In this issue of “Small Molecule Highlights” we bring you 5 new small molecules out of recent drug discovery journals. These molecules target a diverse selection of targets, including the SIK 1/2/3, RORγ, PAPD 5/7, KRAS (G12D), and SOS1. Enjoy!
GLPG3312: The team at Galapagos took aim at developing a pan-SIK (SIK1/2/3) inhibitor to complement its pipeline of small molecules targeting inflammatory diseases (Filgotinib, a.k.a. Jyseleca, targeting JAK1 has been approved for the treatment of rheumatoid arthritis and ulcerative colitis). After a high throughput screen of their internal kinase focused library (42,000 compounds), the medicinal chemistry team discovered a potent SIK3 compound as a starting point. Fortuitously, this initial compound contained a wealth of handles for modification, facilitating optimization of the scaffold. Multiple rounds of SAR, highlighted by the incorporation of amido-cyclopropane and difluoromethoxy substituents to increase potency and selectivity for SIK1/2/3, followed by lead optimization/evaluation where required to arrive at clinical candidate GLPG3312. A close congener of the compound was co-crystallized with SIK3, providing valuable insights into the selection of the lead compound and potentially informing future projects (more below). The co-crystal (PDB ID: 8OKU; atomic coordinates were unavailable at the time of writing) is the first reported x-ray structure of a SIK protein family member. In biochemical assays (ADP-Glo), GLP3312 exhibited potent inhibition of all three SIKs (2.0/0.7/0.6 nM for SIK1/SIK2/SIK3, respectively) and good selectivity (DDR1, LIMK1, MAP3K20, and RIPK2 were the only kinases to show > 80% inhibition @ 1 uM in the Eurofins Kinome Panel). Pharmacokinetic characterization of the compound revealed low mouse liver microsome and hepatocyte clearance (4.76 L/h/kg and <1.75 4.76 L/h/kg, respectively) and respectable oral bioavailability in mouse, rat, and dog (F = 60/41.4/45.5% in mouse/rat/dog, respectively). In vivo efficacy was assessed in an LPS challenge model in Balb/c mice where GLPG3312 was shown to have a dose-dependent effect on serum TNFα and IL-10 concentrations (77% decrease in TNFα and 3.1-fold increase in IL-10 was observed at 3 mg/kg). The authors indicated that GLPG3312 was progressed to a phase 1 clinical trial (NCT03800472) looking at two separate formulations/exposure regimens in healthy individuals. Also teased was GLPG3970; a novel selective SIK2/SIK3 inhibitor candidate.
JTE-151: There is increasing evidence supporting the role of IL-17 and Th17 cells in chronic inflammation, with several pharmaceutical companies investing in modalities targeting this pathway. Biologics targeting IL-17 have garnered a bulk of the attention/success, however, inherent pharmacokinetic limitations have shifted attention onto retinoic acid receptor-related orphan receptor gamma (RORγ) as a promising target for small molecule intervention. The challenge, nuclear receptors, such as RORγ, are heavily hydrophobic leading to selectivity and solubility issues for designed ligands/inhibitors/agonists. The authors took a pragmatic approach towards the development of JTE-151 by incorporating “drug-likeness” as a major design consideration and candidate selection criteria. As such, non-typical chemotypes, rarely seen in nuclear receptor development programs, were considered, and SAR decisions were heavily influenced by ligand efficiency (LE) and sp3 carbon content. Every appendage, including the iso-oxazole core, was surveyed, and optimized to arrive at JTE-151. Interestingly, the co-crystal of the compound with RORγ (PDB: 8X7E) shows a unique binding orientation, with the hydrophobic portion of the molecule pulled back in an almost U-shape conformation and the carboxylic acid group (positioned at the bottom of the U) projecting outside of the pocket towards solvent. Compound efficacy was first assessed in a cellular luciferase activity assay (CHO-K1 cells) were JTE-151 exhibited low nanomolar inhibition (EC50 = 13 nM) of RORγ. Specificity was demonstrated in a panel of closely related and often hit nuclear receptors (100-fold selectivity was observed). In pharmacokinetic studies, JTE-151 displayed low clearance (CL = 0.11 L/h/kg) in rats and good overall bioavailability in mouse and dog (F = 98 and 64% in mouse and dog, respectively). In vivo, the compound was shown to dose-dependently decrease the severity of paralysis in a mouse model of experimental autoimmune encephalomyelitis (EAE). In toxicology studies, an initially observed hERG liability (patch-clamp assay) was ruled out as insignificant in further cardiac telemetry studies in conscious dog (no QTcF elongation at doses of 100 mg/kg p.o. BID). Measures of genotoxicity CHL cells and a GLP-complaint toxicity study in rats and dog did not reveal any development limiting toxicity. Recently, JTE-151 was assessed in clinical trials with results from a phase I study being reported shortly. The authors offered a small preview of the results by disclosing dose tolerance results. JTE-151 was well tolerated in healthy individuals (30 – 1600 mg doses) with no observable plateauing of plasma concentration within the dose range tested. Also, no severe adverse effect/events were observed at the highest dose of 1600 mg/kg. It will be interesting to see how far JTE-151 progresses.
AB-452: What a difference an N makes. The medicinal chemistry team at Arbutus Biopharma were looking to develop a small molecule therapeutic to reduce plasma concentrations of Hepatitis B soluble antigen (HBsAg) to mitigate T-cell exhaustion in HBV infected individuals. Using RG7834 (Roche) as a starting point, the team considered a simple expansion of the central 6-membered ring to a 7-membered oxazepine. While a concise SAR of catechol appended substituents led to two potential candidates, undesirable PK profiles in rat and a substantial hERG liability halted further development. In parallel, the team explored the incorporation/replacement of the terminal catechol ring with a variety of heteroaromatics. An SAR covering pyridines (various regioisomers), pyridones, thiophenes, and thiazoles led the team to a very minor modification of the parent scaffold, a substitution of an aromatic carbon with nitrogen. Introduction of the pyridine had no effect on the potency or cytotoxicity of AB-452 compared to RG7834 (EC50 = 1.2 nM and CC50 > 50 μM, respectively). A marked difference was observed in PK and toxicity studies where AB-452 was shown to have 2-fold greater oral exposure (rat and dog) compared to RG7834 and no effect on cardiac telemetry measurement (ECG) at 10 μM concentration. Given the improved PK and safety profile of AB-452 over RG7834, efficacy in a murine in vivo model of HBV infection was assessed. In the study, a dose-dependant decrease in serum and liver HBsAg, as well as Liver HBV RNA, was observed after 7 days (3/10/30 mg/kg, p.o., once daily). The authors quickly glossed over results from a preclinical safety study where signs of peripheral neuropathy were observed after chronic dosing in rat and dog. Similar results in monkey were observed for RG7834, indicating an issue with the overall scaffold rather than the modifications inherent to AB-452. Further optimization of the scaffold to circumvent the observed neuropathy was intimated by the authors.
8o: The KRAS G12D mutation is the most common oncogenic variant of the protein and a prevalent escape mechanism from G12C targeted therapeutics (i.e. sotorasib and adagrasib). As the G12D mutation has an aspartic acid in place of the targeted cysteine, thus eliminating the possibility of covalent modification, the creators of adagrasib (Mirati Therapeutics) have successfully re-tooled the compound into a selective reversible inhibitor of G12D protein (MRTX1133). As MRTX1133 is currently in phase 1 clinical trials, with results yet to be reported, there is concern over rapidly acquired drug resistance, similar to what was observed with G12C inhibitors. As such, targeted degradation is being looked at as a complementary therapeutic strategy for individuals harboring KRAS G12D. To develop a PROTAC for G12D, the authors maintained the pyridopyrimidine core and Asp 12 bicyclo amine recognition substituent of MRTX1133, however, the solvent exposed 5,5-fused bicylic amino group was replaced with a simpler prolinol substituent, similar to that of adagrasib. Swap to the prolinol was a pragmatic design decision as it offered an easily tapped handle for application of the linker and VHL recruitment substituents and had the appropriate exit vector. The original naphthalene group was slightly modified, with an ethyl group replacing the alkyne. This might have been done to counter chemical atropism concerns, almost a certainty in such a congested scaffold. A survey of various linkers identified n-butanol as the best substituent to form the ternary complex. Rigid heterocyclic linkers were also sampled but found to be less effective. The authors didn’t waver from their choice of VHL ligand, stating inspiration from LC-2, a G12C targeting covalent PROTAC based, to support their design. However, with multiple players in the space, including Arvinas, Erasca, and Shanghai Pharma, their SAR choices may have been guided, in part, by chemical white space and IP strategy. The in vitro validation of 8o was comprehensive and exhaustive to say the least. To start, cooperativity measurement looking at the formation of binary (PROTAC + KRAS G12D) vs ternary (PROTAC + KRAS G12D + E3 ligase) was used to evaluate the efficiency of ternary complex formation. Cooperativity measurements were compared to cellular degradation results to deconvolute cellular uptake of compound and inform further SAR decisions. Degradation selectivity across 7 cell lines encompassing KRAS G12D, G12C, G12V, G12S, G12R, and G13D mutants, as well as a WT cell line, was demonstrated at 0.1 and 1 μM. Proteomics experiments were conducted to probe off-target effects more completely. Of the 7510 detected proteins, only DUSP4 and KRT5/9/14 were significantly depleted. Downregulation of DUSP4 was later proven in the article to be dependant on KRAS degradation. Efficacy in cancer relevant cell lines was assessed where 8o exhibited dose-dependent degradation of KRAS G12D in SNU-1, HPAF-II, AGS, and PANC 04.03 cells with corresponding DC50 values of 19.77, 52.96, 7.49, and 87.8 nM, respectively. Compound 8o also exerted an inhibitory effect on the ERK signaling pathway, with durable inhibition of ERK phosphorylation being observed 72 hours post washout in AsPC-1 cells. Pharmacokinetic characterization in Balb/c mice revealed good plasma exposure (AUC0–∞ = 5057 ± 546 h·ng/mL) and long serum half-life (T1/2 = 8.70 ± 2.47 h) after subcutaneous injection. This uncommon mode of delivery raises concerns about the bioavailability of the compound and its potential for oral administration. Pharmacodynamic evaluation in AsPC-1 mouse xenograft study confirmed efficacy of 8o in an in vivo setting. In the PD model, KRAS G12D was significantly depleted 24 hours post dose, with protein levels not fully recovered after 72 hours. Also, tumor growth was inhibited by a respectable 68% over a 22-day chronic dosing (50 mg/kg, QD, s.c.) study with no signs of toxicity. In the same study, MRTX1133 inhibited tumor growth by 89%, albeit, employing a different method of administration (30 mg/kg, BD, i.p.). Taken as a whole, 8o looks to be a promising lead compound in need of further PK optimization to become a potential treatment option for KRAS G12D driven pathologies.
Compound 4: Staying with the theme of KRAS pathway modulation via PROTACs, a collaboration between Shanghai Zelgen Pharma-Tech and the East China University of Science and Technology have reported a new SOS1 targeting PROTAC. SOS1 (Son of Sevenless 1) plays a key role in the KRAS signalling pathway by facilitating nucleotide release from the inactive KRAS-GDP complex. As a therapeutic target, SOS1 inhibition/depletion has been viewed as a strategy to circumvent resistance mutations arising from drugs targeting KRAS directly. Indeed, there are multiple clinical trials looking at SOS1 inhibitors in combination with KRAS G12C therapeutics, however, no PROTACs have been evaluated in combination studies or as a monotherapy in a clinical setting. The medicinal chemistry team decided to use BI3406 (Boehringer-Ingelheim) as a starting scaffold, and quickly analyzed options for appending the E3 ligase ligand. Fortunately, the tetrahydrofuran substituent was ideally placed (solvent exposed), however, it lacked a viable attachment point for the E3 ligand. As such, the team opted to go with a methylamino cyclopropane as similar molecules have been shown to inhibit formation of the KRAS G12C/SOS1 complex. A survey of various linkers indicated that conformationally rigid and heteroatom containing substituents were not tolerated, leading the team to select a 6-carbon aliphatic chain as the ideal linker. Further SAR centered around the CRBN ligand (E3 ligase ligand) and the pendant trifluoromethyl aniline revealed little room for modification. Compound 4 exhibited low nanomolar antiproliferative properties (IC50 = 5 nM) and potent SOS1 degradation in NCI-H358 cells (DC50 = 13 nM, Dmax = 88%). Furthermore, cell proliferation was attenuated in several other cell lines expressing different KRAS mutations (GP2D(G12D), NCI-H441(G12V), and DLD-1(G13D)). Pharmacokinetic characterization of 4 revealed a long plasma half-life (T1/2 = 4.05 h) and good plasma exposure (AUC0–∞ = 4378 h·ng/mL) following intraperitoneal administration. In vivo tumor control was demonstrated in a H358 xenograft study in BALB/c mice. Compound 4 was shown to inhibit tumor growth by almost 60% (30 mg/kg, i.p., 21-days), outperforming BI3406 (30 mg/kg, p.o.) by nearly 2-fold. Collectively, the data suggest compound 4 to be an interesting clinical development candidate.