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Supplementary Figure 10 - PDF file 333K, Analysis of multiple EGFR-related signaling pathways in parental NCI-H1975 and COR resistant clones by Western blotting

Supplementary Figure 6 - PDF file 112K, Impact of CO-1686, erlotinib and afatinib administration on mouse body-weight

Supplementary Figure 9 - PDF file 168K, NCI-H1975 parental and CO-1686 resistant clones cluster into distinct groups using an RNA-based EMT signature

Supplementary Figure 3 - PDF file 295K, Activity of CO-1686 against rare lung-cancer associated EGFR mutants

PDF - 252K, Supplemental Table 1. Mass spectrometry on EGFR-L858R/T790M protein. Supplemental Table 2. EGFR modulation in A431, H1975 and HCC827 cells by compound 3. Supplemental Table 3. Kinase selectivity profile of compound 3, afatinib and WZ4002 at 1 ?M. Supplemental Table 4.Multi-dose level PK/PD study in H1975 tumor-bearing mice. Supplemental Figure 1. Signaling inhibition in EGFR wild-type (A431) and mutant EGFR NSCLC cells (H1975 and HCC827). Supplemental Figure 2. Prolonged duration of action on EGFR-L858R/T790M (A) and EGFR-DelE746-A750 (B) proteins. Supplemental Figure 3. Protein degradation (t1/2) of EGFR-L858R/T790M (A) and EGFR-DelE746-A750 (B) in H1975 and HCC827 cells, respectively. Supplemental Figure 4. Prolonged duration of action on EGFR-DelE746-A750 protein by erlotinib. Supplemental Figure 5. Time-dependent inhibition in EGFR mutant H1975 (A) and HCC827 (B) cells.

Supplementary Table 1 - PDF file 114K, Kinase selectivity profiling of CO-1686

Patients with non–small cell lung carcinoma (NSCLC) with activating mutations in epidermal growth factor receptor (EGFR) initially respond well to the EGFR inhibitors erlotinib and gefitinib. However, all patients relapse because of the emergence of drug-resistant mutations, with T790M mutations accounting for approximately 60% of all resistance. Second-generation irreversible EGFR inhibitors are effective against T790M mutations in vitro, but retain affinity for wild-type EGFR (EGFRWT). These inhibitors have not provided compelling clinical benefit in T790M-positive patients, apparently because of dose-limiting toxicities associated with inhibition of EGFRWT. Thus, there is an urgent clinical need for therapeutics that overcome T790M drug resistance while sparing EGFRWT. Here, we describe a lead optimization program that led to the discovery of four potent irreversible 2,4-diaminopyrimidine compounds that are EGFR mutant (EGFRmut) selective and have been designed to have low affinity for EGFRWT. Pharmacokinetic and pharmacodynamic studies in H1975 tumor–bearing mice showed that exposure was dose proportional resulting in dose-dependent EGFR modulation. Importantly, evaluation of normal lung tissue from the same animals showed no inhibition of EGFRWT. Of all the compounds tested, compound 3 displayed the best efficacy in EGFRL858R/T790M-driven tumors. Compound 3, now renamed CO-1686, is currently in a phase I/II clinical trial in patients with EGFRmut-advanced NSCLC that have received prior EGFR-directed therapy. Mol Cancer Ther; 13(6); 1468–79. ©2014 AACR.

Supplementary Figure 7 - PDF file 352K, H&E and immunohistochemical (Ki67) staining of tumors derived from EGFR mutant GEM models

Supplementary Figure 8 - PDF file 215K, Photomicrographs of CO-1686 resistant NCI-H1975 cell (COR) clones and quantification of EGFR siRNA knockdown in COR clones

Supplementary Figure 4 - PDF file 103K, Pharmacokinetic analysis of CO-1686 in nu/nu mice

Supplementary Materials and Methods - PDF file 179K, Supplemental materials and methods for Walter et al. manuscript

Supplementary Figure 5 - PDF file 134K, Schedule dependence of CO-1686 antitumor activity in NCI-H1975 mode

Supplementary Table 2 - PDF file 113K, Mutant EGFR cell panel profiling of CO-1686

PDF - 91K, Supplemental Materials and Methods and Legends for Supplementary Figures 1 through 5.

Supplementary Table 3 - PDF file 119K, Comparison of differentially expressed genes in COR cell lines with the EMT signature of Byers et al. (2013)

Supplementary Figure 2 - PDF file 111K, CO-1686 inhibits cell growth in EGFR-mutant erlotinib-resistant cell lines

Introduction: The chromatin factor BRD9 is a genetic dependency in some cancers, often referred to as SMARCB1-perturbed cancers. Two types of genetic alterations result in SMARCB1 perturbation: SS18-SSX gene fusion and SMARCB1 loss-of-function mutations. In synovial sarcoma, a rare and aggressive soft tissue malignancy comprising approximately 10% of all soft tissue sarcomas, the presence of the SS18-SSX fusion gene drives the disruption of SMARCB1 function and leads to a synthetic lethal dependence on BRD9. In SMARCB1-null solid tumors, for example malignant rhabdoid tumors (MRT), poorly differentiated chordomas, and epithelioid sarcomas, the absence of SMARCB1 protein results in a similar BRD9 dependence. Thus, in SMARCB1-perturbed cancers, including synovial sarcoma and SMARCB1-null cancers, degradation of BRD9 is hypothesized to result in an anticancer effect. CFT8634 is an orally bioavailable heterobifunctional degrader that induces ternary complex formation with BRD9 and an E3 ligase, leading to the ubiquitination of BRD9 and its subsequent degradation by the proteasome. Results: Here we describe the chemical structure of CFT8634 and an overview of the medicinal chemistry path leading to its discovery. In vitro, CFT8634 promotes rapid, potent, deep, and selective degradation of BRD9 with a half-maximal degradation concentration (DC50) of 2 nM in a synovial sarcoma cell line. In long-term growth assays, CFT8634 is effective at impairing cell growth in a concentration-dependent manner specifically in SMARCB1-perturbed contexts. In vivo, oral dosing of CFT8634 in xenograft models of SMARCB1-perturbed cancers leads to robust and dose-dependent degradation of BRD9, which translates to significant and dose-dependent inhibition of tumor growth in preclinical xenograft models. Conclusion: The preclinical data presented herein support the clinical development of CFT8634 for the treatment of synovial sarcoma and SMARCB1-null tumors. Citation Format: Katrina L. Jackson, Roman V. Agafonov, Mark W. Carlson, Prasoon Chaturvedi, David Cocozziello, Kyle Cole, Richard Deibler, Scott J. Eron, Andrew Good, Ashley A. Hart, Minsheng He, Christina S. Henderson, Hongwei Huang, Marta Isasa, R. Jason Kirby, Linda Lee, Michelle Mahler, Moses Moustakim, Christopher G. Nasveschuk, Michael Palmer, Laura L. Poling, Roy M. Pollock, Matthew Schnaderbeck, Stan Spence, Gesine K. Veits, Jeremy L. Yap, Ning Yin, Rhamy Zeid, Adam S. Crystal, Andrew J. Phillips, Stewart L. Fisher. The discovery and characterization of CFT8634: A potent and selective degrader of BRD9 for the treatment of SMARCB1-perturbed cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND09.

Introduction: CFT7455 is a highly potent and novel Ikaros Family Zinc Finger Protein 1/3 (IKZF1/3) degrader. In xenograft models, CFT7455 has more potent IKZF1/3 degradation compared to other degraders. Early observations from the FIH clinical trial (NCT04756726) along with supporting translational studies are presented here. Methods: Pre-clinical studies comparing CFT7455 and CC-92480 in both in vitro and xenograft models were performed. The pre-clinical studies’ results coincided with observations from the on-going clinical trial. The clinical trial is an open-label, Phase 1/2, multi-center, FIH study in heavily pretreated relapsed/refractory (R/R) MM and non-Hodgkin’s lymphoma (NHL) patients evaluating safety, tolerability, and PK of CFT7455. Eligible MM patients are R/R to therapy and are not candidates for regimens known to provide clinical benefit. A starting dose of 50 μg QD 21 days on/7 days off (21/7) in 28-day cycles was administered. Results: CFT7455 and CC-92480 showed similar cereblon binding profiles and in vitro IKZF1/3 degradation kinetics, translating into sub-nanomolar GI50 values in proliferation assays across a panel of MM cell lines. In the NCI-H929 xenograft model, 100 μg/kg/day of CFT7455 resulted in durable tumor regressions, while 1000 μg/kg/day of CC-92480 gave tumor stasis. Similar results were seen in a systemic model of MM, MM1.S. Both compounds achieved >95% IKZF3 degradation in tumors 4h post dose. At 48h post dose, CFT7455 was more effective than CC-92480 in maintaining IKZF3 degradation (65% vs. 6% respectively). When levels of CFT7455 and CC-92480 in plasma and tumor were compared, CFT7455 concentrations were > DC80 in tumor 48h post dose, while CC-92480 levels were undetectable in tumor and plasma, demonstrating CFT7455 has longer exposure resulting in sustained IKZF1/3 degradation in pre-clinical models. In cohort A, 5 heavily pre-treated MM patients (pts) received single agent CFT7455. 4 pts have received at least 3 cycles, with 2 pts receiving 5 cycles. Neutropenia (grade 4) was observed in 3/5 pts without coincident fever or infection. Additionally, a 2-4 fold accumulation in plasma CFT7455 exposure at steady state was observed. Early pharmacodynamic (PD) data demonstrates deep persistent degradation of IKZF3 (~100%) and serum free light chain reduction (up to 72%) in response to treatment. Stable disease has been observed in 34 pts, suggesting clinical benefit. Conclusions: While CFT7455 showed clinical benefit at 50 ug with deep target degradation, neutropenia was dose limiting. PK/PD modeling suggests alternative dosing regimens may result in increased tolerability with preserved efficacy, and evaluation of them is underway. Updated results will be presented at the meeting. Citation Format: Sagar Lonial, Shambavi Richard, Jeffrey V Matous, Andrew J. Yee, Urvi Shah, Neha Mehta-Shah, Thomas Martin, Eli Muchtar, Sikander Ailawadhi, Paul G. Richardson, Manisha Bhutani, Samantha Perino, Jason Kirby, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, Matthew Schnaderbeck, Michael R. Palmer, Cathleen Gorman, Oliver Schoenborn-Kellenberger, Amanda Hoerres, Stewart L. Fisher, Roy M. Pollock, Adam Crystal, Michelle Mahler, Jesus Bardeja. Pharmacokinetic (PK) profile of a novel IKZF1/3 degrader, CFT7455, enables significant potency advantage over other IKZF1/3 degraders in models of multiple myeloma (MM) and the results of the initial treatment cohort from a first-in-human (FIH) phase 1/2 study of CFT7455 in MM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT186.

Introduction: Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 inhibits the growth of multiple myeloma (MM) cells, confirming their dependency on IKZF1/3. IMiDs (lenalidomide, pomalidomide) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. However, most patients treated with lenalidomide or pomalidomide eventually develop progressive disease due to acquired resistance, underscoring the unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high binding affinity to cereblon (CRBN), rapid and deep IKZF1/3 degradation, and potent dose-dependent efficacy in vivo. Results: A series of novel benzoimidazolone-based CRBN ligands with potent binding affinity were discovered and their binding modes were informed by CRBN co-crystal structures. Although the benzoimidazolone-based CRBN binders did not exhibit IKZF1/3 degradation activity, structural insights into their unique binding modes and knowledge of the IKZF1/3 degradation pharmacophore were combined to enable identification of a novel benzoisoindolone-based ligand that exhibited a 10-fold potency increase in biochemical CRBN binding and a 30-fold potency increase in H929 MM cell growth inhibition when compared to lenalidomide. Additional rounds of structure-based drug design, degradation and phenotypic profiling led to the discovery of CFT7455, a highly potent, selective and orally bioavailable degrader of IKZF1/3. CFT7455 demonstrated an 800 and 1600-fold improvement in CRBN binding compared to pomalidomide in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced >75% degradation of IKZF1 within 1.5 hrs. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines, as well as H929 cells made resistant to IMiDs. In vivo, CFT7455 catalyzed deep and durable degradation of IKZF3, translating into potent antitumor activity in multiple myeloma xenograft models. CFT7455 also retained its activity in models resistant or insensitive to clinically approved IMiDs as single agent or in combination with standard of care agent dexamethasone. Conclusion: Overall, CFT7455 is a next generation IKZF1/3 degrader, with improved potency and anticancer efficacy in preclinical models compared to existing IMiDs. These features make CFT7455 an exciting drug candidate, as a single agent or for use in combination. CFT7455 is currently being studied in a Ph1 clinical trial. Citation Format: James A. Henderson, Scott J. Eron, Andrew Good, R Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Ashley A. Hart, Christina S. Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Adam S. Crystal, Roy M. Pollock, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. The discovery and characterization of CFT7455: A potent and selective degrader of IKZF1/3 for the treatment of relapsed/refractory multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND13.

In addition to the therapeutic applicability of targeted protein degradation (TPD), the modality also harbors unique properties that enable the development of innovative chemical biology tools to interrogate complex biology. TPD offers an all-chemical strategy capable of the potent, durable, selective, reversible, and time-resolved control of the levels of a given target protein in both in vitro and in vivo contexts. These properties are particularly well-suited for enabling the precise perturbation of a given gene to understand its biology, identify dependencies/vulnerabilities in disease contexts, and as a strategy to control gene therapies. To leverage these elegant properties, we developed the AchillesTag (aTAG) degradation system to serve as a tool in target identification and validation efforts. The aTAG degradation system provides a novel degradation tag based on the MTH1 protein paired with three fully validated bifunctional degraders with both in vitro and in vivo applicability. We catalog the development of the aTAG system from selection and validation of the novel MTH1 aTAG, alongside a comprehensive SAR campaign to identify high performing tool degraders. To demonstrate the utility of the aTAG system to dissect a complex biological system, we apply the technology to the control of Chimeric Antigen Receptor (CAR) activity. Using aTAG, we demonstrate the ability to potently and selectively control CAR protein levels, resulting in the exquisite rheostat control of CAR mediated T-cell activity. Furthermore, we showcase the in vivo application of the system via degradation of the aTAG-fused CAR protein in a human xenograft model. The aTAG degradation system provides a complete chemical biology tool to aid foundational target validation efforts that inspire drug discovery campaigns towards therapeutic applicability.

Targeted protein degraders are an emerging modality. Their properties fall outside the traditional small-molecule property space and are in the 'beyond rule of 5' space. Consequently, drug discovery programs focused on developing orally bioavailable degraders are expected to face complex drug metabolism and pharmacokinetics (DMPK) challenges compared with traditional small molecules. Nevertheless, little information is available on the DMPK optimization of oral degraders. Therefore, in this review, we discuss our experience of these DMPK optimization challenges and present methodologies and strategies to overcome the hurdles dealing with this new small-molecule modality, specifically in developing oral degraders to treat cancer.

Introduction Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 in MM cells inhibits growth, confirming their dependency on IKZF1/3. IMiDs (lenalidomide[len], pomalidomide[pom]) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. Most patients treated with len or pom eventually develop progressive disease due to acquired resistance, underscoring an unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high affinity cereblon (CRBN) binding, rapid IKZF1/3 degradation, and potent in vivo efficacy. Results CFT7455 demonstrated an 800 to 1600-fold improvement in CRBN binding compared to pom in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced >75% degradation of IKZF1 within 1.5 hr. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines as well as H929 cells made resistant to IMiDs. In RPMI-8226 MM mice xenografts, CFT7455 (0.1 mg/kg/day) resulted in deep, durable degradation of IKZF3 (21% and 9.5% of vehicle levels at 4 and 24 hr, respectively). Protein levels for IRF4, an essential TF in MM, declined over 7 days with daily CFT7455 treatment to 8% of vehicle levels. Dose dependent efficacy ranged from 0.003-0.1 mg/kg/day, with tumor regression evident at doses ≥0.01 mg/kg/day. Pom was inactive in this model at a human equivalent dose (3 mg/kg/day), with no observed tumor shrinkage in these mice following 17 days of dosing. Switching pom treatment to CFT7455 (0.1 mg/kg/day) on Day 18 led to tumor regression in 67% of animals on Day 28 and 100% tumor regression on Day 35, demonstrating that CFT7455 penetrates large tissues and is efficacious in rapidly growing, IMiD resistant tumors. In the H929 tumor xenograft model, administration of CFT7455 (0.1 mg/kg/day) promoted tumor regression (95% tumor growth inhibition by 7 days); dosing was stopped after 21 days. On Day 63, half the tumors remained below their starting tumor volume. Additionally, CFT7455 demonstrated durable tumor regression in the aggressive MM1.S systemic MM tumor model. In mice bearing RPMI-8226 xenograft tumors, the combination of CFT7455 (QD) and dexamethasone (QW) was more active, and demonstrated a significant survival improvement, compared to either agent alone. Conclusions CFT7455 is a highly potent, catalytic degrader of IKZF1/3, with marked antitumor activity as a single agent and in combination with dexamethasone. Importantly, CFT7455 retains activity in models resistant or insensitive to IMiDs. These results warrant investigation of CFT7455 as a therapeutic approach for MM and a clinical study is planned. Citation Format: James A. Henderson, R. Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Scott J. Eron, Andrew Good, Ashley A. Hart, Christina Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Roy M. Pollock, Adam S. Crystal, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. CFT7455: A novel, IKZF1/3 degrader that demonstrates potent tumor regression in IMiD-resistant multiple myeloma (MM) xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB007.

In this study, we have developed a type B gelatin nanoparticle based siRNA delivery system for silencing of intestinal transglutaminase-2 (TG2) and interleukin-15 (IL-15) genes in cultured human intestinal epithelial cells (Caco-2) and murine alveolar macrophage cells (J774A.1). Small interfering RNA (siRNA) targeting the TG2 or IL-15 gene was encapsulated within gelatin nanoparticles using ethanol-water solvent displacement method. Size, charge, and morphology of gelatin nanoparticles were evaluated using a Zetasizer instrument and transmission electron microscopy. siRNA encapsulation efficiency was determined using an siRNA specific stem-loop quantitative polymerase chain reaction (qPCR) assay. Cellular uptake of siRNA-containing gelatin nanoparticles was determined using fluorescent microscopy and stem-loop qPCR assay. siRNA loading in the RISC (RNA-induced silencing complex) was determined by immunoprecipitation of argonaute 2 (AGO2) protein followed by stem-loop qPCR for siRNA quantification. Gene expression analysis of TG2, IL-15, and the proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), was performed using qPCR assays. Efficacy of silencing TG2 and IL-15 knockdown was evaluated in an in vitro model of celiac disease by utilizing immunogenic α-gliadin peptide p31-43 in cultured J774A.1 cells. siRNA-containing gelatin nanoparticles were spherical in shape with mean particle size and charge of 217 ± 8.39 nm and -6.2 ± 0.95 mV, respectively. siRNA loading efficiency within gelatin nanoparticles was found to be 89.3 ± 3.05%. Evaluations of cellular uptake using fluorescent microscopy showed rapid internalization of gelatin nanoparticles within 2 h of dosing, with cytosolic localization of delivered siRNA in Caco-2 cells. Gelatin nanoparticles showed greater intracellular siRNA exposure with a longer half-life, when compared to Lipofectamine-mediated siRNA delivery. Approximately 0.1% of total intracellular siRNA was associated in the RISC complex. A maximum knockdown of 60% was observed at 72 h post siRNA treatment for both TG2 and IL-15 genes, which corresponded to ∼200 copies of RISC associated siRNA. Further, efficacy of gelatin nanoparticle mediated knockdown of TG2 and IL-15 mRNA was tested in an in vitro model of celiac disease. Significant suppression in the levels of proinflammatory cytokines (TNF-α and IFN-γ) was observed in p31-43 stimulated J774A.1 cells upon either IL-15 or IL-15 + TG2 siRNA treatment. The results from this study indicate that gelatin nanoparticle mediated TG2 and IL-15 siRNA gene silencing is a very promising approach for the treatment of celiac disease.

Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in autoimmune disease and B cell malignancies. Bruton's tyrosine kinase (Btk) plays a crucial role in B cell development and activation through the BCR signaling pathway and represents a new target for diseases characterized by inappropriate B cell activity. N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292) is a highly selective, covalent Btk inhibitor and a sensitive and quantitative assay that measures CC-292-Btk engagement has been developed. This translational pharmacodynamic assay has accompanied CC-292 through each step of drug discovery and development. These studies demonstrate the quantity of Btk bound by CC-292 correlates with the efficacy of CC-292 in vitro and in the collagen-induced arthritis model of autoimmune disease. Recently, CC-292 has entered human clinical trials with a trial design that has provided rapid insight into safety, pharmacokinetics, and pharmacodynamics. This first-in-human healthy volunteer trial has demonstrated that a single oral dose of 2 mg/kg CC-292 consistently engaged all circulating Btk protein and provides the basis for rational dose selection in future clinical trials. This targeted covalent drug design approach has enabled the discovery and early clinical development of CC-292 and has provided support for Btk as a valuable drug target for B-cell mediated disorders.

PI3Kα has been identified as an oncogene in human tumors. By use of rational drug design, a targeted covalent inhibitor 3 (CNX-1351) was created that potently and specifically inhibits PI3Kα. We demonstrate, using mass spectrometry and X-ray crystallography, that the selective inhibitor covalently modifies PI3Kα on cysteine 862 (C862), an amino acid unique to the α isoform, and that PI3Kβ, -γ, and -δ are not covalently modified. 3 is able to potently (EC(50) < 100 nM) and specifically inhibit signaling in PI3Kα-dependent cancer cell lines, and this leads to a potent antiproliferative effect (GI(50) < 100 nM). A covalent probe, 8 (CNX-1220), which selectively bonds to PI3Kα, was used to investigate the duration of occupancy of 3 with PI3Kα in vivo. This is the first report of a PI3Kα-selective inhibitor, and these data demonstrate the biological impact of selectively targeting PI3Kα.

To test the hypothesis that human milk fucosyloligosaccharides are part of an innate immune system, we addressed whether their expression (1) depends on maternal genotype and (2) protects breastfed infants from pathogens. Thus the relationship between maternal Lewis blood group type and milk oligosaccharide expression and between variable oligosaccharide expression and risk of diarrhea in their infants was studied in a cohort of 93 Mexican breastfeeding mother-infant pairs. Milk of the 67 Le(a-b+) mothers contained more LNF-II (Le(a)) and 3-FL (Le(x)) (oligosaccharides whose fucose is exclusively alpha 1,3- or alpha 1,4-linked) than milk from the 24 Le(a-b-) mothers; milk from Le(a-b-) mothers contained more LNF-I (H-1) and 2'-FL (H-2), whose fucose is exclusively alpha 1,2-linked. The pattern of oligosaccharides varied among milk samples; in each milk sample, the pattern was summarized as a ratio of 2-linked to non-2-linked fucosyloligosaccharides. Milks with the highest ratios were produced primarily by Le(a-b-) mothers; those with the lowest ratios were produced exclusively by Le(a-b+) mothers (p

Patients with non–small cell lung carcinoma (NSCLC) with activating mutations in epidermal growth factor receptor (EGFR) initially respond well to the EGFR inhibitors erlotinib and gefitinib. However, all patients relapse because of the emergence of drug-resistant mutations, with T790M mutations accounting for approximately 60% of all resistance. Second-generation irreversible EGFR inhibitors are effective against T790M mutations in vitro, but retain affinity for wild-type EGFR (EGFRWT). These inhibitors have not provided compelling clinical benefit in T790M-positive patients, apparently because of dose-limiting toxicities associated with inhibition of EGFRWT. Thus, there is an urgent clinical need for therapeutics that overcome T790M drug resistance while sparing EGFRWT. Here, we describe a lead optimization program that led to the discovery of four potent irreversible 2,4-diaminopyrimidine compounds that are EGFR mutant (EGFRmut) selective and have been designed to have low affinity for EGFRWT. Pharmacokinetic and pharmacodynamic studies in H1975 tumor–bearing mice showed that exposure was dose proportional resulting in dose-dependent EGFR modulation. Importantly, evaluation of normal lung tissue from the same animals showed no inhibition of EGFRWT. Of all the compounds tested, compound 3 displayed the best efficacy in EGFRL858R/T790M-driven tumors. Compound 3, now renamed CO-1686, is currently in a phase I/II clinical trial in patients with EGFRmut-advanced NSCLC that have received prior EGFR-directed therapy. Mol Cancer Ther; 13(6); 1468–79. ©2014 AACR.

Designing selective inhibitors of proteases has proven problematic, in part because pharmacophores that confer potency exploit the conserved catalytic apparatus. We developed a fundamentally different approach by designing irreversible inhibitors that target noncatalytic cysteines that are structurally unique to a target in a protein family. We have successfully applied this approach to the important therapeutic target HCV protease, which has broad implications for the design of other selective protease inhibitors.

Human milk is rich in oligosaccharides, some of which inhibit toxins and pathogens involved in diseases of infants. To investigate qualitative and quantitative individual variation of human milk oligosaccharides, a sensitive method for routine identification and quantification of intact milk oligosaccharides was developed and applied to milk samples from 50 donors. The isolated, reduced neutral oligosaccharide fractions were perbenzoylated, resolved by reversed-phase HPLC, and detected at 229 nm. This method resolves most structural isomers and does not require stringent removal of lactose. Peaks were detected at the low nanogram (pmol) level and peak areas were linear from 1 to 1000 micrograms for a standard oligosaccharide. Oligosaccharide samples equivalent to 1 microliter of human milk give optimum chromatographic separation and resolution. The method gives quantitative results comparable to those obtained with classic total sugar analyses, and has an average coefficient of variation of 13%. The 12 major peaks in human milk coeluted with authentic oligosaccharide standards ranging from tri- to octasaccharides, and their identities were confirmed by mass spectrometry. Significant individual variation exists in oligosaccharide profiles; almost 70% of samples contained 2'-fucosyllactose and lacto-N-fucopentaose I as the major oligosaccharides; for the remainder, the major oligosaccharides were 3-fucosytlactose and lacto-N-fucopentaose-II or lacto-N-fucopentaose-III. This method can be used to investigate the extent and biological significance of oligosaccharide variation in human milk.

Patients with non–small cell lung cancer (NSCLC) with activating EGF receptor (EGFR) mutations initially respond to first-generation reversible EGFR tyrosine kinase inhibitors. However, clinical efficacy is limited by acquired resistance, frequently driven by the EGFRT790M mutation. CO-1686 is a novel, irreversible, and orally delivered kinase inhibitor that specifically targets the mutant forms of EGFR, including T790M, while exhibiting minimal activity toward the wild-type (WT) receptor. Oral administration of CO-1686 as single agent induces tumor regression in EGFR-mutated NSCLC tumor xenograft and transgenic models. Minimal activity of CO-1686 against the WT EGFR receptor was observed. In NSCLC cells with acquired resistance to CO-1686 in vitro, there was no evidence of additional mutations or amplification of the EGFR gene, but resistant cells exhibited signs of epithelial–mesenchymal transition and demonstrated increased sensitivity to AKT inhibitors. These results suggest that CO-1686 may offer a novel therapeutic option for patients with mutant EGFR NSCLC. Significance: We report the preclinical development of a novel covalent inhibitor, CO-1686, that irreversibly and selectively inhibits mutant EGFR, in particular the T790M drug-resistance mutation, in NSCLC models. CO-1686 is the first drug of its class in clinical development for the treatment of T790M-positive NSCLC, potentially offering potent inhibition of mutant EGFR while avoiding the on-target toxicity observed with inhibition of the WT EGFR. Cancer Discov; 3(12); 1404–15. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 1317

The neutral glycolipids of milk, a small fraction of the total lipids, are of potential biological importance. The simultaneous quantitation of the simple (less than five sugars) glycosphingolipids of human milk samples was achieved by high-pressure liquid chromatography. The samples, representing various stages of lactation, parity of the nursing child, and age of the mother, contained similar glycolipid patterns, but with varying individual glycolipid concentrations. The cerebrosides are major glycosphingolipids of human milk: the non-hydroxylated fatty acid (NFA)-containing species are present at 1.8 microM, and the hydroxylated and/or short-chain fatty acid-containing species (HFA) are present at 1.7 microM; NFA lactosylceramide is present at 931 nM. The cerebrosides appear to be primarily galactosylceramides (galactocerebrosides); glucosylceramides (glucocerebrosides) are a minor component. Globotriaosylceramide (Gb3) is found at 50 nM and 73 nM for the NFA and HFA species, respectively, while globoside (Gb4) is found at 45 nM and 46 nM for the NFA and HFA species. Bovine milk glycosphingolipids differ from those of human milk, with bovine milk containing mainly NFA glucosylceramide (8 microM) and NFA lactosylceramide (17 microM); bovine milk contains little Gb3 or Gb4.

Two fucose-containing pentasaccharides were isolated from goat's milk using a Bio-Gel P-4 column, followed by reverse-phase C-18 high-performance liquid chromatography. The structures of the pentasaccharides as characterized by high-field 1H-n.m.r. spectroscopy and enzymatic digestion were found to be [see text] and [see text].

A key liability in transitioning a new chemical entity (NCE) to a development candidate is NCE-related inhibition (or induction) of cytochrome P450 enzymes, a superfamily of heme-containing oxygenases that are the major route of first-pass metabolism for the majority of marketed drugs. The drawback of a drug/NCE that modulates CYP450 enzyme activity occurs when the compound is co-administered with another drug that relies on the same P450 enzyme for its metabolism. This could result in overdose of the second drug in the case of inhibition, or more rapid metabolism of one or both drugs accompanied by loss of efficacy in the case of enzyme induction. Screening for the inhibition of CYP450 enzymes is now routine in the early stages of evaluating NCEs. This unit describes two inhibition assays using traditional and fluorescent substrates.

Introduction: Uncontrolled complement activation plays a pivotal role in a variety of disorders such as PNH and aHUS. ALN-CC5 is a subcutaneous (SC) investigational RNAi therapeutic targeting complement C5 (C5). In preclinical studies, ALN-CC5 has demonstrated decreased terminal complement activity. Based on the literature, preventing the generation of the terminal complex protects against intravascular hemolysis and complement-mediated tissue damage. The purpose of this study is to evaluate the safety and tolerability of ALN-CC5 in normal healthy volunteers. Material and methods: A multi-centered, placebo controlled, double blind phase 1 clinical study in healthy volunteers is ongoing. Several cohorts of healthy volunteers in Part A, a single ascending dose study and Part B, a weekly multiple ascending dose study have been completed. Primary endpoints are safety and tolerability. Secondary endpoints are pharmacokinetics, reduction of circulating C5, reduction in hemolytic, CAP and CCP activity. Results: In Part A, 20 healthy volunteers were randomized (1:3) to placebo or single SC dose of 50, 200, 400, 600 or 900mg of ALN-CC5 and followed for at least 70 days. In Part B, 12 healthy volunteers were randomized (1:3) to placebo or 5 weekly doses of 100, 200 or 400mg of ALN-CC5. No SAEs or study discontinuations occurred and overall ALN-CC5 was considered safe and generally well tolerated. A dose dependent and 94% mean maximum C5 knockdown was achieved following weekly administration. Updated safety and tolerability data as well as C5 knockdown, and changes in CAP, CCP and hemolytic activity from the study will be presented. Conclusion: Collectively, these initial results suggest that the use of a novel RNAi therapeutic targeting C5 is a promising approach for inhibiting complement in PNH, aHUS and other complement-mediated diseases. The subcutaneous route of administration and infrequent dosing make this a potentially encouraging therapy. Disclosures Hill: Alnylam: Consultancy. Off Label Use: ALN-CC5 is an investigational RNAi therapeutic targeting complement C5.. Borodovsky:Alnylam Pharmaceuticals: Employment, Equity Ownership. Kawahata:Alnylam Pharmaceuticals: Employment, Equity Ownership. Mclean:Alnylam Pharmaceuticals: Employment, Equity Ownership. Powell:Alnylam Pharmaceuticals: Employment, Equity Ownership. Chaturvedi:Alnylam Pharmaceuticals: Employment, Equity Ownership. Warner:Alnylam Pharmaceuticals: Employment, Equity Ownership. Garg:Alnylam Pharmaceuticals: Employment, Equity Ownership. Sorensen:Alnylam Pharmaceuticals: Employment, Equity Ownership.

Objective To determine the association between maternal milk levels of 2-linked fucosylated oligosaccharide and prevention of diarrhea as a result of Campylobacter, caliciviruses, and diarrhea of all causes in breast-fed infants. Study design Data and banked samples were analyzed from 93 breast-feeding mother-infant pairs who were prospectively studied during 1988-1991 from birth to 2 years with infant feeding and diarrhea data collected weekly; diarrhea was diagnosed by a study physician. Milk samples obtained 1 to 5 weeks postpartum were analyzed for oligosaccharide content. Data were analyzed by Poisson regression. Results Total 2-linked fucosyloligosaccharide in maternal milk ranged from 0.8 to 20.8 mmol/L (50%-92% of milk oligosaccharide). Moderate-to-severe diarrhea of all causes (n = 77 cases) occurred less often ( P = .001) in infants whose milk contained high levels of total 2-linked fucosyloligosaccharide as a percent of milk oligosaccharide. Campylobacter diarrhea (n = 31 cases) occurred less often ( P = .004) in infants whose mother's milk contained high levels of 2′-FL, a specific 2-linked fucosyloligosaccharide, and calicivirus diarrhea (n = 16 cases) occurred less often ( P = .012) in infants whose mother's milk contained high levels of lacto- N -difucohexaose (LDFH-I), another 2-linked fucosyloligosaccharide. Conclusion This study provides novel evidence suggesting that human milk oligosaccharides are clinically relevant to protection against infant diarrhea.

Human milk contains a variety of bioactive agents, including oligosaccharides, which are part of the innate defense system (Hanson et al. 1991; Zopf & Roth 1996; Newburg et al. 1998; Erney et al. 2000; Hamosh 2001; Chaturvedi et al. 2001). Oligosaccharides are the third largest solid constituent of human milk after lactose and lipids (Stahl et al. 1994; Zopf & Roth 1996; Erney et al. 2000; Chaturvedi et al. 2001; Hamosh 2001). α 1,2-Linked fucosylated oligosaccharides in human milk have been shown to inhibit host ligand binding to Campylobacter, caliciviruses, and other enteric pathogens (Newburg et al. 1990; Crane et al. 1994; Marionneau et al. 2002; Ruiz-Palacios et al. 2003; Huang et al. 2003). Significant variation exists between mothers in the quantity of α 1,2-linked fucosylated oligosaccharides in their milk. We conducted this study in breastfed infants to determine the clinical relevance of the observed variation in human milk fucosylated oligosaccharides in relation to protection offered infants against diarrhea due to Campylobacter jejuni and caliciviruses.

Sulfated glycolipids (SG) and glycosaminoglycans (GAG) present on the surface of colonic, vaginal epithelial, and neuroglial cells bind to HIV gpl20, suggesting that these glycoconjugates may have a role in HIV infection. The major goal of our study was to test the ability of SG and GAG from human milk to inhibit HIV-1 infection in vitro. SG and GAG were purified from pooled human milk and characterized by high pressure liquid chromatography (HPLC) and mass spectrometry. Eight different preparations of SG and one of GAG were tested for inhibition of infection. Two laboratory isolates, HIV-1Ada (macrophage-tropic virus) and HIV-1SF2 (lymphotropic virus), were used for inhibition assays using peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM). Inhibition assays were performed by preincubation of serial dilutions of glycoconjugates with each virus before infecting the monolayer of cultured MDM and PBMC. After 4 days, HIV p24 antigen was quantified by enzyme immunoassay (EIA) in culture supernatants. Significant inhibition of viral infectivity was defined as >80% reduction in p24 concentration. GAG showed a low inhibitory effect (8–44%) in HIV-1 infection of PBMC. Table 1 shows the minimal amounts of the most active SG fractions, expressed as volumes of human milk from which they were extracted, that inhibit >80% HIV-1 infection.

Breastfed infants have a lower risk of diarrhea than infants fed artificial formula. Human milk components, including oligosaccharides, inhibit pathogens in vitro and are postulated to protect infants from disease. Most human milk oligosaccharides are fucosylated. The fucose terminus may be connected by an a 1,2 linkage catalyzed by a fucosyltransferase produced by the secretor gene (FUT2), or by an α1,3 or α1,4 linkage catalyzed by fucosyltransferases produced by the Lewis gene (FUT3) or other α1,3 transferase genes (FUT4,5,6,7,9) of this family. The secretor and Lewis genes also control expression of the Lewis blood group type (Erney et al. 2000, Henry et al. 1995). Some women are nonsecretors, i.e., they do not secrete 2-linked fucosyloligosaccharides into their milk and other bodily fluids. Even among secretors, however, the expression of milk fucosyloligosaccharides varies significantly (Chaturvedi et al. 2001, Erney et al. 2000, Thurl et al. 1997, Viverge et al. 1990). Enterotoxigenic Escherichia coli that produce stable toxin (ST-E. coli) is a common cause of diarrhea; the ability of stable toxin to cause diarrhea is inhibited by human milk α1,2-linked fucosyloligosaccharides in vitro and in vivo.

Specific human milk oligosaccharides inhibit specific microbial pathogens.1 As soluble homologs or analogs of host receptors for pathogens, milk oligosaccharides, especially fucosylated neutral oligosaccharides, may act as decoys to protect infants against disease.2-4 To study a relationship between specific human milk oligosaccharides and disease in breast-fed infants, it is necessary to know the levels of specific oligosaccharides present in milks of individuals during lactation. Variation in total oligosaccharides, specific α1,2-linked oligosaccharides, and activities of fucosidases and fucosyltransferases has been reported.5, 6This study measures variation in individual fucosylated oligosaccharides in milks of individual mothers over the course of lactation.

Specific human milk oligosaccharides, especially fucosylated neutral oligosaccharides, protect infants against specific microbial pathogens. To study the concentrations of individual neutral oligosaccharides during lactation, a total of 84 milk samples were obtained from 12 women at 7 time periods during weeks 1-49 postpartum. The neutral oligosaccharides from each sample were isolated, perbenzoylated, resolved, and quantified by reversed-phase high-performance liquid chromatography. The resultant oligosaccharide peaks, identified by co-elution with authentic standards and mass spectrometry, ranged in size from tri- to octasaccharides. The total concentration of oligosaccharides declined over the course of lactation; the mean concentration at 1 year was less than half that in the first few weeks postpartum. One of the 12 donors produced milk fucosyloligosaccharides that were essentially devoid of alpha1,2 linkages (but contained alpha1,3- and alpha1,4-linked fucose) until late in lactation, consistent with the nonsecretor phenotype. In milk samples from the remaining 11 donors, fucosyloligosaccharides containing alpha1,2-linked fucose were prevalent, and their profiles were distinct from those of fucosyloligosaccharides devoid of alpha1,2-linked fucose. The ratio of alpha1,2-linked oligosaccharide concentrations to oligosaccharides devoid of alpha1,2-linked fucose changed during the first year of lactation from 5:1 to 1:1. Furthermore, the absolute and the relative concentrations of individual oligosaccharides varied substantially, both between individual donors and over the course of lactation for each individual. The patterns of milk oligosaccharides among individuals suggest the existence of many genotype subpopulations. This variation in individual oligosaccharide concentrations suggests that the protective activities of human milk could also vary among individuals and during lactation.

Hemolytic uremic syndrome (HUS) is associated with intestinal infection by enterohemorrhagic Escherichia coli strains that produce Shiga toxins. Globotriaosylceramide (Gb 3 ) is the functional receptor for Shiga toxin, and tumor necrosis factor alpha (TNF-α) upregulates Gb 3 in both human macrovascular umbilical vein endothelial cells and human microvascular brain endothelial cells. TNF-α treatment enhanced Shiga toxin binding and sensitivity to toxin. This upregulation was specific for Gb 3 species containing normal fatty acids (NFA). Central nervous system (CNS) pathology in HUS could involve cytokine-stimulated elevation of endothelial NFA-Gb 3 levels. Differential expression of Gb 3 species may be a critical determinant of Shiga toxin toxicity and of CNS involvement in HUS.

Hemolytic uremic syndrome, a serious complication of Shiga toxin—associated diarrhea, is rare before 6 months of age. Immunologic and nonimmunologic factors present in human milk may partially explain this observation. In prior studies, we have demonstrated that human milk contains Gb3the receptor for the B subunit of Shiga toxin, and also contains secretory IgA (sIgA) against the toxin. We therefore sought to determine the relative importance of milk glycolipid and toxin-specific sIgA in toxin binding. We studied two populations that differed in their frequency of exposure to Shiga toxin. Human milk samples obtained from healthy donors from Boston and Buenos Aires were separated by centrifugation into aqueous (antibody enriched) and cream (glycosphingolipid enriched) fractions. An emulsion of equal volumes of aqueous phase or cream layer of each sample and purified Shiga toxin was incubated, and the amount of free toxin present in each was determined by enzyme immunoassay. The cream layers bound 85% ± 2 (mean ± SE) (Argentina milk samples) and 86% ± 1 (Boston milk samples) of Shiga toxin. In contrast, the soluble fraction in samples from Buenos Aires, a population expected to frequently have antibodies to Shiga toxin, bound more toxin (48% ± 2) than did this fraction in samples from Boston, an area where toxin exposure is infrequent (30% ± 3) (P < 0.0001). Toxin-binding lipids present in human milk are biologically active and may contribute to the putative protective effect of human milk In a population frequently exposed to Shiga toxins (Argentina), protection may be due to both immune (sIgA), and non-immune (lipid) factors present in human milk In a population infrequently exposed to Shiga toxins, cream fraction—associated glycolipids represent the major toxin binding activity in human milk.

Human milk contains large amounts of many oligosaccharides, most of which are fucosylated; several inhibit pathogenic bacteria, viruses, and toxins that cause disease in humans. Although bovine milk is known to have much less and many fewer types of oligosaccharides, no studies heretofore have indicated whether the amount or complexity of human milk oligosaccharides is unique to our species. Toward this end, a comparison was made of the major individual oligosaccharides in milk specimens from a variety of species, including the great apes. The neutral compounds, which represent the bulk of oligosaccharides in human milk, were isolated, perbenzoylated, resolved by high performance liquid chromatography (HPLC), and detected at 229nm. Ambiguous structures were determined by mass spectrometry. All milk specimens contained lactose, although levels were quite low in bear and kangaroo milk. The types of oligosaccharides in milk specimens from the primates resembled those of human milk, but the amounts, especially of the larger molecules, were markedly lower. The relative amounts of oligosaccharides in the bonobo changed over the course of lactation, as they do in humans. Marine mammals generally had few oligosaccharides in their milk other than 2'-fucosyllactose. Grizzly and black bear milk specimens contained a wide range of oligosaccharides, many of which had novel, fucosylated structures. Milk specimens from humans, bears, and marsupials had the greatest quantity of, and the most complex, neutral oligosaccharides. Although human milk contained more oligosaccharide than did milk specimens from the other species studied, the presence of appreciable amounts of complex oligosaccharides was not unique to humans. This finding suggests that in animal milk specimens, as in human milk, neutral fucosylated oligosaccharides potentially offer protection from pathogens to offspring with immature immune systems.

Several human milk oligosaccharides inhibit human pathogens in vitro and in animal models. In an infant, the ability of these oligosaccharides to offer protection from enteric pathogens would require that they withstand structural modification as they pass through the alimentary canal or are absorbed and excreted in urine. We investigated the fate of human milk oligosaccharides during transit through the alimentary canal by determining the degree to which breast-fed infants’ urine and fecal oligosaccharides resembled those of their mothers’ milk Oligosaccharide profiles of milk from 16 breast-feeding mothers were compared with profiles of stool and urine from their infants. Results were compared with endogenous oligosaccharide profiles obtained from the urine and feces of age-, parity-, and gender-matched formula-fed infants. In all cases, oligosaccharides were extracted, purified, reduced, and separated into acidic and neutral species; the latter were perbenzoylated and subjected to reversed-phase high-performance liquid chromatography. Structures were determined by mass spectrometry after debenzoylation. Among breast-fed infants, concentrations of oligosaccharides were higher in feces than in mothers’ milk, and much higher in feces than in urine. Urinary and fecal oligosaccharides from breast-fed infants resembled those in their mothers’ milk Those from formula-fed infants did not resemble human milk oligosaccharides, were found at much lower concentrations, and probably resulted from remodeling of intestinal glycoconjugates or from intestinal bacteria. Most of the human milk oligosaccharides survived transit through the gut, and some were absorbed and then excreted into the urine intact, implying that inhibition of intestinal and urinary pathogens by human milk oligosaccharides is quite likely in breast-fed infants.