C22 at 200 μM compared with the effect of Deltarasin.*

#### **Figure 5.**

*Compounds D14 and C22 decrease the activation of Ras in the MIA PaCa-2 cell line promoting the decrease of tumor growth. (A) Ras activation decreases by more than 50% in the MIA PaCa-2 cell line treated with D14 and C22. (B) and (C) Compounds D14 and C22 decrease tumor growth in subcutaneous xenograft models, using 20 mg/kg and 10 mg/kg intraperitoneally for 15 days.*

were treated with 49.65, 99.3, and 148.9 μM of compound D14, and with 494 nM of Gemcitabine. It was found that the treatment with compound D14 was able to break up the pancreatospheres formed by BxPC3 and MIA PaCa-2 more efficiently than the first-line treatment with Gemcitabine (**Figure 7**).

*KRas4BG12C/D/PDE6δ Heterodimeric Molecular Complex: A Target Molecular Multicomplex… DOI: http://dx.doi.org/10.5772/intechopen.93402*

#### **Figure 6.**

*CD44, CD24, and ESA immunophenotype in 3D cultures of BxPC3, PANC-1, and MIA PaCa-2. The expression of these markers is crucial for the identification of the cancerous trunk population.*

#### **Figure 7.**

*Morphological visualization of the effect of compound D14 on the viability of BxPC3 and MIA PaCa-2 in 2D and 3D; the result is better than with Gemcitabine, which is the first-line treatment for PDAC.*
