Supplemental Figure Legends for Supplemental Figures 1 through 12.

Methods used for Supplemental Data only: WST-1 cell proliferation assay, Ki-67 mmunohistochemical analysis

Supplemental Figure 1: Larger legend table for symbols in fig 1A. Supplemental Figure 2: The expression of BIRC5, CDKN1A, CXCL1 and IER3 correlates with expression of C9b, but not C9a, in human NSCLC tissues. Supplemental Figure 3: Down-regulation of RIP1 abolishes the effect of C9b on IκBα level.. Supplemental Figure 4: The role of C9b in the NF-κB pathway is translational to other cell lines, H838 and HBEpC. Supplemental Figure 5: C9b activates the canonical NF-κB pathway in H838 cells and HBEpCs. Supplemental Figure 6: C9b level inversely correlates with IκBα level in NSCLC cells. Supplemental Figure 7: Down-regulation of cIAP2 has no effect on NF-κB activation and does not inhibit the effect of C9b on the NF-κB pathway. Supplemental Figure 8: Cleavage of C9b is not important for interaction with cIAP1. Supplemental Figure 9: Tumors formed from NSCLC cells stably expressing WT C9b have higher Ki-67 expression (cell proliferation) than tumors formed from control cells or cells expressing IBM-mutated (Mut) C9b. Supplemental Figure 10: The interaction with cIAP1 mediates the enhancing effect of C9b on the cell viability and survival/AIG of NSCLC cells and HBEpCs. Supplemental Figure 11: Suppression of the canonical NF-κB pathway by ectopically expressing the suppressor, IκBα, increases NIK level. Supplemental Figure 12: Down-regulation of NIK does not affect the survival/AIG of NSCLC cells. Supplemental Table 1: qPCR primers for NF-κB target genes utilized in the study Supplemental Table 2: List of NF-κB-related genes that expression was affected by down-regulation of C9b in A549 cells or up-regulation of C9b in HBEpC cells (identified by deep RNA sequencing). Supplemental Table 3: List of NF-κB target genes that expression is contrastingly affected by down-regulation of C9b in A549 cells versus by up-regulation of C9b in HBEpCs. Supplemental Table 4: NSCLC cell lines used in Figure 2D or Supplemental Figure 6. Supplemental Table 5: Sequences of negative control si-RNA and caspase-9a or -9b si-RNA utilized in the study.

We have developed a chiral route toward the synthesis of muscarinic M4 agonists that was enabled by the biocatalytic synthesis of the key spirocyclic diamine building blocks 10 and 12. Using these bifunctional compounds we were able to optimize a synthetic sequence toward a collection of advanced intermediates for further elaboration. These advanced intermediates were then used as starting points for early medicinal chemistry and the identification of selective M1/M4 agonists.

An imidazolone → triazolone replacement addressed the limited passive permeability of a series of protein arginine methyl transferase 5 (PRMT5) inhibitors. This increase in passive permeability was unexpected given the increase in the hydrogen bond acceptor (HBA) count and topological polar surface area (TPSA), two descriptors that are typically inversely correlated with permeability. Quantum mechanics (QM) calculations revealed that this unusual effect was due to an electronically driven disconnect between TPSA and 3D-PSA, which manifests in a reduction in overall HBA strength as indicated by the HBA moment descriptor from COSMO-RS (conductor-like screening model for real solvation). HBA moment was subsequently deployed as a design parameter leading to the discovery of inhibitors with not only improved passive permeability but also reduced P-glycoprotein (P-gp) transport. Our case study suggests that hidden polarity as quantified by TPSA-3DPSA can be rationally designed through QM calculations.

This article summarizes the evolution of the screening deck at the Novartis Institutes for BioMedical Research (NIBR). Historically, the screening deck was an assembly of all available compounds. In 2015, we designed a first deck to facilitate access to diverse subsets with optimized properties. We allocated the compounds as plated subsets on a 2D grid with property based ranking in one dimension and increasing structural redundancy in the other. The learnings from the 2015 screening deck were applied to the design of a next generation in 2019. We found that using traditional leadlikeness criteria (mainly MW, clogP) reduces the hit rates of attractive chemical starting points in subset screening. Consequently, the 2019 deck relies on solubility and permeability to select preferred compounds. The 2019 design also uses NIBR's experimental assay data and inferred biological activity profiles in addition to structural diversity to define redundancy across the compound sets.

SHP2 is a nonreceptor protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also plays an important role in the programed cell death pathway (PD-1/PD-L1). As an oncoprotein as well as a potential immunomodulator, controlling SHP2 activity is of high therapeutic interest. As part of our comprehensive program targeting SHP2, we identified multiple allosteric binding modes of inhibition and optimized numerous chemical scaffolds in parallel. In this drug annotation report, we detail the identification and optimization of the pyrazine class of allosteric SHP2 inhibitors. Structure and property based drug design enabled the identification of protein-ligand interactions, potent cellular inhibition, control of physicochemical, pharmaceutical and selectivity properties, and potent in vivo antitumor activity. These studies culminated in the discovery of TN0155, (3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine (1), a highly potent, selective, orally efficacious, and first-in-class SHP2 inhibitor currently in clinical trials for cancer.

SHP2 is a nonreceptor protein tyrosine phosphatase within the mitogen-activated protein kinase (MAPK) pathway controlling cell growth, differentiation, and oncogenic transformation. SHP2 also participates in the programed cell death pathway (PD-1/PD-L1) governing immune surveillance. Small-molecule inhibition of SHP2 has been widely investigated, including in our previous reports describing SHP099 (2), which binds to a tunnel-like allosteric binding site. To broaden our approach to allosteric inhibition of SHP2, we conducted additional hit finding, evaluation, and structure-based scaffold morphing. These studies, reported here in the first of two papers, led to the identification of multiple 5,6-fused bicyclic scaffolds that bind to the same allosteric tunnel as 2. We demonstrate the structural diversity permitted by the tunnel pharmacophore and culminated in the identification of pyrazolopyrimidinones (e.g., SHP389, 1) that modulate MAPK signaling in vivo. These studies also served as the basis for further scaffold morphing and optimization, detailed in the following manuscript.

Two decades have passed since the rule of five ushered in the concept of "drug-like" properties. Attempts to quantify, correlate, and categorize molecules based on Ro5 parameters evolved into the introduction of efficiency metrics with far reaching consequences in decision making by industry leaders and scientists seeking to discover new medicines. Examination of oral drug parameters approved before and after the original Ro5 analysis demonstrates that some parameters such as clogP and HBD remained constant while the cutoffs for parameters such as molecular weight and HBA have increased substantially over the past 20 years. The time dependent increase in the molecular weight of oral drugs during the past 20 years provides compelling evidence to disprove the hypothesis that molecular weight is a "drug-like" property. This analysis does not validate parameters that have not changed as being "drug-like" but instead calls into question the entire hypothesis that "drug-like" properties exist.

Protein tyrosine phosphatase SHP2 is an oncoprotein associated with cancer as well as a potential immune modulator due to its role in the programmed cell death PD-L1/PD-1 pathway. Small molecule inhibition of SHP2 has been widely investigated including our previous reports describing SHP099, which binds to a tunnel-like allosteric binding site. To broaden our approach to allosteric inhibition of SHP2, we conducted additional hit finding, evaluation, and structure-based scaffold morphing. These studies led to the identification of multiple, potent inhibitor chemotypes, an additional and distinct allosteric binding site, and the identification of SHP394, an orally efficacious inhibitor of SHP2 with improved potency and enhanced pharmacokinetic properties. Overall, this work improves upon our previously described allosteric inhibitors, and exemplifies and extends the range of permissible chemical templates that inhibit SHP2 via the allosteric mechanism. Citation Format: Matthew J. LaMarche, Jeff Bagdanoff, Mike Acker, Ying-Nan Chen, Homan Chan, Michael Dore, Brant Firestone, Michelle Fodor, Jorge Garcia-Fortanet, Murphy Hentemann, Mitsunori Kato, Robert Koenig, Laura La Bonte, Shumei Liu, Movarid Mohseni, Rukundo Ntaganda, Patrick Sarver, Troy Smith, Martin Sendzik, Travis Stams, Stan Spence, Chris Towler, Hongyun Wang, Ping Wang, Sarah Williams, Zhouliang Chen, Huaixiang Hao, Gang Liu, Chen Liu, Eric McNeill, Bing Yu. Allosteric SHP2 phosphatase inhibition: Multiple mechanisms and chemotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-005.

SHP2 is a cytoplasmic protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell proliferation, differentiation, and survival. Recently, we reported an allosteric mechanism of inhibition that stabilizes the auto-inhibited conformation of SHP2. SHP099 (1) was identified and characterized as a moderately potent, orally bioavailable, allosteric small molecule inhibitor, which binds to a tunnel-like pocket formed by the confluence of three domains of SHP2. In this report, we describe further screening strategies that enabled the identification of a second, distinct small molecule allosteric site. SHP244 (2) was identified as a weak inhibitor of SHP2 with modest thermal stabilization of the enzyme. X-ray crystallography revealed that 2 binds and stabilizes the inactive, closed conformation of SHP2, at a distinct, previously unexplored binding site-a cleft formed at the interface of the N-terminal SH2 and PTP domains. Derivatization of 2 using structure-based design resulted in an increase in SHP2 thermal stabilization, biochemical inhibition, and subsequent MAPK pathway modulation. Downregulation of DUSP6 mRNA, a downstream MAPK pathway marker, was observed in KYSE-520 cancer cells. Remarkably, simultaneous occupation of both allosteric sites by 1 and 2 was possible, as characterized by cooperative biochemical inhibition experiments and X-ray crystallography. Combining an allosteric site 1 inhibitor with an allosteric site 2 inhibitor led to enhanced pharmacological pathway inhibition in cells. This work illustrates a rare example of dual allosteric targeted protein inhibition, demonstrates screening methodology and tactics to identify allosteric inhibitors, and enables further interrogation of SHP2 in cancer and related pathologies.

Abstract SHP2 is a cytoplasmic non-receptor tyrosine phosphatase involved in the propagation of extracellular signaling through receptor tyrosine kinases. Aberrant SHP2 activity has been identified as a driver in multiple cancers and SHP2 has also been implicated in the PD-1/PD-L1-mediated exhaustion of effector T-cells, leading to immune system evasion of tumors. Recently, we reported the identification of SHP099, an allosteric inhibitor of SHP2 with in in vivo efficacy against multiple RTK-driven tumor xenograft models. Here we report the use of alternate screening paradigms to identify a novel allosteric inhibitor which binds to a previously uncharacterized pocket on SHP2. Like SHP099, the second allosteric inhibitor stabilizes a closed conformation of SHP2, which blocks access to the phosphatase active site. Structure based drug design led to improvements in potency, and combination studies in biochemical, biophysical and cellular assays confirm dual occupation of SHP099 and the second allosteric molecule, resulting in improved potency. This work highlights a rare opportunity for dual occupation of inhibitors for a single target and provides additional tools for the exploration of SHP2 biology. Citation Format: Michelle Fodor, Edmund Price, Ping Wang, Hengyu Lu, Andreea Argintaru, Zhouliang Chen, Meir Glick, Huai-Xiang Hao, Mitsunori Kato, Robert Koenig, Jonathan R. LaRochelle, Gang Liu, Eric McNeill, Dyuti Majumdar, Gisele Nishiguchi, Lawrence Perez, Greg Paris, Christopher Quinn, Timothy Ramsey, Martin Sendzik, Michael Shultz, Sarah Williams, Travis Stams, Stephen C. Blacklow, Matthew J. LaMarche, Michael G. Acker. Simultaneous inhibition of SHP2 phosphatase at two allosteric sites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2808.

Abstract The non-receptor protein tyrosine phosphatase (PTP) SHP2 is an important component of RTK signaling in response to growth factor stimulus and sits just upstream of the RAS-MAPK signaling cascade. The first oncogenic phosphatase to be identified, SHP2 is dysregulated in multiple human diseases including the developmental disorders Noonan and Leopard syndromes, as well as leukemia, lung cancer and neuroblastoma where aberrant activity of SHP2 leads to uncontrolled MAPK signaling. Cancer-associated activating mutations in SHP2 impart an “auto-on” state of the enzyme, boosting basal activity by shifting the equilibrium away from the auto-inhibited state. Reduction of SHP2 activity through genetic knockdown suppresses tumor growth, validating SHP2 as a target for cancer therapy. SHP099, a recently reported potent and selective allosteric inhibitor of SHP2, stabilizes the auto-inhibited form of SHP2 through interactions with the N-terminal SH2 and C-terminal PTP domains of the protein. SHP099 suppresses MAPK signaling in RTK amplified cancers resulting in suppressed proliferation in vitro and inhibition of tumor growth in mouse tumor xenograft models. Together, these data demonstrate the therapeutic potential of SHP2 inhibition in the treatment of cancer and other RAS/MAPK-linked diseases. Citation Format: Michael G. Acker, Ying-Nan P. Chen, Matthew J. LaMarche, Ho Man Chan, Peter Fekkes, Jorge Garcia-Fortanet, Jonathan R. LaRochelle, Brandon Antonakos, Christine Hiu-Tung Chen, Zhuoliang Chen, Vesselina G. Cooke, Jason R. Dobson, Zhan Deng, Fei Feng, Brant Firestone, Michelle Fodor, Cary Fridrich, Hui Gao, Huai-Xiang Hao, Jaison Jacob, Samuel Ho, Kathy Hsiao, Zhao B. Kang, Rajesh Karki, Mitsunori Kato, Jay Larrow, Laura R. La Bonte, Gang Liu, Shumei Liu, Dyuti Majumdar, Matthew J. Meyer, Mark Palermo, Minying Pu, Edmund Price, Subarna Shakya, Michael D. Shultz, Kavitha Venkatesan, Ping Wang, Markus Warmuth, Sarah Williams, Guizhi Yang, Jing Yuan, Ji-Hu Zhang, Ping Zhu, Stephen C. Blacklow, Timothy Ramsey, Nicholas J. Keen, William R. Sellers, Travis Stams, Pascal D. Fortin. Conformational activation and allosteric inhibition of SHP2 in RTK-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2084. doi:10.1158/1538-7445.AM2017-2084

Overexpression and somatic heterozygous mutations of EZH2, the catalytic subunit of polycomb repressive complex 2 (PRC2), are associated with several tumor types. EZH2 inhibitor, EPZ-6438 (tazemetostat), demonstrated clinical efficacy in patients with acceptable safety profile as monotherapy. EED, another subunit of PRC2 complex, is essential for its histone methyltransferase activity through direct binding to trimethylated lysine 27 on histone 3 (H3K27Me3). Herein we disclose the discovery of a first-in-class potent, selective, and orally bioavailable EED inhibitor compound 43 (EED226). Guided by X-ray crystallography, compound 43 was discovered by fragmentation and regrowth of compound 7, a PRC2 HTS hit that directly binds EED. The ensuing scaffold hopping followed by multiparameter optimization led to the discovery of 43. Compound 43 induces robust and sustained tumor regression in EZH2MUT preclinical DLBCL model. For the first time we demonstrate that specific and direct inhibition of EED can be effective as an anticancer strategy.

SmNACE is a NAD catabolizing enzyme expressed on the outer tegument of S. mansoni, a human parasite that is one of the major agents of the neglected tropical disease schistosomiasis. Recently, we identified aroylhydrazone derivatives capable of inhibiting the recombinant form of the enzyme with variable potency (IC50 ranging from 88 μM to 33 nM). In the present study, we investigated the mechanism of action of the least potent micromolar inhibitor (compound 1) and the most potent nanomolar inhibitor (compound 2) in the series on both the recombinant and native SmNACE enzymes. Using mass spectroscopy, spectrophotometry, and activity assays under different experimental conditions, we demonstrated that the >3 log gain in potency against recombinant SmNACE by this class of compounds is dependent on the formation of a coordination complex with metal cations, such as Ni(II), Zn(II), and Fe(II), that are loaded on the protein surface. Testing the compounds on live parasites, we observed that only the weak micromolar compound 1 was active on the native enzyme. We showed that S. mansoni effectively sequesters the metal from the coordination complex, resulting in the loss of inhibitory activity of the potent nanomolar compound 2. Importantly, the modeling of the transition complex between Zn(II) and compound 2 enabled the discovery of a new metal-independent aroylhydrazone analogue, which is now the most potent and selective inhibitor of native SmNACE known.