PI3Ka

Conformational Dynamics-Based Design Drives Selectivity for H1047R over WT and Other Isoforms [1]
To improve the potency and PI3Kα H1047R selectivity of [1], we used free energy perturbation calculations and X-ray structures of compounds bound to WT and H1047R to identify analogues. We initially made modifications to the bisaminopyridine core and peripheral toluene and methylamide groups. Bisaminopyridine replacement with an aminoisoxindole core [3] (Supplementary Text) resulted in a 9× IC50 improvement for PI3Kα H1047R and a 2× improvement in H1047R selectivity (Table 1). Further replacement of the toluene and methylamide groups of [3] with 2-chloro-5-fluorophenyl and [1,2,4]triazolo[1,5-a]pyridine-5-carbonitrile groups, respectively, resulted in RLY-2608, which has additional improvement in potency against PI3Kα H1047R and further improvement in selectivity (Table 1). These affinity improvements of the aminoisoxindole core likely stem from more effective space filling of the pocket, including a more extensive interaction with Y1021, along with a stronger hydrogen bond to the backbone of D1018 (1.9 Å in [3] vs. 2.5 Å in [2]). The 5-fluoro substitution in the final compound, RLY-2608, likewise results in improved packing of the hydrophobic pocket in which the phenyl group sits, whereas the 2-chloro substitution introduces a halogen-bond with the backbone carbonyl of E1012 (Fig. 3E). [1]
RLY-2608 is highly selective over isoforms (Supplementary Fig. S5) and does not detectibly inhibit other kinases (Supplementary Table S1). Furthermore, we observe comparable levels of biochemical selectivity for the helical mutants, E542K and E545K, following preincubation with RLY-2608. Also, notably, the pan-mutant selectivity profile of RLY-2608 was comparable whether using liposomes or the soluble lipid substrate dic8-PIP2 (Supplementary Table S3). This newly developed allosteric inhibitor should be able to inhibit the oncogenic activity of mutant kinase while avoiding the negative signaling consequences that come with inhibiting WT and other isoforms.

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RLY-2608 Is Active in Cells against Tail and Helical Domain Mutations [1]
To assess the selectivity of RLY-2608, we utilized an isogenic MCF10A cell line model system, where the expression of a single mutant allele is responsible for signaling. Treatment with an orthosteric molecule inhibits phosphorylation of AKT, a downstream marker of PI3K activation, equally between the different cell lines. However, RLY-2608 preferentially modulates signaling in the mutant cell lines (Fig. 4A). Additionally, we observed mutant selectivity when an endogenously mutant cancer cell line (T47D) was treated alongside a WT, but PI3K-dependent cell line (SKB3), whereas alpelisib equally inhibited both (Fig. 4B). These findings were extended to a panel of cancer cell lines, which harbor mutations spanning three hotspot residues in PIK3CA and represent various indications (Supplementary Fig. S6A and S6B). RLY-2608 led to inhibition of signaling and proliferation, which correlated with the inherent PI3Kα cellular dependency, as assessed by the activity of alpelisib, an orthosteric inhibitor. The ability of RLY-2608 to inhibit kinase domain and helical mutants across a variety of mutated PIK3CA cellular models suggests that these variants exhibit similar coupling between p85α dissociation and cryptic pocket opening.

RLY-2608 Inhibits Tumors in Animal Models with Reduced Impact on Insulin Levels [1]
To assess the activity of RLY-2608 in vivo, we implanted mice with hormone receptor positive (HR+) breast cancer cells (T47D) that are kinase domain mutant (H1047R; Fig. 4C). After tumor growth reached 200 mm3, we orally administered the orthosteric inhibitor alpelisib or RLY-2608. In both models, RLY-2608 (100 mg/kg b.i.d.) led to significant tumor growth inhibition, resulting in stasis or regression similar to treatment with alpelisib. The activity of RLY-2608 was also assessed against HR+ breast cancer cells (MCF7) that are helical domain mutant (E545K; Fig. 4D). Significant tumor regression was observed when mice were treated with RLY-2608 alone at 100 mg/kg. Tumor regression was further potentiated when RLY-2608 was combined with a clinically relevant dose of fulvestrant (estrogen receptor degrader) and in a triple combination with both fulvestrant and ribociclib (a cyclin-dependent kinase 4/6 inhibitor), clinical standard of care in this indication. Additionally, similar tumor regression was observed in ER+/HER2− patient-derived xenografts, mutant in the kinase (ST1056: H1047R) or helical domain (ST986: E542K) using RLY-2608 alone at 100 mg/kg. Improved efficacy was also observed in combination with fulvestrant (Fig. 4E). Notably, these results indicate that RLY-2608 is active in vivo and can inhibit growth for tumors that are dependent on a variety of PI3Kα-activating mutations in a tolerated manner (Supplementary Fig. S7). Because a major complication for alpelisib observed clinically is dose-limiting hyperglycemia and hyperinsulinemia (17), we monitored insulin and insulin C-peptide levels as surrogate markers of glucose, which can be reliably measured in the tumor-bearing animals (Fig. 4F). Although alpelisib exhibited a substantial increase in both markers in the hours after treatment, even at the highest doses of RLY-2608 these markers remained lower than alpelisib treatment, with levels closer to baseline. Collectively, these studies indicate that RLY-2608 can maximally inhibit tumors driven by mutations impacting the regulation of PI3Kα while mitigating the hyperglycemia adverse effect that results from inhibiting WT protein.

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Proof-of-Concept Clinical Activity [1]
In the ongoing dose-escalation portion of the ReDiscover first-in-human study of v (NCT05216432), activity has been observed in patients with both PIK3CA kinase and helical domain mutant breast cancer without adverse events related to WT PI3Kα inhibition.

Case Study 1 [1]
Patient A is a 58-year-old female with metastatic (liver, lung, bone, LN) ER+/PR+, HER2 low breast cancer who progressed after 12 prior lines of therapy (endocrine, chemotherapy, and targeted therapy, including trastuzumab deruxtecan). Genomic analysis by tissue and circulating tumor DNA (ctDNA) showed activating PIK3CA H1047R and E453K mutations. The patient met enrollment eligibility criteria for the monotherapy arm of the study and was assigned to receive RLY-2608 400 mg p.o. b.i.d. Within 4 weeks of therapy, the patient had a marked improvement in bone pain. Furthermore, tumor marker evaluation at 4 weeks compared with baseline showed a reduction of CA15-3 (1598 to 933 U/mL) and CEA (68 to 24 U/mL). Radiographic evaluation after 8 weeks (C3D1) showed partial response with a decrease in multiple liver, lung, and soft-tissue target lesions [−36% in target lesions (TL) compared with baseline by RECIST 1.1].

Case Study 2 [1]
Clinical activity was also demonstrated in patients with helical mutations. Patient B is a 66-year-old female with metastatic (bone, soft tissue) ER+/PR+/HER2− breast cancer who progressed on standard of care with fulvestrant and palbociclib combination therapy. She was referred for clinical trial evaluation. Genomic analysis demonstrated a PIK3CA E542K mutation. The patient met enrollment eligibility criteria for the combination arm of the study and was assigned to receive RLY-2608 600 mg p.o. b.i.d. plus fulvestrant. Tumor marker evaluation at 4 weeks compared with baseline showed a reduction of CA15-3 (192 to 119 U/mL) and CEA (22 to 12 U/mL). Radiographic evaluation after 8 weeks (C3D1) showed partial response with disappearance of the soft-tissue target lesion (−100% in TL compared with baseline by RECIST 1.1). [1]

In summary, these patient cases provide proof of concept that RLY-2608 is a first-in-class isoform- and mutant-selective allosteric inhibitor of PI3Kα, which can induce clinical responses in monotherapy, or in combination with fulvestrant, in breast cancers bearing kinase or helical mutations, with minimal impact on glucose homeostasis.

PI3Kα Enzyme Activity and Inhibition Assays [1]
To 1536-well plates containing the appropriate volume of 10 mmol/L inhibitor in DMSO dispensed from a 384-well low dead-volume plate using an Echo555, 2 nmol/L enzyme in assay buffer (50 mmol/L HEPES pH 7.4, 50 mmol/L NaCl, 6 mmol/L MgCl2, 5 mmol/L DTT, and 0.03% CHAPS in distilled water) was added and allowed to incubate with inhibitors for 2 hours at room temperature. Next, a substrate solution containing 20 μmol/L PI(4,5)P2 diC8 and 200 μmol/L ATP in substrate buffer (50 mmol/L HEPES pH 7.4, 50 mmol/L NaCl, 5 mmol/L DTT, and 0.03% CHAPS in distilled water) was added 1:1 to the enzyme solution, and PIP2 phosphorylation was allowed to proceed for 1 hour at room temperature. The resulting ADP product concentration was then measured using the ADP-Glo Kinase Assay, according to the manufacturer's instructions, with luminescence quantified using an Envision plate reader with a 0.1 second ultrasensitive luminescence protocol. Luminescence values were normalized, per-plate, to neutral and full-inhibition controls using the following equation:
where x is the luminescence value of the sample well, m− is the mean luminescence value of the neutral control wells (DMSO vehicle in assay buffer), and m+ is the mean luminescence value of the inhibitor control wells (GDC-0032 at a concentration of 100 nmol/L). Lastly, normalized concentration-response data were fit to a sigmoidal (four-parameter logistic) linear regression equation using GraphPad Prism to calculate inhibitor IC50 values.

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Liposome Preparation and PI3Kα Inhibition Measurements [1]
The following three lipids, 18:1 (Δ9-Cis) PE (DOPE, Avanti 850725),18:1 PS, and 18:1 PI(4,5)P2, were combined in a 75:23:2 molar ratio, and liposomes with a diameter of 100 nm were prepared via physical extrusion according to manufacturer recommendations. Liposome size and polydispersity were measured by dynamic light scattering on a Zetasizer. PI3Kα enzyme inhibition was measured using ADP-Glo, as described previously, with the PIP2 substrate substituted with a liposome solution that contained PIP2 at a concentration of 25 μmol/L in the enzyme reaction.

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Structure Determination [1]
All crystallographic data were collected at 100 K at the following wavelengths WT 1–1053 apo: 0.979 Å, WT 1–1053 + [2]: 1.000 Å, H1047R 1–1053 apo: 1.033 Å, H1047R 1–1053 + [1]: 1.116 Å, H1047R 1–1053 + [2]: 1.116 Å, H1047R 1–1053 + [3]: 1.033 Å, WT 1–1053 + RLY-2608: 1.116 Å. Data were indexed, integrated, and scaled using autoPROC (Global Phasing). An initial structure was solved by molecular replacement using PDBID 2RD0 as a search model. Subsequent structures were solved by molecular replacement using structures presented herein. For all cases, the molecular replacement was performed using Phaser-MR4), as implemented in Phenix. Final models were generated through iterative rounds of manual building in Coot and ISOLDE with refinement using phenix.refine. The CryoEM model, starting from the WT 1–1053 X-ray structure presented herein, was likewise generated through iterative manual refinement in Coot and ISOLDE and real-space refinement with Phenix.

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Refinement Information [1]
Ramachandran, sidechain, and clashscore statistics were as follows. WT 1–1053 apo: 96.6% favored, 3.2% allowed, 0.16% outliers, 0.86% rotamer outliers, All-atom clashscore: 1.87. WT 1–1053 + [2]: 96.1% favored, 3.8% allowed, 0.1% outliers, 1.3% rotamer outliers, All-atom clashscore: 1.43. WT 1–1053 + RLY-2608: 95.4% favored, 4.66% allowed, 0% outliers, 0.94% rotamer outliers, All-atom clashscore: 1.85. H1047R 1–1053 apo: 96.6% favored, 3.2% allowed, 0.2% outliers, 0.95% rotamer outliers, All-atom clashscore: 2.08. H1047R 1–1053 + [1]: 97.0% favored, 2.9% allowed, 0.1% outliers, 1.36% rotamer outliers, All-atom clashscore: 1.38. H1047R 1–1053 + [2]: 96.0% favored, 4.0% allowed, 0% outliers, 1.02% rotamer outliers, All-atom clashscore: 1.56. H1047R 1–1053 + [3]: 95.6% favored, 4.3% allowed, 0% outliers, 1.79% rotamer outliers, All-atom clashscore: 1.66.

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DNA-Encoded Library Selection and Enrichment Scoring [1]
Full-length PI3Kα (WT, H1047R, E542K, and E545K) bearing N-terminal 8xHis tags were immobilized using Ni-NTA agarose in all selections. Data analysis and enrichment values were obtained as previously described.

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Surface Plasmon Resonance Spectroscopy [1]
Surface plasmon resonance experiments were performed on a Biacore S200 instrument at 25°C using analysis buffer compromised of 20 mmol/L HEPES pH 7.5, 150 mmol/L NaCl, 0.005% Tween20 (v/v), 1 mmol/L MgCl2, 1 mmol/L TCEP and 2% (v/v) DMSO. CM5 chips were preconditioned with 2 ×6 seconds pulses of 100 mmol/L HCl, 50 mmol/L NaOH, and 0.5% (v/v) SDS at flow of 100 μL/minutes. Approximately 6,000 response units (RU) of PI3Kα were immobilized onto the biosensor surface using amine coupling chemistry. Immobilization steps at a flow of 10 μL/minute: 7 minutes 400 mmol/L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/100 mmol/L N-hydroxysuccinimide activation, 1–2 minutes 20 μg/mL PI3Kα in 10 mmol/L MES pH 6.0 with 0.03% CHAPS, 7 minutes 1 M ethanolamine HCl, pH 8.5. Using single-cycle kinetics compound was injected over the PI3Kα surface at increasing concentrations (top concentration 20 μmol/L, 3-fold dilution series, 5 concentrations) at a flow rate of 50 μL/minute. The association was set to 180 seconds followed by 4-minute dissociation. The raw data were processed using Biacore S200 Evaluation Software v1.1, and the data were fitted kinetically to a 1:1 binding model that included a mass transfer limitation. Binding measurements were conducted 2 times to calculate a standard deviation.

Cell Culture [1]
The mutant hemizygous lines were engineered in the immortalized, insulin growth factor-responsive breast epithelial cell line, MCF10A heterozygous background. Cell line generation was performed by targeting the PIK3CA locus via nonhomologous end joining repair of Cas9 nuclease mediated double-strand DNA breaks in one targeting round resulting in a 2 bp deletion in exon 9 of transcript PIK3CA-201 (ENST00000263967.4) leading to a premature stop codon of the WT allele as verified by analysis of cDNA and gDNA.

T47D, NCIH1048, SKBR3, MCF7, ME180, MCF7, EFM19, CAL33, CAL51 (DSMZ), GP2D, OAW42, MFE280, and OVISE were purchased from commercial vendors. Cell lines were authenticated by short-tandem repeat DNA profiling by the cell line bank. They were all provided after testing Mycoplasma-free and were routinely tested while in culture. All cell lines were cultured for less than 1-month post-thaw and used within 10 passages from receipt. Cell lines were cultured at 37°C in 5% CO2 humidified air, in media recommended by the vendor.

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Pharmacodynamic Assay [1]
Cells were seeded in 12 μL of media in a 384-well plate. After 24 hours of incubation at 37°C, 5% CO2, cells were treated with DMSO or test compound in an additional 12.5 nL for 2 hours at 37°C, 5% CO2. Phospho AKT (Ser473) cellular HTRF assay was carried out per the manufacturer's instructions. Data were fit to a sigmoidal four-parameter curve to determine IC50.

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Proliferation [1]
Cells were seeded in 40 μL media into a 384-well, clear-bottom plate, including a day 0 untreated plate to be read after 24 hours. After 24 hours of incubation at 37°C, 5% CO2, cells were treated with DMSO or test compound in additional 40 nL for 120 hours at 37°C, 5% CO2. Following incubation, plates and CellTiter-Glo 2.0 were equilibrated to room temperature for 30 minutes. 30 μL CellTiter-Glo 2.0 was added to all wells. Plates were placed on shaker (protected from light) at room temperature for 30 minutes and read on an EnVision plate reader. Data were normalized by subtracting day 0 values from all treated sample measurements followed by normalization to DMSO controls and conversion to percent viability. A sigmoidal four-parameter curve was used to determine the IC50.

Xenograft Studies [1]
Female Balb/c nude mice at 6–8 weeks of age were inoculated subcutaneously on the flank with 2 × 107 T47D or 1.5 × 107 MCF cells in 0.1 mL of 1:1 mixture of 1640 RPMI: BD Matrigel. For MCF7-inoculated animals, a 17-beta Estradiol tablet (0.5 mg, 90-day release) was implanted subcutaneously in the left flank. Patient-derived xenograft studies were conducted at START. Tumor fragments (∼70 mg) were implanted subcutaneously in athymic nude mice. Animals were supplemented with exogenous estradiol ad libitum via drinking water throughout the study duration. Treatment was initiated when tumors reached a volume of about 200 mm3. Tumors were measured twice weekly in two dimensions using a caliper. Tumor volume was expressed in mm3 using the formula: V = 0.5 a × b2, where a and b are the long and short diameters of the tumor, respectively.

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ReDiscover Phase I/II Study [1]
ReDiscover is a global, open-label, phase I/II, first-in-human study (NCT05216432) of RLY-2608 in advanced cancer patients. Key objectives of phase I are to define the maximum tolerated dose and recommended phase II dose in monotherapy and in combination with fulvestrant, as well as the safety profile, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity in patients with PIK3CA mutations and unresectable or metastatic solid tumors (monotherapy) or metastatic breast cancer (combination).
The key objectives of phase II are to define the overall response rate and duration of response per RECIST 1.1 for patients with advanced PIK3CA mutated solid tumors (monotherapy) or breast cancer (combination). Additional phase II objectives are to assess the safety, pharmacokinetics, and pharmacodynamics of RLY-2608 at the RP2D. The study was initiated in December 2021 and phase I dose escalation is ongoing concurrently with phase II dose expansion at the first selected RP2D of RLY-2608 600 mg b.i.d. with fulvestrant.
The study was conducted in accordance with the Declaration of Helsinki and was reviewed and approved by the institutional review board of each clinical site. Written informed consent was obtained from all patients before study entry. Patients eligible for study participation were ≥18 years old; had Eastern Cooperative Oncology Group performance status 0–1; no prior treatment with PI3Ka inhibitors; no type 1 or 2 diabetes; no uncontrolled CNS metastases and adequate cardiac function. Additional enrollment criteria are provided in Supplementary Appendix S1. RLY-2608 was administered orally, twice daily, in 4-week cycles. Adverse events were graded per Common Terminology Criteria for Adverse Events (CTCAE) 5.0. The response was evaluated per RECIST version 1.1. Levels of ctDNA in plasma were assessed by next-generation sequencing, using 74-gene Guardant360 CDx.

[1]. Discovery and clinical proof-of-concept of RLY-2608, a first-in-class mutant-selective allosteric PI3Ka inhibitor that decouples anti-tumor activity from hyperinsulinemia. Cancer Discov. 2023 Nov 2.

PIK3CA (PI3Kα) is a lipid kinase commonly mutated in cancer, including ∼40% of hormone receptor–positive breast cancer. The most frequently observed mutants occur in the kinase and helical domains. Orthosteric PI3Kα inhibitors suffer from poor selectivity leading to undesirable side effects, most prominently hyperglycemia due to inhibition of wild-type (WT) PI3Kα. Here, we used molecular dynamics simulations and cryo-electron microscopy to identify an allosteric network that provides an explanation for how mutations favor PI3Kα activation. A DNA-encoded library screen leveraging electron microscopy-optimized constructs, differential enrichment, and an orthosteric-blocking compound led to the identification of RLY-2608, a first-in-class allosteric mutant-selective inhibitor of PI3Kα. RLY-2608 inhibited tumor growth in PIK3CA-mutant xenograft models with minimal impact on insulin, a marker of dysregulated glucose homeostasis. RLY-2608 elicited objective tumor responses in two patients diagnosed with advanced hormone receptor–positive breast cancer with kinase or helical domain PIK3CA mutations, with no observed WT PI3Kα-related toxicities. Significance: Treatments for PIK3CA-mutant cancers are limited by toxicities associated with the inhibition of WT PI3Kα. Molecular dynamics, cryo-electron microscopy, and DNA-encoded libraries were used to develop RLY-2608, a first-in-class inhibitor that demonstrates mutant selectivity in patients. This marks the advance of clinical mutant-selective inhibition that overcomes limitations of orthosteric PI3Kα inhibitors. [1]

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Pan-mutant-selective PI3K-alpha Inhibitor RLY-2608 is an orally bioavailable, pan-mutant selective inhibitor of the class I phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit alpha (phosphoinositide 3-kinase alpha; PIK3CA; PI3K p110alpha), with potential antineoplastic activity. Upon oral administration, pan-mutant selective PI3K-alpha inhibitor RLY-2608 selectively targets and allosterically binds to PIK3CA mutated forms, thereby preventing the activity of PIK3CA mutants. This prevents mutant PIK3CA-mediated activation of the PI3K/Akt (protein kinase B)/mammalian target of rapamycin (mTOR) pathway. This results in both apoptosis and growth inhibition in PIK3CA-mutant expressing tumor cells. By specifically targeting PIK3CA mutants, RLY-2608 may be more efficacious and less toxic than PI3K-alpha inhibitors that also inhibit the wild-type (WT) form. Dysregulation of the PI3K/Akt/mTOR pathway is often found in solid tumors and results in the promotion of tumor cell growth, survival, and resistance to chemo- and radio-therapy. PIK3CA, one of the most frequently mutated oncogenes, encodes the p110-alpha catalytic subunit of the class I PI3K. [1]

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The two patient vignettes described here demonstrate clinical proof of concept in metastatic HR+ HER2− breast cancer and the potential to have improved tolerability and efficacy with pan-mutant- and isoform-selective PI3Kα inhibition with the allosteric inhibitor RLY-2608. These two examples highlight patients who achieved confirmed responses with RLY-2608 treatment without adverse events that are associated with WT PI3Kα inhibition. These examples also demonstrate activity in a variety of contexts: in kinase and helical mutations, in earlier and later lines of therapy, and in monotherapy and combination with fulvestrant.

Two decades after the discovery of oncogenic PIK3CA mutations, our collective understating of optimal PI3Kα targeting continues to evolve. Allosteric mutant-selective agents provide a novel advantage of a broader therapeutic index over older generation PI3Kα inhibitors. To this end, the past few years have seen intensifying research activities in developing mutant-selective PI3Kα inhibitors. RLY-2608 is a first-in-class PI3Kα inhibitor that demonstrates mutant-selective efficacy in the clinic. These features have the potential to improve outcomes in patients with PIK3CA-mutant tumors, in monotherapy, and in combination with other targeted therapies. The ongoing phase I/II ReDiscover trial (NCT05216432), studying RLY-2608 in monotherapy, in doublet with fulvestrant, and in triplet with CDK4/6 inhibitor and fulvestrant, will further define the potential benefit of RLY-2608 in patients with advanced PIK3CA-mutant solid tumors and breast cancer.

More broadly, the problem of isoform and mutant selectivity is common across many targets in oncology. Here, we have developed an approach to overcoming this problem via allosteric, rather than orthosteric, inhibitor discovery. In our study, it was essential to use cryoEM and molecular dynamics to discover differences in the conformational dynamics of mutant and WT. Next, we identified inhibitors that impinge on the discovered allosteric network using a DEL screen. Importantly, the conditions of the DEL screen must be biased to magnify the differences between mutant and WT protein. In our case, blocking the orthosteric site was essential to avoid molecules that target the active site, and using full-length protein was essential because the tail plays a key role in the equilibrium governing cryptic pocket opening. Due to the favorable properties of allosteric inhibitors for gaining specificity, we expect the integrated use of cryoEM, MD, and DEL screening that we have leveraged here can help attack many other important targets in the future. [1]

C29H14CLF5N6O2

608.905481815338

608.078

C, 57.20; H, 2.32; Cl, 5.82; F, 15.60; N, 13.80; O, 5.25

2733573-94-7

166822065

White to off-white solid powder

5.5

2

10

4

43

1120

1

C1=CC(=C(C=C1F)[C@H]2C3=C(C=C(C=C3NC(=O)C4=CC(=CC(=C4)F)C(F)(F)F)C5=C(N6C(=NC=N6)C=C5)C#N)C(=O)N2)Cl

VYWRYBZVVSPTQN-SANMLTNESA-N

InChI=1S/C29H14ClF5N6O2/c30-21-3-1-16(31)10-19(21)26-25-20(28(43)40-26)7-13(18-2-4-24-37-12-38-41(24)23(18)11-36)8-22(25)39-27(42)14-5-15(29(33,34)35)9-17(32)6-14/h1-10,12,26H,(H,39,42)(H,40,43)/t26-/m0/s1

N-[(3R)-3-(2-chloro-5-fluorophenyl)-6-(5-cyano-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-1-oxo-2,3-dihydroisoindol-4-yl]-3-fluoro-5-(trifluoromethyl)benzamide

RLY-2608; N-[(3R)-3-(2-chloro-5-fluorophenyl)-6-(5-cyano-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-1-oxo-2,3-dihydroisoindol-4-yl]-3-fluoro-5-(trifluoromethyl)benzamide; N-((3R)-3-(2-chloro-5-fluorophenyl)-6-(5-cyano-(1,2,4)triazolo(1,5-a)pyridin-6-yl)-1-oxo-2,3-dihydroisoindol-4-yl)-3-fluoro-5-(trifluoromethyl)benzamide; PI3Ka Inhibitor RLY-2608; RLY 2608; Pan-mutant PI3Ka Inhibitor RLY-2608; Mutant-selective PI3Ka Inhibitor RLY-2608; ...; 2733573-94-7;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~164 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)