Discovery of Selective LRRK2 Inhibitors Guided by Computational Analysis and Molecular Modeling

Mutations in the genetic sequence of leucine-rich repeat kinase 2 (LRRK2) have been linked to increased LRRK2 activity and risk for the development of Parkinson’s disease (PD). Potent and selective small molecules capable of inhibiting the kinase activity of LRRK2 will be important tools for establishing a link between the kinase activity of LRRK2 and PD. In the absence of LRRK2 kinase domain crystal structures, a LRRK2 homology model was developed that provided robust guidance in the hit-to-lead optimization of small molecule LRRK2 inhibitors. Through a combination of molecular modeling, sequence analysis, and matched molecular pair (MMP) activity cliff analysis, a potent and selective lead inhibitor was discovered. The selectivity of this compound could be understood using the LRRK2 homology model, and application of this learning to a series of 2,4-diaminopyrimidine inhibitors in a scaffold hopping exercise led to the identification of highly potent and selective LRRK2 inhibitors that were also brain penetrable

Discovery of 5‑Azaindazole (GNE-955) as a Potent Pan-Pim Inhibitor with Optimized Bioavailability

Pim kinases have been identified as promising therapeutic targets for hematologic–oncology indications, including multiple myeloma and certain leukemia. Here, we describe our continued efforts in optimizing a lead series by improving bioavailability while maintaining high inhibitory potency against all three Pim kinase isoforms. The discovery of extensive intestinal metabolism and major metabolites helped refine our design strategy, and we observed that optimizing the pharmacokinetic properties first and potency second was a more successful approach than the reverse. In the resulting work, novel analogs such as <b>20</b> (GNE-955) were discovered bearing 5-azaindazole core with noncanonical hydrogen bonding to the hinge

Combination of MEK and ERK inhibitors more potently suppresses <i>Kras</i> mutant PDAC and NSCLC GEMM MAPK signaling and tumor progression.

<p>In PDAC GEMM, combination of cobimetinib and GDC-0994 (cobi at 5 mg/kg, PO, QD + GDC-0994 at 60 mg/kg, PO, QD) more potently (<b>a</b>) reduced MAPK-target gene expression at (6 hr post-last dose following 3 days treatment, n = 4/group); vehicle (black), Cobimetinib (green), GDC-0994 (blue), combination (green); vehicle vs. combo for all genes ****p<0.0001 (red line), n.s., not significant for vehicle vs. GDC-0994 (green line) or Cobi (blue line), Mann-Whitney. (<b>b)</b>, Combination treatment improved responses in PDAC model increasing tumor regressions as measured by ultrasound (d7). (<b>c</b>) Long-term combo treatment significantly reduced tumor growth rate in PDAC tumors. (vehicle (black) n = 13, Cobimetinib (green) n = 9, GDC-0994 (blue) n = 8, Combination (red) n = 12; ****p = 0.001) (<b>d</b>) Long-term combo treatment significantly improved progression free survival in PDAC. Log-rank test, ***p = 0.0023. (<b>e</b>) Combination of cobimetinib and GDC-0994 (cobi at 5 mg/kg, PO, QD + GDC-0994 at 60 mg/kg, PO, QD) more potently reduced MAPK target gene expression at (6 hr post-last dose following 3 days treatment, n = 4/group) in NSCLC tumors; vehicle (black), Cobimetinib (green), GDC-0994 (blue), combination (red); vehicle vs. combo. Mann-Whitney, ****p≤0.0001 for all genes (red line); vehicle vs. GDC-0994 *p<0.05 for <i>SPRY4</i>, <i>DUSP6</i>, <i>ETV4</i>, <i>FST</i>, <i>EPHA2</i>, and <i>PHLDA1</i> (green line); vehicle vs. Cobi *p<0.05 for <i>DUSP6</i>, <i>ETV4</i>, <i>FST</i>, <i>EPHA2</i>, and <i>PHLDA1</i> (blue line); all others n.s., not significant. (<b>f</b>) Combination treatment in the NSCLC model increased tumor regressions as measured by micro computed tomography (μCT) (d14) (*p<0.001). (<b>g</b>) Long-term combo treatment significantly reduced tumor growth rate in NSCLC tumors (vehicle n = 15, Cobimetinib n = 14, GDC-0994 n = 15, Combination, n = 15; *p<0.05, ****p = 0.0001). (<b>h</b>) Reduced tumor growth translated to statistically significant improved progression free survival in NSCLC model (Log-rank test, ***p = 0.004).</p

Combination of MEK and ERK inhibitors results in stronger suppression of <i>KRAS</i> mutant tumor growth due to improved suppression of MAPK output.

<p>(<b>a</b>) Combination of cobimetinib and GDC-0994 demonstrates significantly greater anti-tumor activity in multiple <i>KRAS</i> mutant tumor models A549 and NCI-H2122 (cobimetinib at 5 mg/kg, PO, QD + GDC-0994 at 60 mg/kg, PO, QD) compared to single agent (upper panels). Mean tumor volume is plotted ± SEM (n = 10 mice per group). Study was terminated on day 20. All treatments were tolerated with minimal body weight loss (lower panels), One way ANOVA, * p<0.05, ** p<0.01, *** p<0.005, ****p<0.001. (<b>b</b>) A549 (NSCLC, <i>KRAS</i>^<i>G12S</i>) tumor-bearing mice (n = 3 per time point) were treated with GDC-0994 (60 mg/kg, PO, QDx4), cobimetinib (GDC-0994; 5 mg/kg, PO, QDx4) or the combination and then MAPK target genes expression was assessed in tumor samples (Nanostring^®) and the quantified results are plotted for each individual gene over time. The combination results in deeper, more prolonged suppression of multiple MAPK target genes, including <i>DUSP4</i>, <i>DUSP6</i>, <i>SPRY2</i>, <i>SPRY4</i>, <i>ETV4</i>, and <i>ETV5</i>. Student’s t test at the 24 hr time point, * p<0.05, ** p<0.01, *** p<0.005, ****p<0.001. (<b>c</b>) The combination of cobimetinib and GDC-0994 results in stronger and more prolonged suppression of p-p90RSK/total p90RSK phosphorylation (as determined by quantitative western blot), cyclin D1 and Ki-67, as well as increased induction of cleaved caspase 3 (CC3) (as determined by IHC) in A549 xenograft tumors treated for 4 days (values were quantified from n = 4 mice/time point). Student’s t test at the 24 hr time point, * p<0.05, ** p<0.01, *** p<0.005.</p

MAPK signaling network model simulations predict synergistic activity between cobimetinib and GDC-0994.

<p>(<b>a</b>) MAPK signaling model schematic, wherein the system input Grb2-SOS induces catalysis of Ras-GDP to Ras-GTP, which then catalyzes a phosphorylation cascade from BRAF/CRAF dimers via MEK to ERK, with ppERK as the system output. Three canonical negative feedback mechanisms are considered; inhibitory phosphorylation of MEK and CRAF by ppERK, DUSP-mediated ERK de-phosphorylation, and SPRY-mediated inhibition of Grb2-SOS/RAS signaling. (<b>b</b>, <b>c</b>) Fractional cell viability was predicted in the presence of combined doses of two drugs ranging from 1 to 1000 nM in half-log dilution steps to form a 9x9 matrix; Isobolograms show the predicted effect on cell viability at each dose level (grey lines), with blue line showing 70% effect compared to expected effect for Loewe additivity (red line). Combining GDC-0994 with cobimetinib (<b>b</b>) results in deviation from additivity, combining cobimetinib with itself (<b>c</b>) demonstrates that an additive effect conforms to the expected Loewe model.</p

CRAF plays a key role in pathway reactivation following MEK and ERK inhibition.

<p>(<b>a</b>) CRAF kinase activity measured following single or dual node targeting in A549 (cobimetinib: 0.125μM, GDC-0994: 1.25μM, combination: 0.0625 μM and 0.625 μM, respectively) and HCT116 (cobimetinib: 0.25μM, GDC-0994: 1.25μM, combination 0.125 μM and 0.625 μM, respectively) for 6hr. Quantification of Western data using ImageJ are shown next to the Western blot. This is a representative image from five independent experiments. (<b>b</b>) HCT116 cells transfected with either a non-targeting control (NTC) or CRAF siRNA were treated 24 hr post electroporation with cobimetinib (0.25 μM) or GDC-0994 (1.25 μM) for 6, 24, and 72 hr.</p

Combination of MEK and ERK inhibitors results in stronger suppression of MAPK pathway output and overcomes pathway reactivation.

<p>(<b>a</b>) MAPK target genes <i>DUSP4</i>, <i>DUSP6</i>, <i>SPRY2</i>, <i>SPRY4</i>, <i>ETV1</i>, and <i>ETV5</i> expression following 24hr treatment with the indicated doses in A549 cells. One-way ANOVA, * p<0.05, **p<0.01, *** p<0.005, **** p<0.001. (<b>b</b>) The combination cobimetinib and GDC-0994 in HCT116 and A549 (<i>KRAS</i>^<i>G12S</i>, NSCLC) cells results in stronger reduction of cyclin D1 accumulation, increased p27 levels, increased cleaved PARP levels, and, <b>c</b>, increased cell death at 24 hrs post-treatment at the concentrations indicated. One-way ANOVA, *** p<0.005, **** p<0.001.</p

Dual node targeting with ERK and MEK inhibitors prevents MAPK pathway reactivation and has synergistic activity in <i>KRAS</i> mutant cells.

<p>(<b>a</b>) Calculated GDC-0994 and cobimetinib GI₅₀ (50% growth inhibition) values across all lines tested are significantly correlated (Pearson correlation R = 0.446, <i>p</i> = 1.76 x 10⁻²⁴). Calculated GI₅₀ values from large-scale cell viability screening (474 cell lines) using cobimetinib (Y axis) and GDC-0994 (X axis) in cell lines WT for all <i>RAS</i> and <i>RAF</i> genes, or harboring mutations in any <i>RAS</i> or <i>RAF</i> genes, respectively. (<b>b</b>) Pathway reactivation at the level of p-p90RSK was assessed in <i>KRAS</i> mutant cells, HCT116, using single agent cobimetinib at EC₅₀ concentrations of 0.25 μM or GDC-0994 at 1.25 μM (HCT116 (<i>KRAS</i>^<i>G13D</i>, colorectal)) at indicated time points. <b>c</b>, Pathway reactivation at the level of MAPK target gene transcript in HCT116 cells using the same EC₅₀ concentrations as in <b>b</b> for the indicated time points. (<b>d</b>) Pathway reactivation at the level of p90RSK was assessed in <i>KRAS</i> mutant cells, HCT116, using single agent cobimetinib 0.25 μM, single agent GDC-0994 1.25 μM or combination treatment using 0.125 μM and 0.625 μM, respectively, at indicated time points. The graph to the right shows quantification of the immunoblot on the left for normalized p-p90RSK ([p-p90RSK/actin]/[total p90RSK/actin]) following treatment with cobimetinib (green triangles), GDC-0994 (blue squares) or the combination (red diamonds) in HCT116 cells. (<b>e</b>) HCT116 cells were treated with cobimetinib and GDC-0994 at the indicated concentrations and cell viability was measured after 72 hr of culture (CellTiter-Glo^®). (<b>f</b>) Isobologram analysis of EdU incorporation was utilized to evaluate the combination of cobimetinib and GDC-0994 on HCT116 proliferation. Predicted Loewe additivity is shown in red, whereas fitting of the 50% effect values is plotted in blue.</p

Discovery of (<i>S</i>)‑1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((1-methyl‑1<i>H</i>‑pyrazol-5-yl)amino)pyrimidin-4-yl)pyridin-2(1<i>H</i>)‑one (GDC-0994), an Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibitor in Early Clinical Development

The extracellular signal-regulated kinases ERK1/2 represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that is commonly activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Described herein is the discovery and characterization of GDC-0994 (<b>22</b>), an orally bioavailable small molecule inhibitor selective for ERK kinase activity