**2. Targeted drugs for the inhibition of KRas4B**

The direct inhibition of KRas4B has been a difficult task. Sites susceptible to pharmacological interaction have been identified by means of bioinformatic programs; therefore, compounds such as SCH-53239 and its analogue SCH-54292, which presented low affinity with respect to KRas4B, have been identified. These compounds were designed to interact with the Switch II of KRas4B, competing with the GDP. These compounds have in their chemical structure a hydroxylamine, which is essential for their cytotoxic activity. These compounds present a high level of toxicity in murine models, so they are in the improvement phase [22]. In 2012, several research groups reported a compound called DCAI [22, 23], which interacts with KRas4B at the site located between the α2 helix and β4 loop; this compound was able to inhibit the interaction of SOS1 with KRas4B with an IC50 of 340 μM, having an EC50 of 15.8 μM; therefore, it is so far one of the compounds considered for the treatment of PDAC [22, 23]. Also, different research teams have been working on 11,000 analogues of the DCAI compound in silico, based on the nuclear magnetic resonance (NMR). One of the analogues called VU0460009, showed an IC50 of 240 μM; although the concentration of the mean inhibition decreased, this compound did not have a considerable effect in murine models, so it was not possible to consider it as a candidate for treatment of PDAC [22, 23]. In order to find an organic compound that was capable of inhibiting the activation of KRas4B, studies were conducted to direct a specific molecule to the location site of the KRas4BG12C mutation, which is the most frequent in lung cancer [22]. One of the compounds studied was the so-called SCH-54292 [12], which is capable of binding to the α2 and α3 helices of KRas4B. This compound showed activity only in the cell lines that present KRas4BG12C, and with this finding, the researchers have intended to identify and study the analogues of SCH-54292 with the greatest effect on cancer cell lines [12]. Another group of researchers created a GDP analogue called SML-8-73-1, which could covalently bind to the cysteine of KRas4BG12C without taking into account the affinity of GDP with its binding site in KRas4B. These compounds did not show the expected effect on lung cancer cell lines, so they are in the improvement phase [22]. In recent years, several research groups have been trying to selectively inhibit mutated KRas activation and signaling. One way to prevent the activation of KRas and, therefore, its effector pathways, is through allosteric inhibition. Consequently, several research groups have developed

experimental models based on the *in silico* search for compounds that selectively bind and inhibit KRas. This strategy was carried out with an initial virtual coupling test of a library of compounds based on the reconstructed pocket structure of the switch I of RAS crystal, which resulted in an *in silico* coupling based on the pocket structure. The pocket consists of a hydrophilic part, which is composed of negatively charged residues such as Glu47, Asp48, and Asp 67, and a hydrophobic part, which consists of Leu66, Met77, and Tyr 81; on its surface, it is partially bordered by charged residues such as Lys15 and Asp67. These structural characteristics were used to establish a pharmacophore for the screening of charged residues, such as Asp67 (which corresponds to Asp57 of Ras), located in the lower center of the hydrophilic pocket in order to ensure the specificity of binding and energy. After the coupling analysis, compounds that *in vitro* effectively decreased the activation of Ras as well as its effectors were detected [21]. The development of small molecules that irreversibly bind to the oncogenic mutant KRasG12C allows the interruption of switch-1, and this alters the preference of native nucleotides in order to favor GDP over GTP and, consequently, this prevents its binding with the Raf effector. On the other hand, and using a similar strategy of *in silico* analysis and development of analogues with a favorable balance of ADME attributes (absorption, distribution, metabolism, and excretion), in vivo stability and specificity, in 2018, Janes and collaborators reported the design and characterization of switch-IIP inhibitors of KRasG12C with enhanced potency and pharmacological properties. Using tests based on liquid chromatography-tandem mass spectrometry (LC/MS-MS), compounds that covalently bound to Cys12 of K-Ras were measured directly and quantitatively. Pharmacological inhibition of KRasG12D with compound ARS-1620 suppressed the growth of cancer cells. ARS-1620 exhibited excellent oral bioavailability in mice and sufficient blood stability and, importantly, induces tumor regression through a specific mechanism of action.

Another strategy, proposed by Zeng and collaborators in 2017, is the possibility of designing compounds that incorporate elements of both the switch-IIP and the guanosine pharmacophores, or through the development of bivalent compounds that could recruit ligases to promote the degradation of RAS mediated by ubiquitination. The compounds were prepared with fluorophenyl and piperazinyl substituents and an electrophilic acrylamide warhead attached to the piperazine in order to effectively bind to KRasG12C. Subsequently, the 1\_AM analogue was developed with an amino amide substituent and showed a more complete binding with KRas. In addition, 1\_AM was compared with its serial head [1], and its properties were examined in H358 cells. The 1\_AM inhibitor decreased levels of KRas bound to GTP by ~80% compared to the performance of inhibitor 1, and likewise the decrease of the ERK effector phosphorylated.

An interesting strategy, which has been recently addressed, is the blocking of the interaction of RAS with its effector Raf. A cyclic peptide called cyclosarin 9A5 blocked the RAS-RAF interaction. The amino acids present in cyclosarin such as nal, Fpa, Thr, norleucine (nle), and Trp are critical for binding with KRas. Cyclosarin 9A5 showed improved cell permeability and an affinity for KRas with an IC50 = 0.12 μM. Cyclosarin reduced the proliferation and induced cell death by apoptosis in tumor cells with mutated KRas [24].

Similarly, in 2019, MacCarthy and collaborators used a variety of computational approaches in order to describe four binding sites in K-Ras for allosteric ligands. The new inhibitors bind to the pocket p1 with submicromolar affinity and function primarily by directly inhibiting the interaction of KRas with its effector proteins. This potential inhibitor forms multiple favorable interactions with residues in the pocket p1 of nonmutated KRas (WT) and with residues of the mutants of K-RasG12D, G12C, and Q61H in the active state bound to GTP. In addition, the authors report that the inhibitor of KRas binding to its effectors decreases the

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

levels of phosphorylation of ERK and cRAF in BHK cells that express the mutant of KRasG12D and G12V, which suggests the inhibition of KRas signaling through the MAPK pathway. However, the problem of allosteric KRas inhibitors that prevent interaction with their effectors is that they do not exhibit selectivity toward a particular RAS isoform or KRas WT vs. mutated KRas, which raises the toxicity problem in cells and therefore in healthy organs when they are used in vivo.

Another approach to inhibit the interaction of KRas with its effectors is the search for macromolecules that selectively bind to KRas at an allosteric lobe site that encompasses histidine 95 residue at the interface between the helix α3/loop 7/helix α4. Designed ankyrine repeat proteins (DARPins) K13 and K219 inhibit the interactions with the effectors and the nucleotide exchange of KRas. Similarly, K13 and K19 induce selective inhibition of the RAF/MEK/ERK signaling pathway in cells with the Kras4BG13D mutation but not in cells with other mutated isoforms such as HRasG12V and/or RAS WT. This suggests that K13 and K19 selectively inhibit the function of mutated KRas without affecting cancer cells with RAS WT; however, its toxicity in healthy cells has not been proven [25].
