**2.2 Droplet Digital PCR (ddPCR) and microarrays**

The Droplet Digital PCR (ddPCR) is a recent technology that is commercially available, capitalizing on the use of *Taq* polymerase in a standard PCR reaction in order to amplify a target DNA fragment from a complex sample using pre-validated primers or primer/probe assays [61, 62]. Galbiati et al. have proposed a workflow that combined a microarray assay with ddPCR for both detection and quantification of circulating tumor DNA mutations in colon cancer patients [63]. This approach is useful for the development of reliable non-invasive biomarkers for RAS and BRAF mutations, identifying a target mutation, and providing clinically relevant information. Microarray analysis and ddPCR data have identified mutations in primary breast tumors from female patients treated with adjuvant mono-tamoxifen therapy [64]. Moreover, using microarray and ddPCR, it is observed that epidermal growth factor receptor (EGFR) expression can be used as a prognostic biomarker in patients with oropharyngeal squamous cell carcinoma, as it is associated with smoking status [65]. In another study, microarray analysis of uterine tissue, along with validation using ddPCR has allowed for observing downregulation of genes in pathways of


#### **Table 1.**

*Available techniques for drug discovery.*

the immune response following tetrabromobisphenol A treatment [66]. Moreover, ddPCR analysis of miRNAs identified using a microarray assay has revealed that anti-apoptotic miRNA may be potentially involved in antagonistic effects between the *Alternaria* mycotoxins alternariol and altertoxin *II* in HepG2 cells [67]. In another study using this combined approach, transglutaminase 2 is identified as a novel regulator of the tumor microenvironment in gastric cancer patients, thus serving as a promising target for restricting tumor-promoting inflammation [68].

### **2.3 Undruggable to druggable proteins using microarrays**

In recent years, efforts have been directed towards transforming those proteins that are deemed pharmacologically incapable of being targeted, coined as "undruggable", into "druggable" proteins. Despite the fact that many proteins, such as kinases, that promote cancer development, are capable of serving as drug targets, proteins such as RAS, MYC, and p53 are deemed as "undruggable targets" [69]. Thus, overcoming these "undruggable targets" becomes one of the main challenges for drug discovery. One of the new proposed methods to overcome these challenges is represented by the inhibition of kinase activities of oncogenic proteins using small molecules and antibodies [70]. In one approach, blocking of pathways downstream of a target protein has served as a viable strategy to assess the functional role of a mutation as an oncogenic driver of different types of cancers, and for serving as a valid clinical trial design [71]. In another approach, discovery of hidden allosteric sites is an effective strategy for development of new drug targets, as well as for discovery of allosteric drugs [69].

It is known that *RAS* mutations serve as early genetic events in tumor progression, while sustained expression of *RAS* mutations are deemed necessary for tumor maintenance [72]. Although RAS have been deemed as "undruggable", recent studies have demonstrated that therapies targeting either RAS-activating pathways or RAS effectors pathways combined with direct RAS inhibitors, along with immune checkpoint inhibitors or T-cell targeting methods, *RAS*-mutant tumors are found to be treatable [73]. As the transcription factor MYC promotes cancer progression, small-molecule inhibitors are used to drug the "undruggable" by inducing epigenetic silencing and regulating G-quadruplex structures within the *MYC* promoter [74]. In another example, *p53* is well known as the most frequently altered gene in human cancer, and therefore the p53 mutant protein is deemed as an important undruggable target [75]. Such compounds as p53 reactivation, induction of massive apoptosis-1 (PRIMA-1), and a structural analogue of PRIMA-1, APR-246, have been found to reactivate the mutant p53 protein by converting it to a form with wild-type properties [75].

Using a custom-designed lncRNA microarray, Orilnc1 was identified as a novel nonprotein mediator of RAS/RAF activation, with potential applications as a therapeutic target in RAS/RAF-driven cancers [76]. An Affymetrix microarray revealed coexpression of a mutant β-catenin and K-Ras in mice by targeting β-catenin in hepatocellular cancers [77]. Microarray and pathway enrichment analyses revealed that MYC expression could be downregulated by 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranoside (PGG) in hepatocellular carcinoma [78]. Using a genome-wide microarray analysis, it was reported that targeting c-Myc would unlock novel strategies to combat asthma [79]. β-catenin could be deemed as an anticancer therapeutic target by regulating c-Myc and CDKN1A expression in breast cancer cells [80]. In addition, microarray data identified and characterized novel p53 target genes expressed in hepatocarcinoma cells, and were associated with steroid hormones processing and transfer [81]. Furthermore, it was proposed that there was a novel non-cell-autonomous tumor-suppressive regulation, mediated by p53, playing a key role in maintaining organism homeostasis. Moreover, breast cancer

**59**

range of antibiotics [96].

*Microarrays and NGS for Drug Discovery DOI: http://dx.doi.org/10.5772/intechopen.96657*

as a therapeutic target for cancer [82].

**2.4 Microarrays and drug resistance**

increased apoptosis resistance [86].

metastasis suppressor 1-like (BRMS1L) was found to be upregulated by p53 protein. In addition, p53 inhibited cancer cell invasion and migration, and thus could serve

Resistance to chemotherapy remains a major obstacle to improving a cancer patient's outcome and survival despite significant advances in surgery, radiation therapy, and anticancer treatments. In cancer, drug resistance arises from a complex range of molecular and biochemical processes, such as modifications in DNA repair mechanism, drug uptake, absorption, and metabolism. Recent studies have identified two forms of drug resistance in cancer patients, intrinsic (innate resistance that is present before a patient is exposed to drugs) and acquired (a direct result of chemotherapy). A growing number of microarray studies have exploited the identification of mechanisms involved in both drug response and drug resistance in clinical samples in order to identify biomarkers for drug resistance [83]. For example, microarray analysis has provided a better understanding of circular RNA expression profiles that are associated with gemcitabine resistance in pancreatic cancer cells [84]. In human gastric cancer tissues, a microarray study has revealed that miR-424 regulates cisplatin resistance of gastric cancer [85]. Furthermore, extracellular matrix proteins have been implicated in drug-resistant ovarian cancer cells, thus inhibiting penetration of a drug into cells, as well as contributing to

Of particular interest, new genes associated with drug resistance development in ovarian cancer have been discovered using microarray analysis, wherein 13 genes are found to be upregulated, while nine genes are found to be downregulated [87]. In triple-negative breast cancer cells, notable alterations are observed at both transcriptomic and genomic levels, along with identification of a mutation (*TP53*) associated with drug response [88]. In another study, bioinformatics analyses of microarray datasets have identified neuromedin U (NMU) as a potential gene that confers alectinib resistance in non-small cell lung cancer [89]. Furthermore, expression profiling has allowed for discovery of genes involved in ovarian-drug resistance, wherein these genes are found to be controlled via different signaling pathways, including MAPK– Akt, Wnt, and Notch [90]. In another study, microarray analysis has found that tumor initiation and insulin-like growth factor (IGF)/fibroblast growth factor (FGF)

signaling contribute to sorafenib resistance in hepatocellular carcinoma [91]. As antibiotic resistance has become a global health problem, efforts are underway to identify and screen for new and effective antibiotics. A microarray for 132 gram-negative bacteria has been evaluated to detect genes for resistance to 75 clinically relevant antibiotics [92]. Frye et al. have developed a DNA microarray capable of detecting all antimicrobial resistance genes found at the National Center for Biotechnology [93]. Furthermore, a microarray has been use to identify *Helicobacter pylori* resistance to clarithromycin and levofloxacin, as well as to detect *CYP2C19* polymorphism [94]. It is reported that this microarray can be used for individual therapy detection as it has high specificity, reproducibility, and sensitivity [78]. In another study, an effort has been successfully undertaken to reduce antibiotic susceptibility testing assay time, as well as for rapid determination of minimum inhibitory concentrations of different antibiotics using a nanoliter-sized microchamber/microarray-based microfluidic (N-3 M) platform [95]. More recently, a commercially available microarray (IDENTIBAC AMR-ve) has been developed for determination of antibiotic-resistant clinical isolates of *Klebsiella pneumoniae,* and to identify genes associated with resistance to a wide

*Drug Design - Novel Advances in the Omics Field and Applications*

**2.3 Undruggable to druggable proteins using microarrays**

discovery of allosteric drugs [69].

the immune response following tetrabromobisphenol A treatment [66]. Moreover, ddPCR analysis of miRNAs identified using a microarray assay has revealed that anti-apoptotic miRNA may be potentially involved in antagonistic effects between the *Alternaria* mycotoxins alternariol and altertoxin *II* in HepG2 cells [67]. In another study using this combined approach, transglutaminase 2 is identified as a novel regulator of the tumor microenvironment in gastric cancer patients, thus serving as a promising target for restricting tumor-promoting inflammation [68].

In recent years, efforts have been directed towards transforming those proteins that are deemed pharmacologically incapable of being targeted, coined as "undruggable", into "druggable" proteins. Despite the fact that many proteins, such as kinases, that promote cancer development, are capable of serving as drug targets, proteins such as RAS, MYC, and p53 are deemed as "undruggable targets" [69]. Thus, overcoming these "undruggable targets" becomes one of the main challenges for drug discovery. One of the new proposed methods to overcome these challenges is represented by the inhibition of kinase activities of oncogenic proteins using small molecules and antibodies [70]. In one approach, blocking of pathways downstream of a target protein has served as a viable strategy to assess the functional role of a mutation as an oncogenic driver of different types of cancers, and for serving as a valid clinical trial design [71]. In another approach, discovery of hidden allosteric sites is an effective strategy for development of new drug targets, as well as for

It is known that *RAS* mutations serve as early genetic events in tumor progression, while sustained expression of *RAS* mutations are deemed necessary for tumor maintenance [72]. Although RAS have been deemed as "undruggable", recent studies have demonstrated that therapies targeting either RAS-activating pathways or RAS effectors pathways combined with direct RAS inhibitors, along with immune checkpoint inhibitors or T-cell targeting methods, *RAS*-mutant tumors are found to be treatable [73]. As the transcription factor MYC promotes cancer progression, small-molecule inhibitors are used to drug the "undruggable" by inducing epigenetic silencing and regulating G-quadruplex structures within the *MYC* promoter [74]. In another example, *p53* is well known as the most frequently altered gene in human cancer, and therefore the p53 mutant protein is deemed as an important undruggable target [75]. Such compounds as p53 reactivation, induction of massive apoptosis-1 (PRIMA-1), and a structural analogue of PRIMA-1, APR-246, have been found to reactivate the mutant p53 protein by converting it to a form with wild-type properties [75].

Using a custom-designed lncRNA microarray, Orilnc1 was identified as a novel nonprotein mediator of RAS/RAF activation, with potential applications as a therapeutic target in RAS/RAF-driven cancers [76]. An Affymetrix microarray revealed coexpression of a mutant β-catenin and K-Ras in mice by targeting β-catenin in hepatocellular cancers [77]. Microarray and pathway enrichment analyses revealed that MYC expression could be downregulated by 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranoside (PGG) in hepatocellular carcinoma [78]. Using a genome-wide microarray analysis, it was reported that targeting c-Myc would unlock novel strategies to combat asthma [79]. β-catenin could be deemed as an anticancer therapeutic target by regulating c-Myc and CDKN1A expression in breast cancer cells [80]. In addition, microarray data identified and characterized novel p53 target genes expressed in hepatocarcinoma cells, and were associated with steroid hormones processing and transfer [81]. Furthermore, it was proposed that there was a novel non-cell-autonomous tumor-suppressive regulation, mediated by p53, playing a key role in maintaining organism homeostasis. Moreover, breast cancer

**58**

metastasis suppressor 1-like (BRMS1L) was found to be upregulated by p53 protein. In addition, p53 inhibited cancer cell invasion and migration, and thus could serve as a therapeutic target for cancer [82].
