Structural insights, regulation, and recent advances of RAS inhibitors in the MAPK signaling cascade: a medicinal chemistry perspective

Abstract

The MAPK pathway has four main components: RAS, RAF, MEK, and ERK. Among these, RAS is the most frequently mutated protein and the leading cause of cancer. The three isoforms of the RAS gene are HRAS, NRAS, and KRAS. The KRAS gene is characterized by two splice variants, K-Ras4A and K-Ras4B. The occurrence of cancer often involves a mutation in both KRAS4A and KRAS4B. In this study, we have elucidated the mechanism of the RAS protein complex and the movement of switches I and II. Only two RAS inhibitors, sotorasib and adagrasib, have been approved by the FDA, and several are in clinical trials. This review comprises recent developments in synthetic RAS inhibitors, their unique properties, their importance in inhibiting RAS mutations, and the current challenges in developing new RAS inhibitors. This review will undoubtedly help researchers design novel RAS inhibitors.

Normal and oncogenic RAS signalling pathway along with inhibitors.

1. Introduction

Cancer is one of the leading causes of mortality; with estimates of cancer-related mortality rising day by day, it has become a matter of global concern.¹ Cancer was initially identified as a growing tissue mass, a tumor. Cancer can be defined as a disease of uncontrolled proliferation of transformed cells. In other words, cancer describes a disease in which abnormal cells divide out of control and can assault other tissues. Blood and lymph systems are two major routes via which cancer cells can spread to different body parts.² Several key pathways control the proliferation of cells; one of the significant pathways is the MAPK (mitogen-activated protein kinase) pathway.³ There are four major components of the MAPK pathway: RAS (rat sarcoma virus), RAF (rapidly accelerated fibrosarcoma), MEK (mitogen-activated protein kinase kinase), and ERK (extracellular signal-regulated kinase). Out of these, RAS is the most frequently mutated protein and a major cause of cancer.⁴ RAS signaling is one of the most prominent oncogenic drivers and has a role in tumor genesis. Available RAS pathway inhibitors are used in the treatment of both benign and malignant tumors.³

RAS proteins are guanine nucleotide-binding proteins that are crucial intermediates in cell signaling pathways.⁵ These proteins, also known as guanosine triphosphatases (GTPases), function by hydrolyzing guanosine triphosphate (GTP) and alternately binding to either GTP or guanosine diphosphate (GDP).⁶ The conversion between the inactive GDP-bound state and the active GTP-bound state of RAS is governed by GEFs, which activate RAS by encouraging the exchange of GDP to GTP, and GAPs (GTPase-activating proteins), which catalyze RAS-mediated GTP hydrolysis, inhibit RAS. One of the most often dysregulated pathways in human cancer is the RAS pathway.⁷ The RAS gene is a monomeric membrane-localized G protein of 21 kDa, which serves as a molecular relay connecting the activation of receptor and non-receptor tyrosine kinase to downstream cytoplasmic or nuclear events.⁶ The three most common isoforms of the RAS gene are HRAS (Harvey murine sarcoma virus), NRAS (neuroblastoma murine sarcoma virus), and KRAS (Kirsten murine sarcoma virus). The expression level of KRAS4B is greater than that of KRAS4A in tumors.⁸ Approximately 30% of all human malignancies contain oncogenic mutations in the RAS gene. Mutations in KRAS are common in pancreatic carcinoma, non-small-cell lung cancer, and colorectal cancer. Mutations in HRAS are common in thyroid, bladder, and kidney cancers. Mutations in NRAS are found in melanoma, hepatocellular carcinoma, and hematologic tumors.⁶ Within the RAS gene family, KRAS is the predominant site of mutation, accounting for 75% of RAS-driven malignancies, followed by NRAS (17%) and HRAS (7%).⁹ RAS-driven malignancies, include 95% of pancreatic cancers, 42% of colon cancers, and 20–30% of lung cancers.¹⁰ The exclusive role of KRAS (∼98%) is found in pancreatic ductal adenocarcinoma (PDAC) in RAS mutant cancers, whereas mutations in NRAS dominate (94%) in carcinoma.¹¹

The first oncogene found in human cancer cells was the RAS gene. This included the first somatic mutation in human cancer, the HRAS gene.^8,12 The founding members of the RAS family are HRAS and NRAS of about 150 small GTPases (20–30 kDa), which are separated into five smaller groups based on the relationships in the primary sequence. These small GTPases are modulators of numerous biological processes, including growth, proliferation, survival, apoptosis, senescence, cytoskeletal variability, vesicular trading, differentiation, and gene expression.¹³ Mutationally triggered and potently transforming human RAS genes were discovered in human cancer cell lines in 1982. The discovery of oncogenic genetic elements began with the identification of the Harvey murine sarcoma virus in 1964 and the Kirsten murine sarcoma virus in 1967, which were identified many years later as HRAS and KRAS oncogenes in humans.¹⁴ The G-domain of all RAS proteins harbors the primary oncogenic mutation at conserved amino acid positions G12, G13, and Q61. Distinct variations in the frequency of mutations at given positions are additionally noted among the different RAS isoforms. All KRAS alterations include G12 (83%), G13 (14%), and Q61 (2%) mutations. These mutations occur near γ-phosphate of GTP within the active site.¹⁵ In comparison, NRAS mutations predominantly occur at Q61, with G12 and G13 mutations trailing behind, whereas in HRAS, a more comparable rate of mutation of G12, G13, and Q61 is spotted.⁹ Some other proteins also belong to the RAS superfamily, such as R-Ras, R-heb, Rap, Rin, Ral, and Rit, which are also connected to cell proliferation and differentiation. Because of the involvement of multiple proteins in the RAS signaling pathway, it becomes more challenging to understand.¹⁶

2. Regulation of the RAS pathway

RAS proteins function as molecular switches that show the transition between an inactive GDP (guanosine diphosphate)-bound and an active GTP-bound form, enabling interaction with downstream effectors. When RAS is in active form, it can initiate a downstream signaling pathway.¹⁷ The extracellular stimuli that promote RAS activation include receptor tyrosine kinases (RTKs), non-receptor tyrosine kinases, G protein-coupled receptors, and integrins. Two proteins govern the transition between GDP-bound RAS and GTP-bound RAS: one is guanine nucleotide exchange factors (GEFs) which are responsible for loading RAS with GTP, and the other is GTPase-activating proteins (GAPs) which hydrolyze bound GTP to return RAS to its inactive GDP-bound state.¹⁸ GTP-bound RAS can engage in beneficial interactions with 20 effectors, including Raf, phosphatidyl inositol three kinase (PI3K), and Ral guanine nucleotide-dissociation stimulator (RALGDS), to control different biological reactions such as differentiation, survival, and proliferation.¹⁹ Three different steps control the signaling dynamics of RAS proteins: (i) GTP hydrolysis, (ii) nucleotide binding/unbinding, and (iii) effector binding/unbinding.¹⁷

After the activation of RAS, intracellular tyrosine residues undergo autophosphorylation. This phosphorylated residue interacts with the SH2 domain of GRB2 (growth receptor binding 2) protein. The GRB2 protein has two domains, SH2 and SH3, whereas the SH2 domain is bound to SOS (Son of Sevenless), which releases GEF. This GEF catalyzes the nucleotide exchange of RAS, leading to the replacement of GDP with GTP. The active form of RAS is the prevailing state of oncogenic RAS in RAS mutant cancers.²⁰

Then, active RAS interacts with various downstream effector molecules and shows kinase activity. This results in phosphorylation of RAF (rapidly accelerated fibrosarcoma), which activates MAPK kinase (MEK). Subsequently, this MEK phosphorylates and activates ERK.²¹ ERK then phosphorylates transcription factors belonging to the ETS family. For example, ELK1 controls the expression of c-fos and c-jun, activating AP-1 transcription factors, which consist of FOS-JUN heterodimer. Due to the stimulation of these transcription regulators, expression of D-type cyclins (cell cycle regulatory proteins) takes place, which leads to cell division, proliferation, and metastasis (Fig. 1).⁵

Fig. 1. Regulation of RAS (normal and oncogenic) pathway in MAPK signaling cascade.

3. Molecular structure of RAS protein

The molecular structure of the RAS protein consists of a 6-stranded β-sheet surrounded by five α-helices in a Rossmann-type fold. The three types of RAS proteins include HRAS, NRAS, and KRAS. These isoforms show high similarity in GDP/GTP binding motif elements that are extended up to the N-terminal G domain. The C-terminal (hypervariable region) undergoes post-translational modifications to facilitate the attachment of RAS to the inner leaflet of the plasma membrane. This catalytic domain of RAS protein consists of 1–166 residues containing the P-loop (10–17 residues). The G-domain of all RAS proteins is composed of two switches: switch I (residues 25–40) and switch II (residues 59–75) (Fig. 2). Understanding the dynamics of switch I and II conformations and binding sites is crucial when developing inhibitors targeting oncogenic RAS mutants. With the help of these switches, the two nucleotide-bound states of RAS (RAS-GDP and -GTP) are recognized by regulatory proteins and effectors, which results in interactions between RAS and RAS-binding proteins. The P-loop, along with switches I and II, comprises the active site for GTP hydrolysis and provides an interaction surface for different effectors and regulators.^22–25

Fig. 2. Structure of RAS protein with different binding conformations.

The catalytic domain is composed of two lobes. The first half of the domain is called the effector lobe (residues 1–86), in which active site structural elements are present. This lobe has a 100% identical sequence among all RAS isoforms.^26,27 Another half of the catalytic domain is called the allosteric lobe (residues 87–166). This lobe contains NKXD (residues 116–119) and EXSAK (residues 143–147) motifs (supported by effector lobe residue Phe-28); the earlier one is responsible for recognizing and positioning guanine nucleotide in the active site, while the latter one helps to stabilize nucleotide binding.²⁸

HRAS and NRAS have different intrinsic hydrolytic activities. Research on the molecular dynamics of RAS in both its nucleotide-free and nucleotide-bound states reveals differences in the overall dynamics of HRAS, NRAS, and KRAS. Assumptions that the RAS isoforms have identical active sites and identical kinetics have resulted in dependency on HRAS for linking biochemical and structural properties. Based on the experiments performed on the three wild-type isoforms under similar conditions, an orderly study of the intrinsic hydrolytic reactivity of H-, N-, and KRAS is given. It was found that there are substantial differences in biochemical reactions among the isoforms. The rate constant for hydrolysis of HRAS was found to be 3 times that of KRAS and HRAS. Direct comparison was made of the G-domain sequence by determination of specific locations that vary in H-, N-, and KRAS isoforms. In the allosteric lobe, 17 residues show variation in one or more of the three isoforms gathering close to highly conserved nucleotide binding NKXD and EXSAK motifs of small GTP binding proteins.

Within these 17 residues, just five (29%) of them are unique to KRAS, whereas nine (50%) are unique to NRAS (neuroblastoma RAS viral oncogene homolog) and seven (41%) to HRAS. In HRAS, residue variation assembles at residues 121–122 (loop 8) and 126–128 (helix 4). In NRAS, these variations occur at 94 and 95 (helix 3) and 131 and 132 (helix 4), whereas KRAS has five unique residues scattered all through the allosteric lobe (residues 95, 107, 122, 153, and 163). Also, KRAS lacks two or more variations in sequences. Overall, there are only two positions that show variations in all three isoforms: residue 95 (helix 3) and residue 122 (loop 8).^24,28–31

4. RAS inhibitors in cancer treatment

Various RAS inhibitors are currently undergoing testing in the clinical and preclinical stages. Earlier studies on RAS structure, processing, and signaling pathways in melanoma have created new opportunities for inhibiting RAS function. The activity of RAS can be inhibited by two major approaches: (1) direct binding of the inhibitor to the RAS protein and (2) inhibiting the enzymes involved in post-translational modification of RAS protein.³² Regarding efficacy and safety, those inhibitors that directly target RAS and inhibit its ability to activate the downstream effector cascade have been anticipated to be comparatively better.³³

4.1. FDA-approved RAS inhibitors

The first RAS inhibitor, sotorasib (Lumakras™), developed by Amgen (Table 1), received approval on May 28, 2021, by the US FDA for the treatment of KRAS^G12C-mutated non-small cell lung cancer (NSCLC) in adult patients. In June 2019, the US FDA designated sotorasib as an orphan medication for KRAS^G12C-positive NSCLC and colorectal cancer.³⁴ In December 2020, this drug was permitted as an innovative treatment for KRAS^G12C-mutated NSCLC and got approval in May 2021.³⁵ Sotorasib is administered orally with a dose of 960 mg o.d. Sotorasib forms a covalent bond with a pocket in the switch II region and shows irreversible interaction with cysteine residues of the KRAS^G12C mutant. As a result, the KRAS protein is held in its inactive GDP-bound state, thereby preventing downstream signaling. Under in vitro conditions, sotorasib blocked the nucleotide exchange of the recombinant mutant KRAS^G12C/C118A, which was mediated by SOS1.³⁶ Metabolism of sotorasib occurs through CYP3A4. It is eliminated through the fecal route. The use of sotorasib is recommended with CYP3A inducers and gastric acid-reducing agents.³⁷

Table 1. FDA-approved RAS inhibitors.

Sr. no.	RAS inhibitors	Chemical structure	Mode of action	Targets	Developer	References
1.	Sotorasib (AMG510)		Covalent allosteric inhibitor	KRAS G12C	Amgen	35, 38
2.	Adagrasib (MRTX849)		Covalent allosteric inhibitor	KRAS G12C	Krazati, Mirati Therapeutics	38, 39

The second RAS inhibitor to receive approval was adagrasib (also known as MRTX849) (Table 1), developed by Krazati, Mirati Therapeutics, which received approval on December 12, 2022, in the USA for treating adults with KRAS^G12C-mutated locally advanced or metastatic NSCLC.³⁹ Adagrasib is an orally administered small molecule that binds covalently and irreversibly to KRAS^G12C in its GDP-bound inactive state. The pharmacokinetic properties of this drug include a half-life of 23 hours, central nervous system penetration, and dose dependency. The activity of adagrasib is seen in colorectal, pancreatic, and biliary tract cancers in patients with KRAS^G12C mutated solid tumors. Adagrasib is given as a monotherapy and combined with other NSCLC therapies. In the phase III trial, adagrasib in combination with docetaxel is used to treat NSCLC. In the phase II trial, adagrasib alone and in combination with pembrolizumab is used to treat patients with advanced KRAS^G12C mutated NSCLC.^40,41 The oral dose of adagrasib monotherapy is 600 mg b.d. or given i.v. along with cetuximab (weekly) starting with a dose of 400 milligrams per square meter of body surface area, followed by 250 milligrams per square meter.⁴²

4.2. RAS inhibitors under clinical trial

At present, many RAS inhibitors have been developed and are also in clinical and preclinical stages (Fig. 3).

Fig. 3. Clinical trial RAS inhibitors.

Salirasib (Table 2) was initially developed by Tel Aviv University and Universitaet Wien, then licensed to Concordia Pharmaceuticals.⁴³ Salirasib is a salicylic acid derivative (S-trans, trans-farnesyl thiosalicylic acid). Salirasib imitates farnesylcysteine carboxymethyl ester (a recognition unit for membrane attachment) from the carboxy-terminal, common to all RAS proteins. It acts by competitive inhibition of the attachment of GDP-bound RAS to the plasma membrane, resulting in blockage of RAS signaling.⁴⁴ Salirasib actively inhibits all three isoforms. Safety, efficacy and pharmacokinetic parameters were determined in the phase I trial conducted on Japanese patients for RAS-positive refractory solid tumors. Salirasib was found to be safe and well tolerated, and the trial was advanced to phase II at a dose of 800 mg twice daily.⁴⁵ Salirasib, as a monotherapy and in combination with chemotherapy, reduced tumor growth and cells in NSCLC and lung carcinoma. Initially, an 800 mg dose of salirasib twice daily was given to patients from 1 to 28 of a 35-day cycle. The most common adverse effects were found to be diarrhea, nausea, abdominal pain, and fatigue. After 4 weeks of continuous treatment, after enrolling 10 patients, the initial dose was adjusted to 600 mg twice daily due to toxicity.⁴⁶ The main mediator of salirasib metabolism is the human cytochrome P450(CYP450) 2C subfamily, specifically CYP2C19, 2C9 and 2C8 isozymes.⁴⁴

Table 2. RAS inhibitors under clinical trials.

Sr. no	Name	Phase	Target	Cancer type	Developer	References
1.	Salirasib	Phase II/III	KRASG12C	NSCLC, lung carcinoma	Tel Aviv University and Universitaet Wien	43, 45
2.	Tipifarnib (R115777)	Phase II	HRAS	Head and neck squamous cell carcinoma (HNSCC), urothelial carcinoma	Johnson & Johnson Pharmaceutical	47, 48
3.	ARS-853	Phase II	KRASG12C	Bronchioalveolar carcinoma	Shokat Laboratory	49
4.	ARS-1620	Phase I	KRASG12C	Human lung, pancreatic, and colorectal cancer cell lines	Kura Oncology, J&J and Araxes Pharma	50
5.	Lonafarnib (SCH66336)	Discontinued	RAS	Human colorectal cancer cell line, lung cancer, breast cancer, bladder cancer	Merck & Co.	51
6.	Andrographolide	—	KRASG12V	Cervical and colon cancer	—
7.	Divarasib (GDC-6036)	Phase III	KRASG12C	NSCLC, colorectal cancer	Genentech USA	52, 53
8.	LY-3537982	Phase I	KRASG12C	Advanced solid tumors, pancreatic tumors, endometrial cancer	Eli Lilly and Company	53, 54
9.	BI-1823911	Phase I	KRASG12C	Metastatic colorectal cancer, biliary tract cancer, advanced solid tumors	Boehringer Ingelheim	53, 55
10.	D-1553	Phase I/III	KRASG12C	NSCLC and solid tumors	Inventis	53, 56
11.	JNJ-74699157 (ARS-3248)	Phase I	KRASG12C	NSCLC, metastatic colorectal cancer	Araxes	57, 58
12.	JDQ-443	Phase III	KRASG12C	Advanced solid tumors, metastatic colorectal cancer	Novartis	59, 60
13.	IBI-351	Phase I	KRASG12C	Advanced non-squamous NSCLC	Innovent Biologics	53
14.	BPI-421286	Phase I	KRASG12C	Advanced or metastatic solid tumors	Betta Pharmaceuticals	53, 61
15.	RMC-6236	Phase I/Ib	KRASG12X (G12D,G12V,G12R)	PDAC	Revolution Medicines	62, 63
				NSCLC
16.	MRTX1133	Phase I/II	KRASG12D	PDAC	Mirati Therapeutics	64, 65
17.	BI 1701963	Phase I	KRAS–SOS1	CRC	Boehringer Ingelheim	66
				NSCLC

Tipifarnib, also known as R115777 (Table 2), is a farnesyl transferase (FTase) inhibitor that interrupts HRAS function. The post-translational attachment of farnesyl groups (essential for localization to cell membranes) to signaling proteins is facilitated by FTase. Tipifarnib was developed by Johnson & Johnson Pharmaceutical Research and Development LLC.⁴⁷ The clinical advancement of tipifarnib started in the year 1997.⁶⁷ It is effective in head and neck squamous cell carcinoma (HNSCC). Tipifarnib is a non-peptidomimetic quinolinone derivative that acts by inhibiting farnesyl transferase. Various doses of tipifarnib were administered, including a low dose of 300 mg twice daily for 21 days (28 day schedule) and a high dose of 900 mg orally twice daily for days 1–7 and 15–21 of 28 day cycles. However, several toxic effects were found, such as anemia and peripheral neuropathy, in the initial nine patients, which led to a reduction in dose to 600 mg twice daily. Later, a protocol was made to administer 600 mg twice daily of tipifarnib to improve endurance, which facilitated patients to maintain an effective dose for a longer duration.⁶⁸

ARS 853 (Table 2) is a selective KRAS^G12C inhibitor. It inhibits the activity of mutant KRAS by binding to the mutated cysteine residue in the switch II pocket of KRAS in the GDP-bound state, thereby preventing its activation.⁶⁹ During the trial in mice, ARS853 was found to have short metabolic plasma stability and poor oral bioavailability, which became a significant reason for making ARS853 unsuitable for in vivo studies. KRAS^G12C (H358 cells), upon treatment with ARS853, resulted in considerable loss in KRAS–CRAF interactions and inhibition of downstream MAPK and PI3K signaling pathways. Some other observations, like growth inhibition and an increase in the rate of apoptosis, were also observed in H358 cells.⁷⁰

ARS1620 (switch II pocket binder, Table 2) is another orally available selective KRAS^G12C inhibitor.⁷¹ The first preclinical evidence of KRAS^G12C inhibition in vitro and in vivo was provided by ARS1620.⁷² The flexible linker of ARS853 was replaced by a bicyclic quinazoline ring to synthesize various compounds, which led to the development of ARS1620 containing a fluorophenol hydrophobic binding moiety. It inhibits KRAS by binding covalently to the mutant cysteine and hindering the exchange of GDP for GTP, thereby maintaining the oncoprotein in its inactive form. ARS1620 is 10 times improved in potency, in quantifying RAS engagement, and in biochemical assay when compared to ARS853.⁷³ In the in vivo studies, combination therapy of ARS1620 with EGFR (epidermal growth factor receptor) or AURKA (Aurora A-kinase) small-molecule inhibitors produced a much improved suppression of tumors in comparison to ARS1620 monotherapy.⁷² Also, combination therapy with RTK inhibitors or PI3K inhibitors was found to have enhanced suppression of KRAS^G12C mutant cell lines. ARS1620 is known to inhibit the growth of human lung, pancreatic, and colorectal cancer cell lines.⁷⁴ In a xenograft model with NSCLC and pancreatic adenocarcinoma, ARS 1620 showed remarkable tumor suppression at a dose of 200 mg per day with no toxicity. However, the clinical trial of ARS 1620 was suspended due to its inadequate potency.⁷⁰

Lonafarnib (also known as Zokinvy™, SCH66336) (Table 2) is an orally available farnesyl transferase inhibitor. Merck & Co. discovered it. Lonafarnib is a non-peptidomimetic CAAX competitive, selective FTase inhibitor.⁷⁵ It inhibits RAS activity by blocking the post-translational attachment of the prenyl moiety to the C-terminal cysteine residue.⁷⁶ Lonafarnib is found to disrupt the cell cycle of HCT 116 (human colorectal cell line), H460 (lung cancer), MCF7 (breast cancer) and MIA PaCa-2 (pancreatic cancer) in the G₂–M phase and in the G₁ phase for T24 cells (bladder cancer) with activated HRAS.⁵¹ Combination therapy of lonafarnib with cyclophosphamide, 5-fluorouracil, and vincristine has shown an increase in antitumor activity. Along with Ras inhibition, lonafarnib also possesses some other inhibiting activities, such as inhibition of PI3K/Akt-mediated growth and adhesion-dependent survival pathways and inhibition of the microtubule cytoskeleton. This results in microtubule stabilization and suppression of microtubule dynamics, blocking the prenylation of Rheb (Ras homolog enriched in the brain) and induction of phosphorylation in HNSCC cell lines. This inactivates eEF2, resulting in the suppression of protein synthesis. During phase II clinical trials on 20 patients, lonafarnib was administered orally twice daily at a dose of 400 mg for 7 days for a 21 day cycle. The most common toxic effects observed were leucopenia (hematological) and diarrhea (non-hematological). In the phase II/III clinical trial, 200 mg of lonafarnib was administered twice a day for 28 consecutive days in patients with metastatic colorectal cancer. Side effects were seen, such as fatigue, nausea, and diarrhea.^76–78

Andrographolide (AGP), isolated from Andrographis paniculata (Table 2), is a bicyclic diterpenoid lactone.⁷⁹ Benzylidene derivatives of this drug bind to transient pockets on Kirsten-Ras (K-Ras) and inhibit GEF-induced nucleotide exchange. Several therapeutic properties of AGP are antioxidant, antiviral, and anticancer properties.⁸⁰ Prolonged treatment with AGP leads to reduction in GTP loading and signal transmission by mutant KRAS^G12V.⁸¹ AGP interrupts tumorigenesis by growth suppression, apoptosis induction, angiogenesis inhibition.⁸² AGP primarily inhibits cancerous cells by inducing reactive oxygen species, activating c-Jun N-terminal kinase, and blocking autophagy. AGP has IC₅₀ values reported against leukemia (P388 cell line, 1.0 μg ml⁻¹) and colon cancer (COLO205 cell line, <5 μM) cell lines.⁷⁹ During trials, AGP was used in combination therapy with capecitabine for colorectal cancer. Since 2014, 52 patients have been studied where AGP was given as monotherapy and in combination therapy with capecitabine for CRC, but no practical results were reported.⁸¹

Divarasib (GDC-6036), developed by Genentech, USA (Table 2), is an orally available, covalent KRAS^G12C inhibitor. During phase I trials, the administered dose ranged from 50 to 400 mg in patients having advanced or metastatic solid tumors due to KRAS^G12C mutation. A total of 137 patients were administered divarasib (60 with NSCLC, 55 with CRC, and 22 with other solid tumors).⁵² Divarasib inhibits KRAS activity by reversing oncogenic signaling by trapping the protein in its inactive state. Divarasib (at 400 mg) has encouraged tumor suppression activity against CRC (colorectal cancer). Still, its efficacy became limited due to adaptive feedback reactivation of RAS–MAPK signaling, resulting in resistance to KRAS^G12C inhibition in CRC. Divarasib, in combination therapy with cetuximab, resulted in a decrease in KRAS^G12C levels and had a manageable safety profile and promising clinical activity.⁸³

LY-3537982 (Table 2) is a selective covalent KRAS^G12C inhibitor. Lilly revealed it in the American Association for Cancer Research (AACR) in 2021. In the case of the inhibiting activity of KRAS^G12C in human H358 lung cancer cell lines, the IC₅₀ value of LY-3537982 was 3.35 nM, which was 10 and 25 times more when compared to sotorasib (IC₅₀ = 47.9 nM) and adagrasib (IC₅₀ = 88.9 nM), respectively. LY-3537982 was accepted for clinical trials in July 2021.⁵³ Currently, under phase I clinical trial, LY-3537982 as a monotherapy and in combination therapy is being administered to patients with KRAS^G12C-mediated advanced solid tumors.⁸⁴ LY-3537982 is currently under trial for inhibiting different mutant KRAS cancers such as colorectal cancer, NSCLC, ovarian tumors, advanced solid tumors, pancreatic tumors, and endometrial cancer.⁵⁴

BI-1823911 (Table 2) is a novel, highly selective, covalent, irreversible, orally available KRAS^G12C inhibitor developed by Boehringer Ingelheim. It is used to treat patients with locally advanced solid tumors with KRAS^G12C mutation. It is currently undergoing clinical trials for various cancer types, including metastatic lung cancer, metastatic NSCLC, metastatic colorectal cancer, metastatic pancreatic cancer, adenocarcinoma, biliary tract cancer, and advanced solid tumors. It was approved for clinical trials in July 2021. BI-1823911 is under investigation as a monotherapy and in combination therapy with other inhibitors for treating cancers occurring due to KRAS mutations.⁵⁵ BI-1823911, in combination with BI-1701963 (SOS inhibitor), resulted in the arrest of the cell cycle in the G₁ phase and apoptosis.⁸⁵

D-1553 (Table 2) is a small-molecule covalent inhibitor of KRAS^G12C. Inventis developed it. It is the first drug approved in China that targets KRAS^G12C mutation. Preclinical investigations have shown that D-1553 has vigorous tumor-inhibition activity and exemplary safety. D-1553 was registered for clinical trials in October 2020. At present, D-1553 is in phase I/II trials for the treatment of NSCLC and solid tumors.⁵⁶ According to a report presented at the World Congress on Lung Cancer (WCLC), no dose-limiting toxicity was observed in a trial involving 79 patients with KRAS^G12C-mediated NSCLC.⁸⁶ D-1553 in combination with chemotherapy, MEK inhibitor, or SHP2 (Src homology 2 domain-containing phosphatase 2) inhibitor has resulted in greater potency in suppression of tumor growth in comparison to D-1553 monotherapy.⁸⁷

JNJ-74699157 (ARS-3248, Table 2) is an orally available, selective, covalent KRAS^G12C inhibitor. It was developed by Araxes (Wellspring Bioscience). It was the first company to indulge in the development of a new mutation site in KRAS. ARS-3248 covalently binds to switch II pockets in KRAS protein, resulting in blockage of downstream signaling of KRAS^G12C. Observations made through clinical research state that ARS-3248 is effective in advanced solid tumors, metastatic NSCLC, and metastatic colorectal cancer. The phase I clinical trial of ARS-3248 was started in May 2019 on patients with KRAS^G12C-mediated advanced solid tumors. Ten patients were enrolled in this trial, of which nine were at a dose of 100 mg, and one was at 200 mg. Toxicity, such as increased blood creatinine phosphokinase (CPK), was observed at these two doses.^57,58

JDQ-443 (Table 2) is a selective, covalent, irreversible KRAS^G12C inhibitor. Novartis developed it. JDQ-443 covalently binds to the switch II region of mutant KRAS^G12C, stabilizing it in an inactive GDP-bound state.⁵⁹ It is used in the treatment of advanced solid tumors, metastatic colorectal cancer, and metastatic NSCLC. JDQ-443 entered clinical trials in January 2021.^59,88 JDQ-443 is used as a monotherapy and in combination therapy with TNO155 (SHP2 inhibitor) and also with PD-1 blocker.⁸⁹ In the phase II trial of JDQ-443, 57% of the response rate was observed in patients having advanced NSCLC.⁵³ The phase III trial of JDQ-443 was started in November 2022 by Novartis. This phase was mainly for checking the safety and efficacy of JDQ-443 against TNO-155 in patients having metastatic KRAS^G12C-mutated NSCLC.⁶⁰

IBI-351 (GFH-925, GF-105, Table 2) is a covalent, irreversible KRAS^G12C inhibitor. Innovent Biologics developed it. IBI-351 treats KRAS^G12C-mediated gastrointestinal tumors, NSCLC, and solid tumors. It was registered for clinical trials in August 2022. In phase I trials, combination therapy of IBI-351 with sintilimab is under observation for the treatment of patients with advanced non-squamous NSCLC and in combination with cetuximab for treatment of metastatic colorectal cancer having KRAS^G12C mutation. A total of 55 patients were evaluated, and an overall response rate of 50.9% was observed for CRC and 61.9% for NSCLC.⁵³

BPI-421286 (Table 2) is a potent, selective, covalent, orally available small-molecule KRAS^G12C inhibitor. The IC₅₀ value of BPI-421286 for inhibiting KRAS^G12C was 55 nM.⁶¹ Betta Pharmaceuticals developed it. It is used to treat advanced or metastatic solid tumors arising from the KRAS^G12C mutant gene. According to the preclinical data available, BPI-421286 effectively suppresses the proliferation of tumor cells with the KRAS^G12C mutant gene. BPI-421286 entered a phase I clinical trial in April 2022 for the evaluation of efficacy in patients with advanced solid tumors.⁵³

RMC-6236 (Table 2) is a novel selective, orally available tri-complex (CYPA: RMC-6236:KRAS^G12C) mutant RAS inhibitor. It inhibits both mutant RAS and wild-type RAS in the GTP-bound RAS ON state, which is why it is known as a RASMULTI inhibitor. The mechanism of inhibition involves binding of RMC-6236 to cyclophilin A (CypA), a cellular chaperone protein, which results in interruption in downstream effector interactions. It effectively inhibits mutant KRAS-mediated pancreatic ductal adenocarcinoma (PDAC) and NSCLC. In a phase I/Ib trial, parameters like safety, tolerability, and pharmacokinetics were evaluated.^63,90,91

MRTX1133 (Table 2) is a selective, reversible, potent, non-covalently binding inhibitor of KRAS^G12D. It is the first inhibitor discovered that binds non-covalently to KRAS^G12D. It inhibits the signaling pathway by causing interruption in effector interaction. MRTX1133 binds to the switch II region and stabilizes it by forming a hydrogen bond with Gly60. It can bind with both active and inactive states of KRAS^G12D.^64,92 It is developed by Mirati Therapeutics and is effective in the treatment of PDAC.⁶⁵ The presence of the H95 residue is responsible for the selective nature of MRTX1133 towards KRAS.⁹³ Combination therapy of MRTX1133 with cetuximab resulted in effective inhibition of colorectal cancer having KRAS^G12D mutation.⁹⁴

BI-1701963 (Table 2), an orally available small molecule, is the first SOS1::KRAS inhibitor to enter clinical testing. It binds to SOS1 and hinders interaction with KRAS-GDP, resulting in nucleotide exchange failure. In phase I trials, BI-1701963 as a monotherapy and combination therapy with trametinib (MEK inhibitor) showed good anticancer activity against KRAS mutated colorectal cancer (CRC), non-small cell lung cancer (NSCLC) and pancreatic adenocarcinoma.^95,96

4.3. Recent advancements (pre-clinical studies) in RAS inhibitors

Since 1982, the year of the discovery of RAS mutations, scientists have started exploring the dynamics of RAS genes and various problems related to RAS mutations. To cope with mutation, various scaffolds such as pyrimidine, quinoline, quinazoline, pyrazole, trimethoxyphenyl, indole, pyridine, piperazine, naphthalene, guanidine, phenylpropanoid, etc. and their derivatives were synthesized. Their inhibitory activities were determined against different cell lines such as H358 (NSCLC), H23 (adenocarcinoma), Panc1 (human pancreatic cells), K-562 (human lymphoblastoid), A549 (NSCLC), CT-26 (colon carcinoma), MDA-MB-231 (human breast cancer), and HK2-6 (renal cancer). A summary of the different scaffolds synthesized and their inhibitory activities are given in Fig. 4 and Table 3. Other derivatives of parent scaffolds were synthesized, but only the most potent compound's structure is shown based on cell line activity.

Fig. 4. Various synthesized compounds with parent scaffolds as RAS mutant inhibitors.

Table 3. Different scaffolds and their critical aspects of RAS mutant inhibition.

Cpd. no.	Derivatives	Cell line	Activity	Pin point	References
1.	Pyrrolo[2,3-d]pyrimidine	KRASG12C/SOS1	IC50 0.21 μM	In vivo results showed that the synthesized compounds have satisfactory selectivity, moderate pharmacokinetic characteristics, and good anticancer effects	97
		H358 cell	IC50 0.16 μM
2.	Thieno[2,3-d]pyrimidine	KRASG12D	IC50 2.1 μM	The compound showed the highest anti-proliferative activity against KRASG12D mutated cells	98
		Panc1 cell line
		SW1990 cell line
		CT26 cell line
3.	Pyridopyrimidinone	KRASG12C	IC50 1.87 μM	Showed activity against KRASG12C and PaCa2 cell line	99
		PaCa2 cells	IC50 0.79 μM
4.	Tetrahydropyrimidine	H358 cells	IC50 0.070 μM	Irreversible covalent inhibitor of KRASG12C	100
5.	Quinoline	HCT-116	GI50 1.4 μM	Inhibition of proliferation of CRC cells by degradation of β-catenin and RAS	101
6.	Quinoline	A549 cells	IC50 8.99 μM	Induction of apoptosis in HepG2 cells, cell cycle arrest in S phase, antitumor activity	102
		HepG2 cells	IC50 6.92 μM
		DU145 cells	IC50 7.89 μM
		MCF7 cells	IC50 8.26 μM
7.	4(1H)-Quinolinone and urea	H-358 (KRASG12C)	IC50 0.5 μM	The synthesized compound shows excellent anti-proliferative activity and higher selectivity against KRASG12C in NSCLC	103
		H-23 (KRASG12C)	IC50 1.3 μM
		H-2228 (KRASWT)	IC50 11.6 μM
8.	8-Hydroxyquinoline	HsRce1 cells	IC50 6.9 μM	An effective Rce1 inhibitor in the presence of RAS isoforms, delocalization of RAS isoforms	104
9.	Quinazoline	H358 cell line	IC50 0.46 μM	The compound showed improved anti-cancer activities and selectivity in comparison to ARS-1620 against KRASG12C	105
		H23 cell line	IC50 0.87 μM
10.	Quinazoline	H358 (KRASG12C)	IC50 2.34 μM	High anti-proliferative activity against KRASG12C mutated cancer cell lines	106
11.	Quinazoline	K-562 cells	IC50 1090 ± 170 nM	An inhibitor of RAS–SOS1 interaction	107
		MOLM-13 cells	IC50 995 ± 400 nM
		H358 cells	IC50 3480 ± 100 nM
		Calu-1 cells	IC50 3190 ± 50 nM
12.	Quinazoline	Capan-1 cells	IC50 8.8 ± 2.4 μM	Good antitumor activity and binding affinity with PDEδ	108
13.	Quinazoline	H-358 cells	IC50 1.2 μM	Inhibition of KRASG12C signaling and tumor growth	109
		Calu-1 cells	IC50 2.4 μM
14.	Diazaspiro	KRASG12C	IC50 0.077 μM	The compound showed optimal activity with KRASG12C and a dose-dependent anti-proliferative effect on subcutaneous administration in an NCI-H1373 xenograft mouse model	110
		H1373 ERK	IC50 0.14 μM
15.	Spiro amide	C-RAF	IC50 1.6 nM	Compound showed excellent in vivo efficacy in a RAS mutant Calu6 xenograft mouse model	111
		H358 cell line	GI50 1.5 μM
		A375 cell line	GI50 0.17 μM
16.	Spiropyrrolizidine and piperazine	SW480 cells	IC50 4.17 μM	Potent inhibitor of KRAS expression levels in human CRC	112
		HT29 cells	IC50 6.76 μM
17.	Tetrahydroisoquinoline fused spirooxindoles	HCT116 cells	IC50 1.7 μM	Inhibitor of activation of RAS-GTP and suppressor of downstream signalling	113
		SW620 cells	IC50 3.8 μM
		SW480 cells	IC50 6.5 μM
		Caco-2 cells	IC50 71.8 μM
18.	Pyrano[2,3-c]pyrazole	RalA	IC50 0.22 μM	Potent inhibitor of HCC, HepG2, SMCC-7721, Hep3B cell lines	114
		HepG2 cell line	IC50 2.28 μM
19.	Pyrazolo[3,4-b]pyrazin	LS-180 cells	IC50 2.97 ± 0.75 μM	Exhibits inhibitory effects against the SHP2 enzyme and KRAS mutant CRC both in vitro and in vivo	115
		HCT-116 cells	IC50 2.00 ± 0.71 μM
		SW 1463 cells	IC50 1.84 ± 0.40 μM
20.	Pyrazole	MDA-MB-231 cells	IC50 7.6 μM	GGTase-1 inhibitor	116
21.	Pyrazolo[3,4-d]-pyridazine	Capan-1 cells	IC50 12.4 ± 1.7 μM	Highly active KRAS–PDEδ inhibitor	117
22.	Pyrazole	MDA-MB-231 cells	IC50 2.4 μM	Synthesized compound is a potent geranylgeranyltransferase 1 (GGT1) and farnesyltransferase (FT) inhibition	118
23.	Trimethoxy phenyl	KRASG12V	IC50 7.42 μM	Delocalization of KRAS from plasma membrane and blocking oncogenic signalling	119
24.	Trimethoxyphenyl	KRAS	IC50 7.01 μM	Delocalization of oncogenic KRAS, inhibition of PDAC and NSCLC	120
25.	Indole	KRAS4B	IC50 < 1 μM	Inhibition of prenylated KRAS, blockage of KRAS4B signalling	121
26.	Aminopiperidine indole	HeLa cells	EC50 0.8 μM	Under in vitro conditions, the compound activates the nucleotide exchange process at submicromolar concentrations	122
27.	Indole	MEF cells	IC50 3.8 μM	An effective pan-RAS inhibitor	123
28.	Guanidinobenzimidazole	MCF-7 cells	IC50 0.3 μM	In vivo activity against breast cancer cell line and NSCLC in tumor xenograft models	124
		MDA-MB-231	IC50 1 μM
29.	Guanidyl	HDJ-2 cells	IC50 0.5 μM	Delocalization of KRAS from plasma membrane and association with c-Raf	125
30.	Methoxypyridine	DLD-1 (KRASG13D)	IC50 8 μM	Inhibitor of KRAS and RAS dependent signaling in cancerous cells	126
		HT1080 (NRASQ61K)	IC50 10 μM
31.	Oxy-pyridine	Calu-6	EC50 0.95 μM	Highly kinase selective and potent in cell lines having KRAS mutation	127
32.	Morpholinopyridine	KRASQ61K	IC50 0.47 μM	High kinase selectivity and cellular potency in RAS mutant cell line	128
33.	Piperazine	KRASG12C	EC50 0.32 μM	Acts as an irreversible covalent KRASG12C inhibitor	129
34.	Piperazine	KRAS	IC50 0.3 μM	Causes delocalization of KRAS form plasma membrane	130
		MiaPaCa-2 cells
35.	Piperazinone	A549 cells	IC50 1.3 ± 0.22 μM	Better cytotoxicity in comparison to doxorubicin and potent against cancer cell lines	131
		HT29 cells	IC50 1.8 ± 0.13 μM
		MRC-5 cells	IC50 5.28 ± 0.41 μM
36.	Pyrone	Panc-1 cells	IC50 4.4 μM	In vivo and in vitro effects on RAS mutated pancreatic cancer cells	132
		BxPC-3 cells	IC50 8 μM
37.	Pyranone	HCT-116 cells	IC50 8 μM	Causes impairment of mutated RAS isoforms, antitumor activity	133
		H-460 cells	IC50 15 μM
		MDA-MB-231	IC50 7 μM
		NIH-3T3 (G12V)	IC50 14 μM
38.	Pyrano[2,3-c]pyrazole	H2122 cells	IC50 2.2 μM	Potential Ral inhibitor with improved ADME properties	134
39.	Naphthalene	NIH3T3 (HRASG12V)	IC50 4.73 μM	High cytotoxic effect in mouse embryonic fibroblast cells	135
40.	Naphthalene	hIcmt	IC50 1.5 ± 0.2 μM	Potent inhibitor of human isoprenylcysteine carboxyl methyltransferase (hIcmt) enzyme	136
41.	Triazole and α-hydroxynaphthaldehyde	HepG2 cells	IC50 45.08 μg ml−1	Inhibitor of HRAS protein(121p)	137
		MCF-7 cells	IC50 34.45 μg ml−1
42.	Phenylpropamide	RAS	IC50 2 μM	Selective against ICMT (isoprenylcysteinecarboxyl methyltransferase) enzyme involved in post translational modification of RAS	138
43.	Benzamide	SMMC-7721 cells	IC50 4.93 ± 0.55 μM	Inhibition of RAS signaling pathway, antitumor activity	139
44.	Oxadiazole	KRASG12C	IC50 7 ± 2.4 nM	Showed excellent in vivo efficacy against H-358 cell line	70
45.	Dihydro-α-carboline	A549 cell line	IC50 0.43 ± 0.03 μM	Anti-proliferative activity against NSCLC cell line and inhibition of RalA/B	140
		H1299 cell line	IC50 0.64 ± 0.07 μM
		H460 cell line	IC50 0.93 ± 0.10 μM
		H1975 cell line	IC50 1.54 ± 0.15 μM
46.	Benzofuran	KRASG12V	IC50 0.7 μM	Potent ARF6 and RAS inhibitor	141
47.	Thiourea	A549 cell line	IC50 0.21 μM	Inhibits A549 cell proliferation having KRASG12V mutation	142
48.	Myo-inositol	MDA-MB-231 cells	IC50 22.41 ± 0.80 μM	Synthesized compounds show potential anti-proliferative effect against human breast cancer cells	143
		MCF-7 cells	IC50 35.05 ± 2.77 μM
49.	Naphthyridinone	KRASG12C	IC50 1.3 μM	Potent KRASG12C inhibitor	144
50.	Imidazoline	HK2-6 cells	IC50 2.78 μM	Inhibition of KRAS gene expression in KRAS mutated tumors	145
51.	Biphenyl	KRASQ61H	IC50 4.5 μM	Potent RAS inhibitor	146
52.	Aminobenzoate	KRAS	IC50 3.94 ± 0.03 μM	Selectivity against KRAS only	147
53.	Azobenzene	KRASG12C	IC50 2.140 ± 0.096 μM	Allosteric and photoswitchable inhibitor of the nucleotide binding and activation of K-Ras protein	148
54.	Triazole	PDEδ–RAS	IC50 25.2 ± 4.0 nM	Increase in binding affinity up to 1000-fold in comparison to deltarasin	149
55.	Dihydroanthracene	MDCK cells	IC50 1 μM	Delocalization of oncogenic KRAS, HRAS, and KRAS4A from the plasma membrane	150
56.	Acryloylazetidine	MIA PaCa-2 cells	IC50 0.067 μM	Inhibition of KRASG12C in submicromolar concentration	151
57.	Spirocyclicpyrazolone-ferrocene	PANC-1 cells	IC50 1.6 μM	Inhibitor of RalA and thioredoxin reductase and cell proliferation	152
		HPAF-II cells	IC50 4.8 μM
58.	Oxanthroquinone	MDCK cells	IC50 1 μM	Potent inhibitor of KRAS dependent on pancreatic, colon, and endometrial tumor cell lines	153
59.	Anthrafurandione	Panc-1 cells	IC50 1.6 μM	Potential anticancer agent and targets KRAS mRNA	154
60.	Pyridazinone	PaTu8902	IC50 4.7 μM	Potent KRAS–PDEδ inhibitor and binds to the farnesyl binding pocket of PDEδ with low nanomolar affinity	155
61.	Phenothiazine	FTase	IC50 9 nM	A potent inhibitors of human farnesyltransferase	156
62.	Purin-6(1H)-one	A549 cells	EC50 43.8 μM	Anti-proliferative activity on cell lines having KRASG12C mutation	157
		H23 cells	EC50 47.6 μM
		H358 cells	EC50 26.6 μM

5. Recent strategies to overcome the current challenges of RAS inhibitors based on binding analysis/computational studies of RAS mutants/resistance proteins

Binding analysis/computational study is essential to understanding the approach for direct inhibition of mutant RAS protein. Based on the studies on RAS, it was found that RAS is an allosteric enzyme with four allosteric sites for binding ligands.¹⁵⁸ NMR and X-ray crystallography confirmed the presence of these binding sites. The binding sites are p1, p2, p3, and p4 pockets present near switch regions; p1 is present in the core β-sheet just behind the switch II region and forms a hydrophobic cavity that consists of V7, L56, M67, and K5, D54, T74, Y71 (polar region). V7, V9, G60, F78, M72, Q99, and I100 form the p2 pocket. Near the C terminal pocket, p3 is present, which is polar; it is composed of residues D105, S106, D107, D108, M111, E162, Q165, and H166; the p4 pocket is present behind switch I region having residues like D30, D33, D38, S39, and Y40. The switch region is responsible for RAS activation and interaction with effector proteins. There is a 5 Å deep and 7 Å wide pocket present in the switch I/II interface site, which is composed of α2 helix (K5, R41, D54, T74 residues) and β-sheet (L56 residues). This pocket is the site for inhibition in the case of G12V, G12D, and Q61H mutation. Thus, targeting any of these four sites may affect the binding of RAS to upstream or downstream proteins.^31,159,160 Development of a potent RAS inhibitor requires specific methodologies such as molecular docking, MM-GBSA, ADMET, cytotoxic studies, and simulations.¹⁵⁸

The major problem in targeting RAS mutant genes is “the absence of small pockets on their surface”, which could be the site for binding small molecules. This produces a challenge for the development of effective therapeutic strategies. According to evidence from the literature, the active KRAS protein binds to cyclophilin A (companion protein), forming pockets that can be targeted by small molecules called “inhibitors”.⁵³

According to NMR spectroscopy results, the Kobe family RAS inhibitor (Kobe0065, its analogs Kobe2601 and Kobe2602) binds to RAS-GTP-bound state in close connection with the Lys5, Leu56, Met67, Gln70, Tyr71, and Thr74 side chains of HRAS. All these six residues together comprise a hydrophobic pocket adjacent to the switch I region in which fluorobenzene was incorporated through hydrophobic interaction.¹⁶¹

Another approach for inhibiting mutant RAS is “peptide inhibitors”. H-REV107 peptide, having 10 amino acids, forms a complex with KRAS protein, having 174 amino acids. Formation of three hydrogen bonds takes place between Ser 17 and Ala 59 residues of KRAS and the Tyr 176 and Asp 177 residues of the peptide.¹⁶²

An alternative strategy for inhibiting RAS is aiming for the effector-binding area, which affects RAS-effector and RAS–GEF interaction. In RAS's switch-off (inactive) state, allosterically modifying the switch I and switch II regions may inhibit effector binding. Misleading the RAS function can also be done by targeting SOS-1.¹⁶³

Different scaffold types are synthesized, with different binding sites/targets and variable effects on RAS protein. BI-2852, discovered by structure-based drug design, is a covalent KRAS^G12C inhibitor. It binds between the switch I and II pocket and results in inhibition of GEF, GAP, and interaction between effector and KRAS.¹⁶⁴

The site for binding of small molecules on RAS lies between the α2 helix and the core β-sheet (β1–β3). DCAI (an indole derivative RAS inhibitor) forms a complex with KRAS and shows high adaptability in the binding pocket. The chloro group at the 6th position in DCAI interacts with the hydrophobic pocket. Adjacent to this pocket, the interaction surface of the RAS–SOS complex is present. This interaction is also affected due to RAS–DCAI binding.¹⁶⁵

A peptide inhibitor named KRpep-2d is a potent non-covalent KRASG12D inhibitor. It binds close to switch II and α3 helix in a space of two α-helices. The acetyl oxygen from the N-terminal in KRpep-2d forms an intramolecular hydrogen bond with the nitrogen of Arg19 from the C-terminal. The formation of intramolecular disulfide bonds also takes place between Cys5 and Cys15. KRpep-2d causes inhibition of the RAS–SOS complex.¹⁶⁶ A compound named XY-02-075 (a phosphoryl sulfonamide derivative) forms an intramolecular hydrogen bond between the hydrogen of sulfonamide and phosphonate oxygen with mutant KRAS^G12C. The terminal carbon atom of this compound forms a covalent bond with Cys12 of KRAS protein. XY-02-075 causes inhibition of the nucleotide binding site, leading to interruption of downstream effector interaction.¹⁶⁷ KAL-21404358, a KRAS^G12D inhibitor, binds to the allosteric lobe's P110 site (named on behalf of proline 110 residues). This compound interrupts the KRAS^G12D–B-raf interaction and causes interference in RAF–MEK–ERK and PI3K–AKT signaling pathways.¹⁶⁸

On comparing the structures of RAS-GDP and RAS-GTP, it was found that these two show a high degree of structural similarity. There is a very slight variation (only10 amino acids) in the structure of RAS-GDP and RAS-GTP in the switch I region, and adjacent to this is the switch II region.¹⁶⁹ McCarthy et al. reported that compound 11 (pyrazolopyrimidine derivative) showed an interaction with p1 pocket residues. This compound showed binding affinity towards the catalytic domain. The GTP-bound state has quite a similar affinity with KRAS^G12D, KRAS^G12C, and KRASQ61H mutants. Various techniques like fluorescence polarization, pull-down assay, and FLIM–FRET were used to determine the interruption of compound 63 (2-(4-(2-methyl-3,5-diphenylpyrazolo[1,5-a]pyrimidine-7-yl)piperazine-1-yl)ethan-1-ol) in the interaction of KRAS with RAF, where fluorescence polarization and pull-down assay are used to measure the interaction of KRAS with the RBD of Raf and FLIM–FRET was used to measure the interaction of KRAS with full-length Raf (PDB ID 4DSO).¹⁷⁰

Conclusion

This review aims to highlight the impact and role of mutant RAS oncogenes in the development and growth of tumors, particularly KRAS, the most commonly mutated isoform. This review mainly discussed the RAS signaling pathway, its regulation, the molecular structure of the RAS protein with its binding mechanism, and small-molecule inhibitors. Numerous inhibitors have been identified that specifically target particular components of the RAS pathway. Until now, only two drugs (sotorasib and adagrasib) have been approved by the FDA for the inhibition of oncogenic RAS targeting various solid tumors, while most drugs are under clinical and preclinical testing. Multiple strategies have been reported for the inhibition of mutant RAS, the majority of which cannot distinguish between oncogenic mutant RAS and wild-type RAS. Various drugs are under preclinical and clinical studies and have responded satisfactorily.

To summarize, drug development efforts are centered on the RAS protein kinase, and designing and discovering RAS inhibitors for cancer treatment remains one of the most promising areas of cancer research. There is still a need for detailed analysis based on computational and biological studies of the changes in normal and mutant RAS proteins with reported inhibitors and the selectivity issue of RAS mutations in specific cancers. Due to the problem of selectivity, there are still concerns about toxicity, which is one of the barriers to developing RAS inhibitors. Based on these findings, a molecular understanding of RAS structure and function under physiological conditions could serve as a model for developing RAS inhibitors. This review will serve as a valuable knowledge resource for researchers working in this domain.

Abbreviations

AURKA

Aurora A-kinase

ERK

Extracellular signal regulated kinase

EGFR

Epidermal growth factor receptor

GAP

GTPase-activating protein

GDP

Guanosine diphosphate

GEF

Guanine nucleotide exchange factor

GRB

Growth receptor binding protein

GTP

Guanosine triphosphate

HRAS

Harvey murine sarcoma virus

HNSCC

Head and neck squamous cell carcinoma

ICMT

Isoprenylcysteine carboxyl methyltransferase

KRAS

Kirsten murine sarcoma virus

MAPK

Mitogen activated protein kinase

MEK

Mitogen-activated protein kinase kinase

NSCLC

Non-small cell lung cancer

NRAS

Neuroblastoma RAS viral oncogene homolog

PDE

Phosphodiesterase

PDAC

Pancreatic ductal adenocarcinoma

PI3K

Phosphatidylinositol 3 kinase

RAS

Rat sarcoma viral oncogene

RAF

Rapidly accelerated fibrosarcoma

RALGDS

Ral guanine nucleotide-dissociation stimulator

RTK

Receptor tyrosine kinase

SOS

Son of Sevenless

SHP2

Src homology 2 domain-containing phosphatase 2

SH2

Src homology 2

SH3

Src homology 3

R-RAS

Rat sarcoma viral oncogene homolog R

R-HEB

Ras homolog enriched in brain

Data availability

No primary research results, software or code have been included and no new data were generated or analysed as part of this review.

Author contributions

Conceptualization: Pradeep Kumar. Data collection: Vineet Prajapati, Ankit Kumar Singh. Writing the manuscript: Vineet Prajapati, Ankit Kumar Singh. Sketching of figures and data interpretation: Adarsh Kumar, Harshwardhan Singh. Writing, review and final editing of the manuscript: Prateek Pathak, Amita Verma, Maria Grishina, Vikas Kumar, Habibullah Khalilullah and Pradeep Kumar.

Conflicts of interest

The authors declare no competing interest.