Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a Pyridopyridazinone pan-RAF Kinase Inhibitor

Abstract

Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (7), which features a unique pyrido[2,3-d]pyridazin-8(7H)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations. In vivo evaluation of GNE-9815 in combination with the MEK inhibitor cobimetinib demonstrated synergistic MAPK pathway modulation in an HCT116 xenograft mouse model. To the best of our knowledge, GNE-9815 is among the most highly kinase-selective RAF inhibitors reported to date.

RAS mutations are found in nearly 20% of all cancers, but despite their prevalence and considerable research efforts over the past three decades no effective therapeutic has been approved to date.¹ Mutations of RAS are characterized by an enrichment of its active GTP-bound population, leading to uncontrolled downstream signaling to effector proteins. An effector of particular importance is RAF (consisting of the three isoforms ARAF, BRAF, and CRAF), the downstream kinase member of the MAPK pathway. RAF interaction with GTP-bound RAS drives RAF dimerization and phosphorylation, and activated RAF in turn phosphorylates its substrate MEK1/2 to transmit signaling within the MAPK pathway.

Until recently,² direct therapeutic targeting of RAS has proven challenging; therefore, targeting of inherently more druggable downstream kinases in the MAPK pathway has been of considerable interest over the last decades. However, these efforts have met significant obstacles, in part due to the propensity of RAF inhibitors to paradoxically activate the pathway³ as a result of detrimental negative feedback loops integral to the MAPK pathway.⁴ In our efforts to overcome these obstacles, we recently reported on the synergistic use of Type II RAF inhibitors in combination with MEK inhibitors to target KRAS mutant cancers.⁵ The binding mode of Type II RAF inhibitors obliterates the formation of asymmetric RAF dimers thus thwarting undesirable pathway activation. Conversely, the combination with MEK inhibitors disables negative feedback loops, thereby sensitizing tumors to RAF inhibitors.

We recently reported the discovery of a potent and selective Type II RAF inhibitor, GNE-0749, which showed significant antitumor efficacy in combination with the MEK inhibitor cobimetinib in a HCT116 xenograft tumor model (Figure 1).⁶ Concurrent with this effort, we also investigated novel hinge binder moieties with improved physicochemical properties. Specifically, we sought to modify the 2-aminopyrido[2,3-d]pyrimidine hinge binder of LY3009120⁷ (1) with the goal of reducing hydrogen bond donors and rotatable bonds to support oral dosing (Table 1). Antiproliferative activity was compared in BRAF mutant A375 and KRAS mutant A549 cells along with A549 in the presence of a submaximal concentration (100 nM) of cobimetinib. Significant A549 potency shift in the presence versus absence of cobimetinib cotreatment is indicative of synergistic inhibition.

Figure 1.

GNE-0749, a potent and selective Type II pan-RAF inhibitor.

Table 1. Evaluation of Hinge Binder Motifs.

Beginning with pyrido[2,3-d]pyrimidinone 2, a compound with a hinge binder motif that maintains the same kinase hinge hydrogen bond donor/acceptor as LY3009120 (1), comparable potency was achieved, albeit with poor kinase selectivity (15/26 kinases with >70% inhibition at 0.1 μM vs 11/29 for 1), which may be contributing to the antiproliferative activity. Possessing a more lipophilic 3-aminoisoquinoline heteroarene, compound 3 partially restored kinase selectivity with an increase in biochemical potency. To further improve kinase selectivity, we aimed to minimize the common hinge interactions of 3 and sought to replace the aminoisoquinoline N–H with a polarized C–H, leading to the design of 1-(2H)-phthalazinone 4.⁸ To the best of our knowledge, this is the first example of the use of this heteroarene as a hinge-binding motif in kinase inhibitors. Gratifyingly, phthalazinone 4 exhibited similar biochemical potency to 3, but along with improved kinase selectivity (5/29 kinases with >70% inhibition at 0.1 μM).

Aiming to increase RAF potency in this new series and reduce the number of polar N–H groups, we investigated whether a suitable alternative to the urea moiety could be found (Table 2). Deletion of one of the nitrogen atoms from the urea portion of the molecule afforded benzamide 5 which maintained the biochemical potency of phthalazinone 4 but displayed poor cellular potency (∼6-fold loss). Changing the benzamide to an aryl anilide functional group similar to other type II RAF inhibitors,⁹ RAF kinase cellular potency was restored, while simultaneously lowering log D (compound 6). However, the overall physicochemical properties and microsomal stability were not improved. Finally, we discovered that the introduction of an additional nitrogen atom to the 1-(2H)-phthalazinone ring in compound 6, to provide pyrido[2,3-d]pyridazin-8(7H)-one 7 (GNE-9815), reduced lipophilicity, improved microsomal stability, and imparted a dramatic increase in kinase selectivity such that a panel of 223 kinases found no inhibition > 70% at a concentration of 0.1 μM (besides RAF kinases, Figure 2).¹⁰

Table 2. Discovery of Pan-RAF Inhibitor GNE-9815 (7)^a.

^aMDCK apical → basolateral P_app (apparent permeability, ×10^–6 cm s^–1); efflux = BA/AB; LM = liver microsome CL_hep human/rat/mouse.

Figure 2.

Kinome tree for 7, tested against 226 kinases at a concentration of 0.1 μM.

An X-ray crystal structure of 7 bound in a mutant BRAF construct (see SI Methods) is shown in Figure 3. As expected, the substituted benzamide occupies the lipophilic back pocket of RAF, while the pyrido[2,3-d]pyridazin-8(7H)-one ring system shows interaction with the backbone N–H (3.1 Å) of Cys532 at the hinge of the protein in addition to a nontraditional polarized N-methyl C–H interaction with Cys532 C=O (3.1 Å).⁸ With less discrete polarized hinge contacts, the exquisite selectivity for RAF kinases may be due to van der Waals π-stacking interactions of the heteroaryl hinge binder with Trp531 (unique to ARAF, BRAF, and CRAF), Phe595 (DFG), and Phe583 interactions. The pyrido nitrogen faces the bulk solvent channel and makes little direct contact with the protein; therefore, it is not obvious how the nitrogen imparts such a dramatic increase in selectivity.

Figure 3.

X-ray crystal structure of 7 bound in a mutant BRAF construct (PDB ID 6XLO).

Based on these promising in vitro results, phthalazinone 4 and pyridopyridazinone 7 were progressed to in vivo studies. Pharmacokinetic (PK) data from these compounds in mice are shown in Table 3. Both compounds showed low blood clearance, moderate volume of distribution, and short half-life. However, 7 offered significantly better oral bioavailability upon dosing as a crystalline suspension using a methylcellulose/Tween (MCT) formulation.

Table 3. Property Evaluation of Compounds 4 and 7.

compd	mouse CLba	Vdss,at1/2	F
4	22 mL min–1 kg–1	2.1 L kg–1, 1.3 h	2% (MCT)b
7	17 mL min–1 kg–1	1.7 L kg–1, 1.9 h	37% (MCT)b

^aIV 1 mg kg^–1 10% DMSO, 10% Cremophor EL in saline.

^bPO 5 mg kg^–1.

Given the optimal combination of cellular potency and pharmacokinetic properties, 7 was progressed to further studies. To confirm that the RAF inhibition elicited by 7 combined with MEK inhibition was synergistic in KRAS mutant tumors, we performed pharmacokinetic/pharmacodynamics (PKPD) studies to assess the combination of 7 with cobimetinib. Single-agent treatment with 7 resulted in pathway inhibition as demonstrated by partial inhibition of pRSK between 2 and 24 h, but more robust, albeit transient, inhibition of the downstream MAPK target genes, DUSP6 and SPRY4 (Figure 4). In contrast, cobimetinib alone showed minimal reduction in pRSK at any time point, with modest reduction in downstream expression of the MAPK target genes, DUSP6 and SPRY4. The combination of 7 and cobimetinib afforded increased inhibition of pRSK at 2 and 8 h with rebound at 24 h. The increased pathway inhibition by the combination of 7 and cobimetinib resulted in deeper inhibition of the downstream MAPK target genes DUSP6 and SPRY4 with maximal inhibition observed at 8 h and with a more modest rebound in levels at 24 h, post final dose. This data corroborates earlier findings⁶ that the combination of RAF and MEK inhibition results in increased MAPK pathway suppression in a KRAS mutant tumor setting. The pristine kinase selectivity observed for compound 7 provides further confidence that the observed synergistic effect with the MEK inhibitor cobimetinib is driven exquisitely by RAF inhibition.

Figure 4.

PKPD study for GNE-9815 (7) in HCT116 xenograft mice.

In summary, we have reported here the discovery of pan-RAF inhibitors that avoid paradoxical activation based on a type II kinase binding mode. Limiting the hinge interactions to improve kinome selectivity led to the discovery of compounds with novel 1-(2H)-phthalazinone and pyrido[2,3-d]pyridazin-8(7H)-one kinase hinge-binding motifs. GNE-9815 (7) is a potent pan-RAF inhibitor showing synergistic activity in KRAS mutant A549 and HCT116 cancer cells in combination with the MEK inhibitor cobimetinib. The synergistic pharmacodynamic effects were also demonstrated in HCT116 tumor-bearing mice. The kinase selectivity imparted by the pyrido[2,3-d]pyridazin-8(7H)-one ring system makes GNE-9815 (7) one of the most kinase selective RAF inhibitors reported to date. Based on its favorable property profile, including exquisite kinase selectivity, satisfactory solubility, and rodent oral exposure, we believe that GNE-9815 could become a valuable chemical probe for researchers interested in interrogating RAF biology.

Glossary

Abbreviations Used

CL

clearance

DELFIA

dissociation-enhanced lanthanide fluorescence immunoassay

DFG

Asp-Phe-Gly

DUSP6

dual specificity phosphatase 6 kinase

ERK

extracellular signal-regulated kinase

F

bioavailability

HCT

human colorectal cancer cells

IV

intravenous

KRAS

Kirsten rat sarcoma

log D

log of distribution coefficient

MDCK

Madin–Darby canine kidney cell

MEK

mitogen-activated protein kinase kinase

MAPK

mitogen-activated protein kinase

MCT

methylcellulose-tween

MP

melting point

MS

mass spectrometry

P_app

permeability coefficient

PK

pharmacokinetic

PKPD

pharmacokinetic/pharmacodynamics

PO

per os (oral)

RAF

rapidly accelerated fibrosarcoma

SPRY4

sprouty RTK signaling antagonist 4

TPSA

topological polar surface area

V_dss

volume of distribution

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00063.

• Experimental procedures, compound characterization, NMR and HPLC spectra for compound 7, detailed kinase selectivity panel data for compounds 2–7, crystallographic statistics for protein cocrystal structure of 7 (PDF)

The authors declare the following competing financial interest(s): All authors are employees/former employees of Genentech, Inc. and may hold stock in Roche Holding AG. All studies were funded by Genentech, Inc.

Notes

Coordinates and structure factors for the BRAF domain complex are available in the PDB with accession code 6XLO (7). The authors will release the atomic coordinates and experimental data upon article publication.