Hematopoietic progenitor kinase 1 (HPK1) is implicated as a negative regulator of T-cell receptor-induced T-cell activation. Studies using HPK1 kinase-dead knock-in animals have demonstrated the loss ...of HPK1 kinase activity resulted in an increase in T-cell function and tumor growth inhibition in glioma models. Herein, we describe the discovery of a series of small molecule inhibitors of HPK1. Using a structure-based drug design approach, the kinase selectivity of the molecules was significantly improved by inducing and stabilizing an unusual P-loop folded binding mode. The metabolic liabilities of the initial 7-azaindole high-throughput screening hit were mitigated by addressing a key metabolic soft spot along with physicochemical property-based optimization. The resulting spiro-azaindoline HPK1 inhibitors demonstrated improved in vitro ADME properties and the ability to induce cytokine production in primary human T-cells.
Inhibition of PI3Kδ is considered to be an attractive mechanism for the treatment of inflammatory diseases and leukocyte malignancies. Using a structure-based design approach, we have identified a ...series of potent and selective benzimidazole-based inhibitors of PI3Kδ. These inhibitors do not occupy the selectivity pocket between Trp760 and Met752 that is induced by other families of PI3Kδ inhibitors. Instead, the selectivity of the compounds for inhibition of PI3Kδ relative to other PI3K isoforms appears to be due primarily to the strong interactions these inhibitors are able to make with Trp760 in the PI3Kδ binding pocket. The pharmacokinetic properties and the ability of compound 5 to inhibit the function of B-cells in vivo are described.
Inhibition of phosphoinositide 3-kinase (PI3K) signaling through PI3Kα has received significant attention for its potential in cancer therapy. While the PI3K pathway is a well-established and widely ...pursued target for the treatment of many cancer types due to the high frequency of abnormal PI3K signaling, glioblastoma multiforme (GBM) is particularly relevant because the pathway is implicated in more than 80% of GBM cases. Herein, we report the identification of PI3K inhibitors designed to cross the blood–brain barrier (BBB) to engage their target where GBM tumors reside. We leveraged our historical experience with PI3K inhibitors to identify correlations between physicochemical properties and transporter efflux as well as metabolic stability to focus the selection of molecules for further study.
Na
1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe ...inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 (
) and GDC-0310 (
), selective Na
1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound
. This resulted in the discovery of GDC-0276 (
), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Na
1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 (
), which possesses improved Na
selectivity and pharmacokinetic profile over
.
Glioblastoma (GBM), the most common primary brain tumor in adults, presents a high frequency of alteration in the PI3K pathway. Our objectives were to identify a dual PI3K/mTOR inhibitor optimized to ...cross the blood-brain barrier (BBB) and characterize its brain penetration, pathway modulation in the brain and efficacy in orthotopic xenograft models of GBM.
Physicochemical properties of PI3K inhibitors were optimized using in silico tools, leading to the identification of GNE-317. This compound was tested in cells overexpressing P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP). Following administration to mice, GNE-317 plasma and brain concentrations were determined, and phosphorylated biomarkers (pAkt, p4EBP1, and pS6) were measured to assess PI3K pathway suppression in the brain. GNE-317 efficacy was evaluated in the U87, GS2, and GBM10 orthotopic models of GBM.
GNE-317 was identified as having physicochemical properties predictive of low efflux by P-gp and BCRP. Studies in transfected MDCK cells showed that GNE-317 was not a substrate of either transporter. GNE-317 markedly inhibited the PI3K pathway in mouse brain, causing 40% to 90% suppression of the pAkt and pS6 signals up to 6-hour postdose. GNE-317 was efficacious in the U87, GS2, and GBM10 orthotopic models, achieving tumor growth inhibition of 90% and 50%, and survival benefit, respectively.
These results indicated that specific optimization of PI3K inhibitors to cross the BBB led to potent suppression of the PI3K pathway in healthy brain. The efficacy of GNE-317 in 3 intracranial models of GBM suggested that this compound could be effective in the treatment of GBM.
The sodium channel Na
1.7 has emerged as a promising target for the treatment of pain based on strong genetic validation of its role in nociception. In recent years, a number of aryl and acyl ...sulfonamides have been reported as potent inhibitors of Na
1.7, with high selectivity over the cardiac isoform Na
1.5. Herein, we report on the discovery of a novel series of N-(1,2,4triazolo4,3- apyridin-3-yl)methanesulfonamides as selective Na
1.7 inhibitors. Starting with the crystal structure of an acyl sulfonamide, we rationalized that cyclization to form a fused heterocycle would improve physicochemical properties, in particular lipophilicity. Our design strategy focused on optimization of potency for block of Na
1.7 and human metabolic stability. Lead compounds 10, 13 (GNE-131), and 25 showed excellent potency, good in vitro metabolic stability, and low in vivo clearance in mouse, rat, and dog. Compound 13 also displayed excellent efficacy in a transgenic mouse model of induced pain.
We report on a novel series of aryl sulfonamides that act as nanomolar potent, isoform-selective inhibitors of the human sodium channel hNaV1.7. The optimization of these inhibitors is described. We ...aimed to improve potency against hNaV1.7 while minimizing off-target safety concerns and generated compound 3. This agent displayed significant analgesic effects in rodent models of acute and inflammatory pain and demonstrated that binding to the voltage sensor domain 4 site of NaV1.7 leads to an analgesic effect in vivo. Our findings corroborate the importance of hNaV1.7 as a drug target for the treatment of pain.
Abstract
PI3K inhibitors have shown promise for the treatment of anti-estrogen-resistant breast cancers. Current PI3K inhibitor treatment regimens incompletely and transiently inhibit the pathway in ...carcinomas, and are accompanied by adverse effects in patients. We found that different periods of PI3K inhibition (12, 24, 36 h) potentiated anti-estrogen-induced apoptosis and inhibition of proliferation to similar extents in cultured ER+ cells. We thus hypothesized that short-term, complete inhibition of PI3K will have a greater anti-tumor effect and reduced systemic toxicity than chronic, partial inhibition.
Pharmacokinetic analysis of the orally available pan-PI3K inhibitor GDC-0941 at low (100 mg/kg) and high (800 mg/kg) doses in mice revealed that plasma levels peaked after 15-30 min. (18.6 uM and 20.7 uM, respectively), and decreased to a plateau phase after 1 h that was maintained for 8 h with low dose (6.8-10.7 uM) and 23 h with high dose (7.9-15 uM). We performed MCF-7 tumor pharmacokinetic analyses with low and high doses, and with 2 low doses administered 12 hours apart. Tumor GDC-0941 levels peaked after 9 h (1.6 uM with low-dose; 16.9 uM with high-dose). The second low dose increased tumor drug concentrations to 3.2 uM at 9 h after the second dose, compared to 1.6 uM at 9 h after the first dose. After 48 h, tumor drug concentrations decreased to 0 uM with low dose, and to 4.5 uM with high dose.
Mice bearing MCF-7 tumors were treated with fulvestrant (5 mg/wk). Three days later, GDC-0941 was administered to assess pharmacodynamic effects. Phospho-AKT and -S6 levels (markers of PI3K and mTORC1 activities, respectively) were maximally suppressed after 1 h and 3 h of high- and low-dose treatments, respectively, returned to baseline within 16 h after low-dose treatment, and remained suppressed for 36 h following high-dose treatment. PARP cleavage (marker of apoptosis) occurred within 1 h and 3 h of high- and low-dose treatments, and increased over time. Re-treatment of mice with low-dose GDC-0941 after 12 h induced continued inhibition of PI3K and mTORC1 for 9-12 h, suggesting that BID low-dose treatment may be sufficient to continually inhibit PI3K. Comparison of high-dose and low-dose BID tumors showed that these treatments induced similar amounts of PI3K inhibition and PARP cleavage at 21-24 h.
Mice bearing MCF-7 or fulvestrant-resistant T47D/FR tumors were treated with vehicle, fulvestrant, GDC-0941 (100 mg/kg QD 5 d/wk; 100 mg/kg BID 3 d/wk, 800 mg/kg QW), or combinations of fulvestrant and GDC-0941. Drug combinations induced tumor regression, fulvestrant did not affect tumor growth, high-dose GDC-0941 QW slowed tumor growth, and low-dose GDC-0941 QD or BID appreciably inhibited tumor growth. However, there was no significant difference among doses and schedules of GDC-0941 in the context of a fulvestrant backbone in either tumor model. These data suggest that transient/metronomic (QD, BID) and chronic/infrequent (QW) PI3K inhibition may provide similar anti-tumor efficacy in combination with an anti-estrogen. However, these tumor growth data conflict with cell fate data indicating that high-dose GDC-0941 induced much more apoptosis than low-dose GDC-0941. Ongoing studies will reveal how different schedules of PI3K inhibition shape tumor biology.
Citation Format: Wei Yang, Jennifer R Bean, Lloye Dillon, Laurent Salphati, Jodie Pang, Xiaolin Zhang, Michelle Nannini Pepe, Todd W Miller. Understanding pharmacodynamics and consequences of PI3K inhibition in ER+ breast tumors abstract. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-20-03.
Selective block of NaV1.7 promises to produce non-narcotic analgesic activity without motor or cognitive impairment. Several NaV1.7-selective blockers have been reported, but efficacy in animal pain ...models required high multiples of the IC50 for channel block. Here, we report a target engagement assay using transgenic mice that has enabled the development of a second generation of selective Nav1.7 inhibitors that show robust analgesic activity in inflammatory and neuropathic pain models at low multiples of the IC50. Like earlier arylsulfonamides, these newer acylsulfonamides target a binding site on the surface of voltage sensor domain 4 to achieve high selectivity among sodium channel isoforms and steeply state-dependent block. The improved efficacy correlates with very slow dissociation from the target channel. Chronic dosing increases compound potency about 10-fold, possibly due to reversal of sensitization arising during chronic injury, and provides efficacy that persists long after the compound has cleared from plasma.
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•Aconitine-induced pain in IEM mice enables assay for target engagement of hNaV1.7•Acylsulfonamides inhibit pain at low multiples of the IC50 for inhibition of NaV1.7•Improvement versus arylsulfonamides correlates with longer residency time on NaV1.7•Repeat dosing of acylsulfonamides results in a 10-fold decrease in EC50 for analgesia
Bankar et al. show that acylsulfonamides produce analgesic activity at low multiples of the IC50 for inhibition of NaV1.7, in contrast to studies with arylsulfonamides that required >10 times the IC50. The improvement correlates with longer residency time on the target channels.
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