**3.1 Target identification**

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

in both adults and children [162, 164, 165].

This powerful NGS can be used in several different areas. For example, NGS can be used in clinical settings for identifying genetic variants with high specificity and sensibility, thus allowing for detection of mosaic mutations that could not be previously identified by Sanger sequencing [162]. In the field of microbiology, NGS can be used for identifying and characterizing pathogens, including novel strains or mutants, thereby allowing for linking a pathogen or a new pathogenic strain to an outbreak in a specific region or to a particular individual(s) [163]. The role of NGS in the field of oncology is quite significant, as this technology can be used for pursuing personalized medicine, in particular for developing targeted therapies for specific cancers correlated with individual genetic profiles of patients. Moreover, NGS is highly useful for diagnosis, and for classification of different types of cancer

Furthermore, NGS is highly versatile, primarily for the diversity of analysis that can be undertaken, as well as to numbers and types of biological samples that can be analyzed. A listing of major types of analyses that can be undertaken, as well as of

As for drug discovery, NGS has been successfully used in various areas of drug discovery, beginning with target identification, compound screening,

**Analysis type Purpose(s) Biological sample(s)**

Cell cultures; whole blood; serum; plasma; fresh/frozen tissue; formalin-fixed paraffinembedded tissue

Cell cultures; whole blood; fresh/frozen tissue; formalin-fixed paraffin-embedded tissue

Cell cultures; whole blood; fresh/frozen tissue; formalin-fixed paraffin-embedded tissue

Cell cultures; whole blood; serum; plasma; fresh/frozen tissue

Cell cultures, Whole blood, serum, plasma, fresh/frozen tissue

Cell cultures; fresh/ frozen tissue

Cell cultures; whole blood; fresh/frozen tissue; formalin-fixed paraffin-embedded tissue

Cell cultures; whole blood; fresh/frozen tissue; formalin-fixed paraffin-embedded tissue

set of gene region(s) or SNP(s)

region(s) of DNA (exomes)

genome of an organism(s)

genes present in an organism

sequencing allows for identifying alterations at DNA –binding sites of different transcriptional factor(s) or

Detect duplication(s), deletion(s), translocation(s), or inversion(s) of one or

in CpG, CHG, and CHH regions across a

types of biological samples used in NGS are presented in **Table 2**.

Targeted gene sequencing Identify genetic alteration(s) for a specific

Whole exome sequencing Evaluate variation(s) present in coding

Whole genome sequencing Identify variations present in the whole

miRNAseq Identify miRNAs and their expression level(s)

RNAseq Determine expression levels of whole

CHIPseq Chromatin immunoprecipitation

protein(s)

more genes

Methylation sequencing Evaluate whole methylation pattern(s)

*Types of NGS analysis, purpose(s), and biological samples used.*

genome

**66**

**Table 2.**

Copy number alterations/ variations (CNVs)

In recent years, NGS has been valuable in the identification of different genetic alterations of a pathogen/pathology that can be useful for targeted treatment. The versatility of NGS allows for evaluating genomic regions using genomic analysis, transcriptomics, RNAseq, and miRNA seq in order to identify gene(s) and their regulation(s)/functionality(ies) in response to different disease conditions, which in turn could be used for target identification [169].

Analysis of genetic variant(s) is yet another important approach for identifying mutations in rare diseases, as these could then be used for treatment of such target(s) [170, 171]. Epigenetic studies, such as methylation analysis or CHIP-seq analysis, known to be altered in different pathologies, could also aid in identifying targets for specialized treatments/therapies [172, 173].

NGS has been widely used for gene to target identification for treatment of cancer. As it is well known, the National Comprehensive Cancer Network (NCCN) has several guidelines for NGS target identification used for treatment of various types of cancer. These include targeting genes for lung cancer (*EGFR, ALK, ROS1, BRAF*, and *PDL1*) [174], colorectal cancer (*NRAS/HRAS/KRAS, BRAF*, *HER2*, *MLH1, MSH2, MSH6,* and *PMS2*) [175], breast and ovarian cancers (*BRCA1/2, TP53, STK11, PTEN, CDH1, PALB2*, among others) [176]. By identifying mutations in each of these genes, clinicians are able to treat patients with specific targeted treatments. In Waldenström's macroglobulinemia, NGS has been employed in evaluating genomic variations that could better inform treatment of patients, and that would ultimately lead to better outcomes. It is observed that patients with recurrent somatic mutations in genes of myeloid differentiation factor 88 (*MYD88*) and chemokine receptor type 4 (*CXCR4*) demonstrate different responses to the same treatment, and thus these genes serve as clinical determinants of clinical

### **Figure 4.**

*Various steps involved in drug discovery whereby NGS can be of particular benefit.*

presentation [154]. Therefore, a treatment algorithm can be used, based on the mutational status of a patient, in a clinic to adjust targeted treatment [177].

Although cancer has been the most widely studied disease over the last decade, other diseases have employed this approach to identify improved therapies/treatments for each individual patient. For example, Tshibangu-Kabamba et al. have used NGS for evaluating antimicrobial resistance (AMR) of different strains of *Helicobacter pylori*, as well as for determining antimicrobial susceptibilities of these bacterial strains [155]. Whole genome sequencing has aided in identifying several variants in AMR genes, such as *pbp1A* (T558S, F366L), *gyrA* (A92T, A129T), *gyrB* (R579C), and *rdxA* (R131\_K166del). This has been instrumental in determining susceptibility of these strains to specific drugs [178].

RNA-seq technology has been used for profiling of host, bacteria, and SARS-CoV-2 virus outbreaks in New York City [156]. It is reported that RNA-seq results are similar to those of RT-PCR. In addition, it is observed that SARS-CoV-2 samples seem to carry other types of viruses. Interestingly, it is also observed that there are gene expression dysregulation in viral response pathways, innate immune responses, and interferon signaling that could explain different responses of patients to the same antiviral drugs [179].

In another study, NGS has been used to identify a targeted treatment for a patient suffering from an immune dysregulation syndrome. As a result, a new germline mutation in the *CTLA4* (Cytotoxic T-Lymphocyte Associated Protein 4) gene, susceptible to the drug abatacept, has been identified [180].

#### **3.2 Target to standard of care**

In this step of drug discovery, NGS plays an important role, mainly due to its ability to assess multiple gene alterations within a short period of time. Moreover, the Food and Drug Administration (FDA) has approved NGS testing in clinics. One such example is the case of using a hybrid capture NGS assay for evaluating non-small cell lung cancer in patients. Using this assay, Schatz et al. have diagnosed 417 patients based on both genetic alterations and tumor burden. This approach has made it possible to use specific treatments based on tumor burden values if no actionable genetic alteration is detected [181, 182]. Furthermore, Klowak et al. have used NGS in a pilot study to identify pathogens in neonates suspected of having sepsis. They have proposed an NGS-based protocol for implementation in clinics to accurately and rapidly identify those pathogens affecting neonates, as well as to provide better treatments [183]. Yet in another example, an NGS panel, consisting of seven fusion genes and seven genes with frequent copy number changes, has been used to diagnose 113 sarcoma patients with 97% sensitivity and 100% specificity. This has rendered this gene panel as a highly promising toll for implementing gene targets in standard of care for sarcoma patients [184]. There are several other studies demonstrating the utility of NGS testing in identifying targets that could be actionable by either specific drugs or that could be implemented as specific targets for standard of care for particular diseases [185–189].

#### **3.3 Compound screening**

In recent years, a common method used for compound screening during drug discovery is "encoded library technology" (ELT) [190]. ELT is based on DNA binding to members of a small molecule library of chemical compounds. This DNA tag, serving as an amplifiable identification barcode, is unique to each compound/ organic ligand, thus rendering it possible for its incubation with specific protein targets of interest. Subsequently, these organic compounds/ligands are washed away

**69**

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

compounds [191–194].

screening [199].

**3.4 Undruggable targets and NGS**

development of treatments for various diseases.

validated in multiple preclinical studies [204].

based on their affinities to the target; thus, compounds/ligands with high affinities for the target are enriched, and identified by NGS sequencing of PCR products [190]. This approach allows for both constructing and screening of combinatorial libraries of large volumes, thus facilitating rapid discovery of ligands to various different protein targets. ELT is used in several clinical areas, mainly for cancer, but also for various diseases, as it is a rapid and economical screening system of organic

Recently, Lemke et al. used ELT and virtual computation library screening methods, DNA-encoded chemical libraries (DECL), to identify inhibitors for poly-ADR-ribose polymerase member 10 (*PARP10*). In effect, they integrated DECL screening with structure-based computational methods to streamline the development of leading compounds. Thus, following DECL screening, they observed that a compound with an A82-CONHMe-B54 motif yielded the best result. Therefore, they screened over 10,500 virtual compounds, and selected ten compounds for synthesis. These compounds were assessed for *PARP10* inhibition, and they found two compounds with promising results [195]. In another study, Reidenbach et al. attempted to identify compounds against Prion disease, a neurodegenerative disease with no therapeutic options; however, the only benzimidazole compounds identified demonstrated low affinities [196]. Whereas, Cuozzo et al. screened a DECL library of 225 million compounds, and identified a single compound (X-165) with a high activity against the production of lysophosphatidic acid, and this compound has been approved by the FDA for Phase I Clinical trials [197]. In other examples of using this strategy, Dawadi et al. discovered a thrombin inhibitor using DECL [198], while, Kung et al. identified two compounds that presented inhibition/binders to e Nα-terminal acetyltransferase (Naa50) using ECL library

As mentioned above, an "Undruggable" target is a term given to sets of proteins that cannot be targeted by a specific treatment, yet they can be exploited for the

Among these undruggable targets are non-enzymatic proteins, transcriptional factors, regulatory proteins, and scaffolding proteins [200, 201]. One such undruggable target is the Kristen Rat Sarcoma (KRAS) protein encoding a viral oncogene, detected in non-small cell lung cancer (NSCLC). Recently, KRAS mutations have been successfully targeted using different approaches, such as inhibition of downstream effectors, epigenomic approaches, post-translational modifications, and high-affinity KRAS binders, among others, wherein direct pharmacological inhibition of a *KRAS p.G12C* mutation is deemed possible, thus serving as an effective targeted treatment available for patients with advanced NSCLC [202]. Moreover, other members of the RAS family are deemed as undruggable targets in cancer, and several approaches have been used. Kato et al. have used NGS to evaluate the mutational status of 1937 patients with different cancers, and have observed that over 20% presented *RAS* alterations. Unfortunately, poor overall survival has been observed in spite of various treatment options that are offered; however, a better survival is observed for patients treated using a combined therapy targeting MAPK and non-MAPK pathways [203]. Among other undruggable targets, MYC and TP53 are known to have no enzymatic activities, and are located intracellularly. However, a Phase III trial is undergoing for TP53 using the APR-246 drug for myelodysplastic syndrome, and although there are no current clinical trials for MYC, an anti-MYC compound, OmoMYC, has been

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

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

susceptibility of these strains to specific drugs [178].

patients to the same antiviral drugs [179].

**3.2 Target to standard of care**

**3.3 Compound screening**

presentation [154]. Therefore, a treatment algorithm can be used, based on the mutational status of a patient, in a clinic to adjust targeted treatment [177].

Although cancer has been the most widely studied disease over the last decade, other diseases have employed this approach to identify improved therapies/treatments for each individual patient. For example, Tshibangu-Kabamba et al. have used NGS for evaluating antimicrobial resistance (AMR) of different strains of *Helicobacter pylori*, as well as for determining antimicrobial susceptibilities of these bacterial strains [155]. Whole genome sequencing has aided in identifying several variants in AMR genes, such as *pbp1A* (T558S, F366L), *gyrA* (A92T, A129T), *gyrB* (R579C), and *rdxA* (R131\_K166del). This has been instrumental in determining

RNA-seq technology has been used for profiling of host, bacteria, and SARS-CoV-2 virus outbreaks in New York City [156]. It is reported that RNA-seq results are similar to those of RT-PCR. In addition, it is observed that SARS-CoV-2 samples seem to carry other types of viruses. Interestingly, it is also observed that there are gene expression dysregulation in viral response pathways, innate immune responses, and interferon signaling that could explain different responses of

In another study, NGS has been used to identify a targeted treatment for a patient suffering from an immune dysregulation syndrome. As a result, a new germline mutation in the *CTLA4* (Cytotoxic T-Lymphocyte Associated Protein 4)

In this step of drug discovery, NGS plays an important role, mainly due to its ability to assess multiple gene alterations within a short period of time. Moreover, the Food and Drug Administration (FDA) has approved NGS testing in clinics. One such example is the case of using a hybrid capture NGS assay for evaluating non-small cell lung cancer in patients. Using this assay, Schatz et al. have diagnosed 417 patients based on both genetic alterations and tumor burden. This approach has made it possible to use specific treatments based on tumor burden values if no actionable genetic alteration is detected [181, 182]. Furthermore, Klowak et al. have used NGS in a pilot study to identify pathogens in neonates suspected of having sepsis. They have proposed an NGS-based protocol for implementation in clinics to accurately and rapidly identify those pathogens affecting neonates, as well as to provide better treatments [183]. Yet in another example, an NGS panel, consisting of seven fusion genes and seven genes with frequent copy number changes, has been used to diagnose 113 sarcoma patients with 97% sensitivity and 100% specificity. This has rendered this gene panel as a highly promising toll for implementing gene targets in standard of care for sarcoma patients [184]. There are several other studies demonstrating the utility of NGS testing in identifying targets that could be actionable by either specific drugs or that could be implemented as specific targets

In recent years, a common method used for compound screening during drug discovery is "encoded library technology" (ELT) [190]. ELT is based on DNA binding to members of a small molecule library of chemical compounds. This DNA tag, serving as an amplifiable identification barcode, is unique to each compound/ organic ligand, thus rendering it possible for its incubation with specific protein targets of interest. Subsequently, these organic compounds/ligands are washed away

gene, susceptible to the drug abatacept, has been identified [180].

for standard of care for particular diseases [185–189].

**68**

based on their affinities to the target; thus, compounds/ligands with high affinities for the target are enriched, and identified by NGS sequencing of PCR products [190]. This approach allows for both constructing and screening of combinatorial libraries of large volumes, thus facilitating rapid discovery of ligands to various different protein targets. ELT is used in several clinical areas, mainly for cancer, but also for various diseases, as it is a rapid and economical screening system of organic compounds [191–194].

Recently, Lemke et al. used ELT and virtual computation library screening methods, DNA-encoded chemical libraries (DECL), to identify inhibitors for poly-ADR-ribose polymerase member 10 (*PARP10*). In effect, they integrated DECL screening with structure-based computational methods to streamline the development of leading compounds. Thus, following DECL screening, they observed that a compound with an A82-CONHMe-B54 motif yielded the best result. Therefore, they screened over 10,500 virtual compounds, and selected ten compounds for synthesis. These compounds were assessed for *PARP10* inhibition, and they found two compounds with promising results [195]. In another study, Reidenbach et al. attempted to identify compounds against Prion disease, a neurodegenerative disease with no therapeutic options; however, the only benzimidazole compounds identified demonstrated low affinities [196]. Whereas, Cuozzo et al. screened a DECL library of 225 million compounds, and identified a single compound (X-165) with a high activity against the production of lysophosphatidic acid, and this compound has been approved by the FDA for Phase I Clinical trials [197]. In other examples of using this strategy, Dawadi et al. discovered a thrombin inhibitor using DECL [198], while, Kung et al. identified two compounds that presented inhibition/binders to e Nα-terminal acetyltransferase (Naa50) using ECL library screening [199].
