Abstract

The protein KRAS has for decades been considered a holy grail of cancer drug discovery. For most of that time, it has also been considered undruggable. Since 2018, five compounds have entered the clinic targeting a single mutant form of KRAS, G12C. Here, we review each of these compounds along with additional approaches to targeting this and other mutants.Remaining challenges include expanding the identification of inhibitors to a broader range of known mutants and to con formations of the protein more likely to avoid development of resistance.

Keywords:-KRAS, G12C, G12D, Covalent drugs, Precision oncology, Fragmentbased drug discovery.

Introduction

Among cancer-driving genes, few are as prominent as KRAS. First identified in a murine sarcoma virus in 1982 [1], the gene is now known to be involved in 14.3% of human cancers [2]. The KRAS protein is a small membrane-bound GTPase important for multiple cell signaling functions. It exists in two states. When bound to GDP, it is ‘off’. When GDP is exchanged for GTP (usually in response to various growth stimuli), KRAS is turned on, activating the kinases RAF and PI3K and downstream signaling to promote cell proliferation and survival. KRAS returns to the off state when GTP is hydrolyzed to GDP often via GTPase-activating proteins.Tumors can arise when KRAS signaling gets stuck in the on state. Activation occurs through multiple mechanisms, including constitutive activation of growth factor receptor signaling and most often through activating mutations of KRAS. Each mechanism of activation shifts KRAS toward a GTP-loaded or ‘on’ state, triggering proliferative and survival signals. The central role of KRAS as a molecular switch driving oncogenesis has made it the focus of drug discovery for decades, and more than 20,000 articles have been published on the protein. The scope of this review is on recent developments targeting mutant forms of KRAS. Targeting wild-type KRAS is also being pursued [3,4], but even with a focus on KRAS mutants, only a fraction of the literature can be covered. Several excellent recent general reviews provide more detail [5e7], and there are also recent reviews focused on small-molecule inhibitors [8,9].

Mutations

KRAS is the most frequently mutated oncogene across the spectrum of human cancers, with many tumor types showing >10% mutation frequency. The resulting population of patients with cancer carrying mutant KRAS is staggeringly large and represents some of the deadliest tumor types including colorectal cancer (CRC), lung adenocarcinoma (LUAD), and pancreatic adenocarcinoma (PAAD) (Table 1). Recurrent mutations occur at three major sites, G12, G13,and Q61. More than 90% of KRAS mutations occur at glycine 12. Glycine 12 occurs in the P-loop region of the protein and plays a key role in stabilizing nucleotide binding and is proximal to the switch II pocket (Figure 1).The relative frequency of mutation varies based on the type of cancer and geography. For example, KRAS G12C is most frequent in LUAD, representing approximately half of KRAS mutations, whereas KRAS G12D occurs most frequently in PAAD and CRC, representing approximately two-thirds and half of the KRAS-mutant populations, respectively [2,10]. KRAS G12V is also common in PAAD, CRC, and LUAD [11]. These mutations differentially impact intrinsic GTP hydrolysis, GTPase-activating proteineinduced GTP hydrolysis, SOS-independent nucleotide exchange, and effector association/activation [12]. Across cancer genomic data sets, the frequency of KRAS mutation varies, suggesting a regional influence. This is particularly striking in lung cancer, wherein the occurrence of KRAS mutation ranges from >30% in Europe and the US to 5e8% in India and China [13]. Interestingly, this mirrors the mutation frequency of another famous driver oncogene, epidermal growth factor receptor (EGFR).

Targeting KRASG12C : conventional irreversible inhibitorsWith fewer than 200 amino acids, the KRAS protein is relatively devoid of attractive small-molecule binding sites aside from the nucleotide binding site. An obvious approach to develop KRAS inhibitors would be to design molecules that compete with GTP, as in the approach taken with most kinase inhibitor drugs. Unfortunately, while the affinity of kinases for ATP is generally in the micromolar range, the affinity of KRAS for GTPisin the picomolar range, so designing drugs that can outcompete the high GTP concentrations in cells is not feasible [14].A breakthrough article in 2013 by Ostrem etal [15] from University of California San Francisco (UCSF) demonstrated that targeting KRASG12C selectively is possible. The researchers used Tethering [16] to identify molecules that could covalently bind to the mutant cysteine via a disulfide bond. Crystallography revealed that the molecules bound in a newly formed pocket in the socalled switch II region of the protein and disrupted binding to RAF. Medicinal chemistry led to irreversible covalent inhibitors, and although these molecules were weak, they demonstrated that targeting KRASG12C is possible. This discovery launched a flurry of activity in both academia and the pharmaceutical industry. Indeed, more than three dozen patent applications from 9 different organizations have reported small-molecule inhibitors through the end of 2019 [17]. Araxes Pharmaceuticals licensed and improved the UCSF molecules and published a series of informative patent applications and research articles. The most potent disclosed molecule within this series, ARS-1620, rapidly binds to KRASG12C and is potent in cell lines dependent on this mutant (Table 2) [18]. It also has good oral bioavailability in mice (F > 60%) and causes tumor regression in xenograft models.

Many of the other reported KRASG12C inhibitors clearly build on the ARS-1620 scaffold. For example, AstraZeneca has reported molecules such as compound 25 (Table 2), in which the core structure has been cyclized to conformationally lock the molecule in the biologically active conformation [19]. Addition of a methyl group off the piperazine further improves both potency and pharmacokinetic properties; compound 25 has a bioavailability of 94% in rats and causes tumor regression in MIA PaCa-2 mouse xenograft studies.An independent series of molecules was reported by researchers from Amgen and Carmot Therapeutics [20]. These were developed using a fragment-based approach, in which a small reactive ‘warhead’ was coupled to several thousand fragments and the resulting molecules were tested. Iterative optimization ultimatelyled to compound 1, with potent biochemical and cell activity (Table 2). Unfortunately, the low oral bioavailability and high clearance of this series precluded further development. However, the research revealed that histidine 95 (H95) could adopt an alternative conformation, creating a small subpocket that could be exploited to gain added potency.The first KRASG12C inhibitor to enter the clinic was sotorasib, or AMG 510, from Amgen. Remarkably, trials began in August 2018, less than five years after the initial UCSF publication [21]. The molecule bears similarity to ARS-1620 but also extends into the H95 pocket, thereby gaining added affinity [22]. Treatment of tumor cell line panels representing homozygous KRASG12C, heterozygous KRASG12C, and wild-type KRAS cells demonstrated potent, mutation-dependent inhibition of p-ERK and cell proliferation [23]. However, the potency of AMG 510 ranges ~10-fold against the various KRASG12C cell lines tested, portending the varying patient response observed in the clinic.

In a phase 1 trial, in patients with advanced metastatic cancer with the KRASG12C mutation, sotorasib demonstrated anticancer activity [24]. It was well tolerated at exposures that exceeded levels required to inhibit pERK (EC90) for 24 h. The response rate in patients with non-small cell lung cancer (NSCLC) and CRC disease was 88 and 74%, respectively. However, for most patients, the best response was stable disease, with limited durability. Partial response was observed in 32.2% and 7.1% of patients with NSCLC and CRC, respectively.

The second clinical-stage KRASG12C inhibitor, adagrasib or MRTX849, also bears some resemblance to ARS1620, although it may have independent origins [25,26]. This molecule appears to be the most potent of all disclosed inhibitors at both a biochemical and cellular level. The KI for KRASG12C was calculated to be 3.7 mM, considerably weaker than the nanomolar activity of many drugs. However, the inactivation rate (0.13 s1) is quite rapid, much more so than many irreversible kinase inhibitors [27]. Adagrasib also demonstrates potent inhibition of KRAS signaling and growth inhibition in KRASG12C-mutant cell lines [28]. Similar to sotorasib, adagrasib demonstrated a 100-fold difference in potency across a panel of KRASG12Cmutant cell lines in a 3D proliferation assay. Interestingly, adagrasib was shown to engage KRASG12C equally across the set of cell lines, but inhibition of downstream signaling was variable and correlated with response. Adagrasib is currently being evaluated in phase 1 and more advanced clinical trials. Initial reports from the phase 1 study on patients with NSCLC and CRC suggest similar patterns of efficacy with sotorasib, with objective responses of 45% and 17%, respectively [29].

Three other KRASG12C inhibitors have entered clinical development, although much less is known about them. GDC-6036 began a phase 1 trial in mid-2020, and a few properties were described at a meeting [30,31]. Although the structure symbiotic associations has not been disclosed, a patent application reports a number of highly potent molecules that bear some resemblance to adagrasib.Lilly’s LY3499446 entered clinical development in late 2019 [32], although the trial was halted after less than a year reportedly owing to toxicity [33]. IDO-IN-2 manufacturer The structure of the molecule has not been reported, but a patent application reports extensive characterization of the molecule shown in Table 3, which bears close resemblance to ARS-1620, differing only in the terminal aromatic substituent [34]. Interestingly, a trial of JNJ74699157 (ARS-3248) was also terminated after enrolling only 10 of the originally planned 140 participants [35]. Whether this was also due to toxicity, and whether the toxicity is due to covalent or noncovalent off targets, remains to be seen.

Targeting KRASG12C : other approaches

All KRASG12C inhibitors described previously block interactions with downstream effectors such as RAF. Because the covalent bond formed between the small molecule and protein is
irreversible, the modified KRAS protein should be completely inactivated. Nonetheless, the protein is still likely to be present and so could potentially act as a scaffold for signaling. An interesting recent trend in drug discovery has been targeted protein degradation, in which proteins are specifically targeted for destruction, most commonly by the proteasome [36]. Several groups have reported making small-molecule degraders of KRAS.The first, by researchers at Harvard, reports a small library of molecules based on ARS-1620 coupled to a cereblon ligand [37]. Although these could induce degradation of KRASG12C when fused to green fluorescent protein, they were ineffective against endogenous KRASG12C. In contrast, researchers at Yale constructed a degrader based on adagrasib coupled to a VHL ligand (Table 2) [38]. This did cause degradation of endogenous KRASG12C, although the antiproliferative activity in cells was lower than that of adagrasib alone. Finally, researchers from Arvinas have filed a patent application claiming both cereblon and VHL-based degraders, although biological characterization is limited.

Figure 1

Two ribbon views of KRAS. Left: wild-type KRAS (pdb 6MBT) showing the three major mutation sites G12 (yellow), G13 (green),and Q61 (brown) [46]. The P-loop is shown in purple, switch I is shown in red, and switch II is shown in blue. GDP is shown as sticks, and the magnesium ion is shown as a metallic sphere. Right: overlayed with KRASG12C covalently bound to sotorasib (pdb 6OIM). Note the movements in switch II upon ligand binding.Another more exotic approach to targeting KRAS is exemplified by a patent application from Revolution Medicines (Table 1) [40]. This reports macrocyclic molecules that bind to both KRAS and an endogenous protein such as a cyclophilin, thus acting as molecular glues to block KRAS signaling. Recruitment of this second protein may compensate for the lower affinity of the small-molecule component. Revolution Medicines reports that it is working on molecules that inhibit other mutant forms of KRAS including G12D and G13C.Importantly, some of these molecules are reported to bind to the GTP-bound form of the protein, which could be useful for countering resistance (see the next section).Resistance to KRASG12C inhibitors As with other targeted cancer therapeutic strategies, it is becoming clear that a significant fraction of patients will be resistant to direct inhibition of KRASG12C. Both sotorasib and adagrasib fail to elicit an objective response in most patients treated, and the duration of response in patients who do benefit is yet to be determined [24]. This is not a surprise, given the variability in activity observed in tumor models preclinically. Moreover, current clinical inhibitors target the GDPbound (i.e. inactive) form of the protein. Several studies have now demonstrated that increased receptor tyrosine kinase signaling or upregulation of cell cycle is associated with decreased sensitivity to inhibitors, presumably by increasing the fraction of GTP-bound (or active) KRAS [23,24,28,42].Based on these observations, multiple drug combination strategies have been tested in preclinical models and proven effective. In particular, KRASG12C inhibitors combined with EGFR or SHP2 inhibitors that suppress upstream signals are effective. KRASG12C inhibition combined with cell cycle inhibitors or anti-programmed cell death protein 1 (PD1) immunotherapy also shows promise preclinically. These data have led to multiple have the potential to extend the inhibition to a broader subset of patients (NCT04185883;NCT04613596; NCT03785249;NCT04330664).

Targeting KRASG12D

A tremendous practical advantage of targeting KRASG12C is the presence of the nucleophilic cysteine residue, ideal for covalent inhibition. KRASG12D is also a clinically important mutation, but has historically been more challenging. Indeed, an attempt to append carboxylatereactive warheads onto a scaffold based somewhat on ARS-1620 did not yield potent inhibitors of KRASG12D, although some of them did covalently bind to KRASG12C [43]. Given the challenges of finding covalent warheads for aspartic acid, noncovalent inhibitors may have more traction. Indeed, researchers atUCSFand the University of Tokyo have recently reported a screen of more than a MLT Medicinal Leech Therapy trillion cyclic peptides and identified molecules that block the interaction of the active (GppNHp-bound, a more stable analog of GTP) form of KRASG12D with RAF at high nanomolar concentrations [44]. Unfortunately, these peptidic molecules are not active in cells, likely owing to low permeability.More promisingly, researchers at Mirati have reported that a KRASG12D inhibitor called MRTX1133 is in INDenabling studies and slated to enter clinical development in the first half of 2021. The molecule is reported to be a low nanomolar inhibitor with activity in xenograft models [45].

The future of targeting mutant KRAS In less than a decade, KRAS has gone from an undruggable and unattainable aim to the target being tested in multiple clinical trials with three separate inhibitors, one of which is in a phase 3 trial. While this is exciting progress, it is important to remember that all these molecules target KRASG12C, which represents a fraction of KRASmutant diseases [2]. Moreover, all these molecules target the inactive (GDP-bound) form of the protein, and thus, they are susceptible to resistance mechanisms. A key focus will be developing moleculesthat canalsoblock the active (GTP-bound) form of KRAS.Moreover, it will be important to develop molecules that block mutants besides KRASG12C. Some exciting developments have already been reported for KRASG12D,but there are no reports of potent inhibitors of KRASG12V It remains to be seen whether it will be possible to develop additional mutant-specific inhibitors or a pan-mutant KRAS inhibitor. But given the intensive efforts and increased understanding of this protein, the prospects look bright.