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Supplementary Materials and Methods Supplementary References Tables S1 to S3 and legends Figures S1 to S4 and legends

Src homology 2 domain-containing phosphatase (SHP2) is a phosphatase that mediates signaling downstream of multiple receptor tyrosine kinases (RTK) and is required for full activation of the MAPK pathway. SHP2 inhibition has demonstrated tumor growth inhibition in RTK-activated cancers in preclinical studies. The long-term effectiveness of tyrosine kinase inhibitors such as the EGFR inhibitor (EGFRi), osimertinib, in non–small cell lung cancer (NSCLC) is limited by acquired resistance. Multiple clinically identified mechanisms underlie resistance to osimertinib, including mutations in EGFR that preclude drug binding as well as EGFR-independent activation of the MAPK pathway through alternate RTK (RTK-bypass). It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between multiple resistance mechanisms could restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. Here, we report the discovery of IACS-13909, a specific and potent allosteric inhibitor of SHP2, that suppresses signaling through the MAPK pathway. IACS-13909 potently impeded proliferation of tumors harboring a broad spectrum of activated RTKs as the oncogenic driver. In EGFR-mutant osimertinib-resistant NSCLC models with EGFR-dependent and EGFR-independent resistance mechanisms, IACS-13909, administered as a single agent or in combination with osimertinib, potently suppressed tumor cell proliferation in vitro and caused tumor regression in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFRi-resistant NSCLC.Significance:These findings highlight the discovery of IACS-13909 as a potent, selective inhibitor of SHP2 with drug-like properties, and targeting SHP2 may serve as a therapeutic strategy to overcome tumor resistance to osimertinib.

Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) plays a role in receptor tyrosine kinase (RTK), neurofibromin-1 (NF-1), and Kirsten rat sarcoma virus (KRAS) mutant-driven cancers, as well as in RTK-mediated resistance, making the identification of small-molecule therapeutics that interfere with its function of high interest. Our quest to identify potent, orally bioavailable, and safe SHP2 inhibitors led to the discovery of a promising series of pyrazolopyrimidinones that displayed excellent potency but had a suboptimal in vivo pharmacokinetic (PK) profile. Hypothesis-driven scaffold optimization led us to a series of pyrazolopyrazines with excellent PK properties across species but a narrow human Ether-a-go-go-Related Gene (hERG) window. Subsequent optimization of properties led to the discovery of the pyrimidinone series, in which multiple members possessed excellent potency, optimal in vivo PK across species, and no off-target activities including no hERG liability up to 100 μM. Importantly, compound 30 (IACS-15414) potently suppressed the mitogen-activated protein kinase (MAPK) pathway signaling and tumor growth in RTK-activated and KRASmut xenograft models in vivo.

Indoleamine 2,3-dioxygenase 1 (IDO1), a heme-containing enzyme that mediates the rate-limiting step in the metabolism of l-tryptophan to kynurenine, has been widely explored as a potential immunotherapeutic target in oncology. We developed a class of inhibitors with a conformationally constrained bicyclo[3.1.0]hexane core. These potently inhibited IDO1 in a cellular context by binding to the apoenzyme, as elucidated by biochemical characterization and X-ray crystallography. A SKOV3 tumor model was instrumental in differentiating compounds, leading to the identification of IACS-9779 (62) and IACS-70465 (71). IACS-70465 has excellent cellular potency, a robust pharmacodynamic response, and in a human whole blood assay was more potent than linrodostat (BMS-986205). IACS-9779 with a predicted human efficacious once daily dose below 1 mg/kg to sustain >90% inhibition of IDO1 displayed an acceptable safety margin in rodent toxicology and dog cardiovascular studies to support advancement into preclinical safety evaluation for human development.

Supplemental Digital Content is Available in the Text. Dual leucine zipper kinase inhibition is a promising therapeutic strategy against chemotherapy-induced neurotoxicities including chemotherapy-induced peripheral neuropathy and chemotherapy-induced cognitive impairments., Chemotherapy-induced peripheral neuropathy (CIPN) and chemotherapy-induced cognitive impairments (CICI) are common, often severe neurotoxic side effects of cancer treatment that greatly reduce quality of life of cancer patients and survivors. Currently, there are no Food and Drug Administration-approved agents for the prevention or curative treatment of CIPN or CICI. The dual leucine zipper kinase (DLK) is a key mediator of axonal degeneration that is localized to axons and coordinates the neuronal response to injury. We developed a novel brain-penetrant DLK inhibitor, IACS′8287, which demonstrates potent and highly selective inhibition of DLK in vitro and in vivo. Coadministration of IACS′8287 with the platinum derivative cisplatin prevents mechanical allodynia, loss of intraepidermal nerve fibers in the hind paws, cognitive deficits, and impairments in brain connectivity in mice, all without interfering with the antitumor activity of cisplatin. The protective effects of IACS′8287 are associated with preservation of mitochondrial function in dorsal root ganglion neurons and in brain synaptosomes. In addition, RNA sequencing analysis of dorsal root ganglia reveals modulation of genes involved in neuronal activity and markers for immune cell infiltration by DLK inhibition. These data indicate that CIPN and CICI require DLK signaling in mice, and DLK inhibitors could become an attractive treatment in the clinic when coadministered with cisplatin, and potentially other chemotherapeutic agents, to prevent neurotoxicities as a result of cancer treatment.

Tumor-associated macrophages (TAMs) have a significant presence in the tumor stroma across multiple human malignancies and are believed to be beneficial to tumor growth. Targeting CSF1R has been proposed as a potential therapy to reduce TAMs, especially the protumor, immune-suppressive M2 TAMs. Additionally, the high expression of CSF1R on tumor cells has been associated with poor survival in certain cancers, suggesting tumor dependency and therefore a potential therapeutic target. The CSF1-CSF1R signaling pathway modulates the production, differentiation, and function of TAMs; however, the discovery of selective CSF1R inhibitors devoid of type III kinase activity has proven to be challenging. We discovered a potent, highly selective, and orally bioavailable CSF1R inhibitor, IACS-9439 (1). Treatment with 1 led to a dose-dependent reduction in macrophages, promoted macrophage polarization toward the M1 phenotype, and led to tumor growth inhibition in MC38 and PANC02 syngeneic tumor models.

Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) plays a role in receptor tyrosine kinase (RTK), neurofibromin-1 (NF-1), and Kirsten rat sarcoma virus (KRAS) mutant-driven cancers, as well as in RTK-mediated resistance, making the identification of small-molecule therapeutics that interfere with its function of high interest. Our quest to identify potent, orally bioavailable, and safe SHP2 inhibitors led to the discovery of a promising series of pyrazolopyrimidinones that displayed excellent potency but had a suboptimal

Summary: The plasticity of a preexisting regulatory circuit compromises the effectiveness of targeted therapies, and leveraging genetic vulnerabilities in cancer cells may overcome such adaptations. Hereditary leiomyomatosis renal cell carcinoma (HLRCC) is characterized by oxidative phosphorylation (OXPHOS) deficiency caused by fumarate hydratase (FH) nullizyogosity. To identify metabolic genes that are synthetically lethal with OXPHOS deficiency, we conducted a genetic loss-of-function screen and found that phosphogluconate dehydrogenase (PGD) inhibition robustly blocks the proliferation of FH mutant cancer cells both in vitro and in vivo. Mechanistically, PGD inhibition blocks glycolysis, suppresses reductive carboxylation of glutamine, and increases the NADP+/NADPH ratio to disrupt redox homeostasis. Furthermore, in the OXPHOS-proficient context, blocking OXPHOS using the small-molecule inhibitor IACS-010759 enhances sensitivity to PGD inhibition in vitro and in vivo. Together, our study reveals a dependency on PGD in OXPHOS-deficient tumors that might inform therapeutic intervention in specific patient populations. : Loss-of-function genetics screen reveals a synthetically lethal interaction between OXPHOS inhibition and phosphogluconate dehydrogenase (PGD) inactivation. Sun et al. provide an example of targeting tumor metabolism in a genetically predefined context to maximize therapeutic impact and propose PGD as a therapeutic target for fumarate hydratase-deficient HLRCC. Keywords: synthetic lethality, PGD, OXPHOS, tumor metabolism, metabolic vulnerability, fumarate hydratase, redox homeostasis, functional genomics, hereditary leiomyomatosis renal cell carcinoma, pentose phosphate pathway

The disability, mortality and costs caused by non-vertebral osteoporotic fractures are enormous. Existing osteoporosis therapies are highly effective at reducing vertebral but not non-vertebral fractures. Cortical bone is a major determinant of non-vertebral bone strength. To identify novel osteoporosis drug targets, we phenotyped cortical bone of 3 366 viable mouse strains with global knockouts of druggable genes. Cortical bone thickness was substantially elevated in Notum−/− mice. NOTUM is a secreted WNT lipase and we observed high NOTUM expression in cortical bone and osteoblasts but not osteoclasts. Three orally active small molecules and a neutralizing antibody inhibiting NOTUM lipase activity were developed. They increased cortical bone thickness and strength at multiple skeletal sites in both gonadal intact and ovariectomized rodents by stimulating endocortical bone formation. Thus, inhibition of NOTUM activity is a potential novel anabolic therapy for strengthening cortical bone and preventing non-vertebral fractures., Bone Development: NOTUM inhibition stimulates bone formation NOTUM is an enzyme that inactivates WNT proteins (which play a key role in early tissue development), and inhibiting NOTUM has been found to increase the formation of endocortical bone (within the cortex, the hard exterior of bone) and enhance bone strength. Existing therapies for osteoporosis (condition causing bone to become weak and brittle) are effective in reducing vertebral, but not non-vertebral, fractures. A team headed by Robert Brommage at Lexicon Pharmaceuticals, Texas aimed to identify novel osteoporosis drug targets in mice. Following inhibition of NOTUM activity, the authors observed increased cortical bone thickness and strength at multiple skeletal sites through stimulation of endocortical bone formation. The team concluded that inhibiting NOTUM activity has good potential as a new therapeutic strategy and could be beneficial in preventing non-vertebral osteoporotic fractures.

Src homology 2 domain-containing phosphatase (SHP2) is a phosphatase that mediates signaling downstream of multiple receptor tyrosine kinases (RTK) and is required for full activation of the MAPK pathway. SHP2 inhibition has demonstrated tumor growth inhibition in RTK-activated cancers in preclinical studies. The long-term effectiveness of tyrosine kinase inhibitors such as the EGFR inhibitor (EGFRi), osimertinib, in non–small cell lung cancer (NSCLC) is limited by acquired resistance. Multiple clinically identified mechanisms underlie resistance to osimertinib, including mutations in EGFR that preclude drug binding as well as EGFR-independent activation of the MAPK pathway through alternate RTK (RTK-bypass). It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between multiple resistance mechanisms could restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. Here, we report the discovery of IACS-13909, a specific and potent allosteric inhibitor of SHP2, that suppresses signaling through the MAPK pathway. IACS-13909 potently impeded proliferation of tumors harboring a broad spectrum of activated RTKs as the oncogenic driver. In EGFR-mutant osimertinib-resistant NSCLC models with EGFR-dependent and EGFR-independent resistance mechanisms, IACS-13909, administered as a single agent or in combination with osimertinib, potently suppressed tumor cell proliferation in vitro and caused tumor regression in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFRi-resistant NSCLC. Significance: These findings highlight the discovery of IACS-13909 as a potent, selective inhibitor of SHP2 with drug-like properties, and targeting SHP2 may serve as a therapeutic strategy to overcome tumor resistance to osimertinib.

Src homology 2 domain-containing phosphatase (SHP2), a ubiquitously expressed non-receptor tyrosine phosphatase, plays a critical role in the regulation of the MAPK signaling pathway and cellular proliferation. Activating mutations in SHP2 are associated with the development of multiple malignancies including leukemia, lung cancer and neuroblastoma. In addition, SHP2 promotes the conversion of oncogenic KRAS to its active GTP-bound state and it’s inhibition can enhance efficacy of GDP-KRASG12C inhibitors as well as other MAPK pathway inhibitors (RAF, MEK and ERK) which have suboptimal clinical efficacy as single agents. As a result, inhibition of SHP2 through genetic manipulation or pharmacological means has been shown to suppress tumor growth and presents an attractive potential avenue for the treatment of malignancies as monotherapy or in combination with other MAPK/PI3K inhibitors. Here we describe BBP-398, a potent, orally bioavailable allosteric small molecule inhibitor of SHP2. BBP-398 displays high selectivity against other phosphatases, kinases, GPCRs, transporters and hERG. Predicted human PK properties show good oral bioavailability with half-life of ~12-16 hours enabling continuous daily dosing and optimal therapeutic index in combination with other targeted therapeutics. In cellular assays, BBP-398 demonstrates potent pERK/DUSP6 inhibition and loss of viability across a panel of cell lines with active MAPK signaling, such as mutant EGFR and KRASG12C. In vivo, BBP-398 strongly suppresses RAS-ERK signaling in RTK- or RAS-driven xenografts. In the EGFR-dependent non-small cell lung cancer (NSCLC) HCC827 and esophageal squamous cell carcinoma KYSE-520 xenograft models, BBP-398 drives dose dependent efficacy consistent with the level of target inhibition. Detailed analysis of tumor response shows that efficacy is driven by maintaining better than 50% inhibition of pERK for most of the dosing interval. In addition to its strong single agent activity, BBP-398 also leads to enhanced efficacy in vitro and in vivo when used in combination with targeted therapeutics against driver MAPK genetic alterations, such as KRAS, EGFR or MET. Combination targeting, such as with the GDP-KRASG12C inhibitor sotorasib in the NSCLC NCI-H358 xenograft model, or with the mutant EGFR inhibitor osimertinib in the HCC827 erlotinib resistant (ER) xenograft model, drives strong suppression of MAPK activity and results in tumor regressions. Collectively, these findings highlight that SHP2 inhibition is a promising molecular therapeutic strategy in cancer which can potentially strongly suppress tumor growth as a single agent or in combination with other MAPK pathway inhibitors. Given its preclinical properties and projected favorable clinical pharmacokinetic profile, BBP-398 is currently being evaluated in a Phase 1/1b trial in patients with advanced solid tumors (NCT04528836). Citation Format: James P. Stice, Sofia Donovan, Yuting Sun, Nancy Kohl, Barbara Czako, Faika Mseeh, Paul Leonard, Anna Wade, Justin Lim, Phil Jones, Eli Wallace, Kerstin Sinkevicius, Pedro Beltran. BBP-398, a potent, small molecule inhibitor of SHP2, enhances the response of established NSCLC xenografts to KRASG12C and mutEGFR inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P207.

The increasing number of people afflicted with diabetes throughout the world is a major health issue. Inhibitors of the sodium-dependent glucose cotransporters (SGLT) have appeared as viable therapeutics to control blood glucose levels in diabetic patents. Herein we report the discovery of LX2761, a locally acting SGLT1 inhibitor that is highly potent in vitro and delays intestinal glucose absorption in vivo to improve glycemic control.

LX2761 is a potent sodium/glucose cotransporter 1 inhibitor restricted to the intestinal lumen after oral administration. Studies presented here evaluated the effect of orally administered LX2761 on glycemic control in preclinical models. In healthy mice and rats treated with LX2761, blood glucose excursions were lower and plasma total glucagon-like peptide-1 (GLP-1) levels higher after an oral glucose challenge; these decreased glucose excursions persisted even when the glucose challenge occurred 15 hours after LX2761 dosing in ad lib-fed mice. Further, treating mice with LX2761 and the dipeptidyl-peptidase 4 inhibitor sitagliptin synergistically increased active GLP-1 levels, suggesting increased LX2761-mediated release of GLP-1 into the portal circulation. LX2761 also lowered postprandial glucose, fasting glucose, and hemoglobin A1C, and increased plasma total GLP-1, during long-term treatment of mice with either early- or late-onset streptozotocin-diabetes; in the late-onset cohort, LX2761 treatment improved survival. Mice and rats treated with LX2761 occasionally had diarrhea; this dose-dependent side effect decreased in severity and frequency over time, and LX2761 doses were identified that decreased postprandial glucose excursions without causing diarrhea. Further, the frequency of LX2761-associated diarrhea was greatly decreased in mice either by gradual dose escalation or by pretreatment with resistant starch 4, which is slowly digested to glucose in the colon, a process that primes the colon for glucose metabolism by selecting for glucose-fermenting bacterial species. These data suggest that clinical trials are warranted to determine if LX2761 doses and dosing strategies exist that provide improved glycemic control combined with adequate gastrointestinal tolerability in people living with diabetes.

Structure based design of a novel class of aminopyrimidine MTH1 (MutT homolog 1) inhibitors is described. Optimization led to identification of IACS-4759 (compound 5), a sub-nanomolar inhibitor of MTH1 with excellent cell permeability and good metabolic stability in microsomes. This compound robustly inhibited MTH1 activity in cells and proved to be an excellent tool for interrogation of the utility of MTH1 inhibition in the context of oncology.

Thirty-six patients with type 2 diabetes mellitus (T2DM) were randomized 1:1:1 to receive a once-daily oral dose of placebo or 150 or 300 mg of the dual SGLT1/SGLT2 inhibitor LX4211 for 28 days. Relative to placebo, LX4211 enhanced urinary glucose excretion by inhibiting SGLT2-mediated renal glucose reabsorption; markedly and significantly improved multiple measures of glycemic control, including fasting plasma glucose, oral glucose tolerance, and HbA(1c); and significantly lowered serum triglycerides. LX4211 also mediated trends for lower weight, lower blood pressure, and higher glucagon-like peptide-1 levels. In a follow-up single-dose study in 12 patients with T2DM, LX4211 (300 mg) significantly increased glucagon-like peptide-1 and peptide YY levels relative to pretreatment values, probably by delaying SGLT1-mediated intestinal glucose absorption. In both studies, LX4211 was well tolerated without evidence of increased gastrointestinal side effects. These data support further study of LX4211-mediated dual SGLT1/SGLT2 inhibition as a novel mechanism of action in the treatment of T2DM.

Tumor-associated macrophages (TAMs) are critical drivers of tumor progression and immunosuppression within the tumor microenvironment. The dominant TAM phenotype is broadly characterized as harboring M2-like macrophage properties, which are anti-inflammatory and pro-tumor, as opposed to M1-like macrophages, which possess tumoricidal and pro-inflammatory characteristics. The dependence of M2 TAMs on CSF1 receptor (CSF1R) kinase signaling has made CSF1R a desirable therapeutic target, and the need for highly selective therapies for use in combinations. Through an extensive medicinal chemistry campaign, we identified a series of orally bioavailable, highly potent, exquisitely selective inhibitors of CSF1R (IC50 < 10 nM) with excellent pharmacologic properties that are appropriate for evaluation as a cancer therapy. The aim of our study was to assess the biological impact of our lead CSF1R inhibitor (CSF1Ri) on macrophage populations and the consequent effect on T effector cells. In vitro biochemical activity was evaluated in various kinase assays comparing our CSF1Ri to BLZ945. The compound was evaluated using syngeneic murine models of colorectal cancer (MC38) and pancreatic adenocarcinoma (PANC02). Tumors were immune profiled using NanoString, immunohistochemistry and flow cytometric analysis establishing that there was a depletion of macrophages and a reduction in the relative amount of M2+ (CD206+MHCII-) cells, with a concomitant increase in M1+ (MHCII+CD206-) cells. Myeloid-derived suppressor cells (MDSCs), CD4, and CD8 cell infiltration were also altered with an elevation of cytotoxic immune cells. In conclusion, we have identified and characterized a novel potent and selective inhibitor of CSF1R with highly favorable PK/PD properties, that compares favorably to BLZ945. We also have clear evidence that our novel CSF1R inhibitors modulates TAM infiltration and phenotype altering the immune cell milieu toward a more favorable anti-tumor environment. Citation Format: Erika Suzuki, Jeffrey J. Kovacs, Nakia D. Spencer, Sonal Sonal, Ningping Feng, Angela L. Harris, Robert A. Mullinax, Andy M. Zuniga, Sarah B. Johnson, Mikhila Mahendra, Tin Oo Khor, Faika Mseeh, Zhen Liu, Jason P. Burke, Keith Mikule, Martin Tremblay, Timothy P. Heffernan, Philip Jones, Barbara Czako, Joseph R. Marszalek. Promoting an anti-tumor immune environment with a novel, exquisitely selective CSF1R inhibitor [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 LB-124.

Indoleamine 2,3-dioxygenase (IDO1 and IDO2) and tryptophan dioxygenase (TDO) are heme-containing enzymes that mediate the rate limiting step in the oxidative degradation of L-tryptophan (L-TRP) to kynurenine (KYN) metabolites. Tryptophan catabolism through the KYN metabolic pathway is now recognized as one of many mechanisms involved in tumor cell evasion of the immune surveillance system. Inhibition of the KYN pathway in the tumor microenvironment can lead to improved immune response and tumor growth suppression. Recently, clinical proof of concept of this mechanism has been demonstrated using an Indoleamine 2,3-dioxygenase (IDO1) inhibitor in combination with a PD-1 antagonist in a variety of tumor contexts. Consideration of known low molecular weight heme-co-ordinating ligands identified from the PDB, in conjunction with a virtual screen performed in-silico identified a number of potentially interesting starting points for medicinal chemistry development. Identification of an attractive indazole fragment as a starting point, and expansion into alternative bicyclic cores, resulted in the discovery of a family of imidazopyridines as potent human IDO1 inhibitors with >200 fold selectivity against TDO. Utilizing a structure-based design approach allowed rapid lead optimization that resulted in the identification of IACS-8968. Crystallography studies were conducted, and binding of IACS-8968 to the heme domain of the human IDO1 was confirmed. The homochiral imidazopyridine IACS-8968 displayed cellular IC50= 29 nM in a HeLa cell line expressing human IDO1 and IC50= 21 nM in a PANC02 mouse cell line expressing the murine IDO1 enzyme, showed satisfactory selectivity margin (> 150 fold) versus its CYP450 inhibition profile and good oral bioavailability across species. PK/PD experiments indicated that, at equivalent exposure, IACS-8968 (sodium salt) and epacadostat decreased tumor KYN at comparable levels in CT26 syngeneic mouse model. Citation Format: Alessia Petrocchi, Naphtali J. Reyna, Faika Mseeh, Connor A. Parker, Simon Yu, Quanyun Xu, Ningping Feng, Paul Leonard, Norma Rogers, Jason B. Cross, Angela L. Harris, Yongying Jiang, Tin Oo Khor, Mikhila G. Mahendra, Jihai Pang, Qi Wu, Andy M. Zuniga, Timothy McAfoos, Timothy McAfoos, Matthew M. Hamilton, Joe R. Marszalek, Keith Mikule, Paul Vancutsem, Keith Wilcoxen, Martin Tremblay, Philip Jones, Richard T. Lewis. Discovery of an imidazopyridine series of potent human IDO1 inhibitors with robust target engagement in a preclinical tumor model [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 LB-071.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of oral anti-diabetic agents that improve glycemic control by inhibiting SGLT2-mediated renal glucose reabsorption. Currently available agents increase urinary glucose excretion (UGE) to 50% of filtered glucose when SGLT2 is completely inhibited. This led us to test whether LP-925219, a small molecule dual SGLT1/SGLT2 inhibitor, increases UGE to maximal values in wild-type (WT) mice. We first tested LP-925219 inhibition of glucose transport by HEK293 cells expressing SGLT1 or SGLT2, and then characterized LP-925219 pharmacokinetics. We found that LP-925219 was a potent inhibitor of mouse SGLT1 (IC50 = 22.6 nmol/L) and SGLT2 (IC50 = 0.5 nmol/L), and that a 10 mg/kg oral dose was bioavailable (87%) with a long half-life (7 h). We next delivered LP-925219 by oral gavage to WT, SGLT1 knockout (KO), SGLT2 KO, and SGLT1/SGLT2 double KO (DKO) mice and measured their 24-h UGE. We found that, in vehicle-treated mice, DKO UGE was maximal and SGLT2 KO, SGLT1 KO, and WT UGEs were 30%, 2%, and 0.2% of maximal, respectively; we also found that LP-925219 dosed at 60 mg/kg twice daily increased UGE of SGLT1 KO, SGLT2 KO, and WT mice to DKO UGE levels. These findings show that orally available dual SGLT1/SGLT2 inhibitors can maximize 24-h UGE in mammals, and suggest that such agents merit further evaluation for their potential, in diabetic patients, to achieve better glycemic control than is achieved using selective SGLT2 inhibitors.

Defining the gene products that play an essential role in an organism's functional repertoire is vital to understanding the system level organization of living cells. We used a genetic footprinting technique for a genome-wide assessment of genes required for robust aerobic growth of Escherichia coli in rich media. We identified 620 genes as essential and 3,126 genes as dispensable for growth under these conditions. Functional context analysis of these data allows individual functional assignments to be refined. Evolutionary context analysis demonstrates a significant tendency of essential E. coli genes to be preserved throughout the bacterial kingdom. Projection of these data over metabolic subsystems reveals topologic modules with essential and evolutionarily preserved enzymes with reduced capacity for error tolerance.

Riboflavin kinase (RFK) is an essential enzyme catalyzing the phosphorylation of riboflavin (vitamin B 2 ) to form FMN, an obligatory step in vitamin B 2 utilization and flavin cofactor synthesis. The structure of human RFK revealed a six-stranded antiparallel β barrel core structurally similar to the riboflavin synthase/ferredoxin reductase FAD binding domain fold. The binding site of an intrinsically bound MgADP defines a novel nucleotide binding motif that encompasses a loop, a 3 10 helix, and a reverse turn followed by a short β strand. This active site loop forms an arch with ATP and riboflavin binding at the opposite side and the phosphoryl transfer appears to occur through the hole underneath the arch. The invariant residues Asn36 and Glu86 are implicated in the catalysis.

Novel drug targets are required in order to design new defenses against antibiotic-resistant pathogens. Comparative genomics provides new opportunities for finding optimal targets among previously unexplored cellular functions, based on an understanding of related biological processes in bacterial pathogens and their hosts. We describe an integrated approach to identification and prioritization of broad-spectrum drug targets. Our strategy is based on genetic footprinting in Escherichia coli followed by metabolic context analysis of essential gene orthologs in various species. Genes required for viability of E. coli in rich medium were identified on a whole-genome scale using the genetic footprinting technique. Potential target pathways were deduced from these data and compared with a panel of representative bacterial pathogens by using metabolic reconstructions from genomic data. Conserved and indispensable functions revealed by this analysis potentially represent broad-spectrum antibacterial targets. Further target prioritization involves comparison of the corresponding pathways and individual functions between pathogens and the human host. The most promising targets are validated by direct knockouts in model pathogens. The efficacy of this approach is illustrated using examples from metabolism of adenylate cofactors NAD(P), coenzyme A, and flavin adenine dinucleotide. Several drug targets within these pathways, including three distantly related adenylyltransferases (orthologs of the E. coli genes nadD , coaD , and ribF ), are discussed in detail.

In mammals, melatonin activates melatonin MT 1 and MT 2 receptors. Using site-directed mutagenesis and chemical modification, we investigated the role of conserved cysteines in ligand binding. Dithiothreitol inhibited 2-[ 125 I]iodomelatonin binding to the FLAG-tagged human melatonin MT 2 receptor without affecting ligand affinity. Alanine substitution of Cys 113 or Cys 190 resulted in a loss of specific 2-[ 125 I]iodomelatonin binding, without altering cell surface receptor expression. This suggests that a putative disulfide bond linking Cys 113 and Cys 190 is essential to maintain a proper human melatonin MT 2 receptor conformation for melatonin binding. N -ethylmaleimide alkylation of cysteines inhibited 2-[ 125 I]iodomelatonin binding, decreasing both ligand affinity and receptor density. Alkylation of Cys 140 contributes to changes in ligand affinity, while alkylation of Cys 143 and Cys 219 reduced binding capacity. We suggest that a disulfide bridge is important for the proper structural conformation of the human melatonin MT 2 receptor to bind melatonin. Cysteines located in receptor regions near the ligand binding site and/or G protein coupling region are involved in N -ethylmaleimide-induced changes in affinity and receptor density.

Cyclic GMP-stimulated cyclic nucleotide phosphodiesterase (PDE2) is the second most abundant of this class of enzymes in platelets. PDE2 probably plays an important role in the regulation of elevated intracellular concentrations of cAMP and cGMP in platelets inhibited by prostacyclin and/or nitric oxide. The cAMP and cGMP PDEs have catalytic domains with 28–40% identity, but vary in their substrate specificity and affinity. As a first step toward the goal of identifying important amino acids in the substrate binding site pocket, we have employed the affinity analog 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine-3′5′ cyclic monophosphate (8-BDB-TcAMP) to inactivate PDE2 and observe the pattern of protection by substrates and their products. Incubation of purified platelet PDE2 with 8-BDB-TcAMP (2–10 mM) resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.013 min−1 mM−1. Both substrates, cAMP and cGMP, as well as the products of hydrolysis by PDE2, AMP and GMP, exhibited concentration-dependent protection against inhibition by 8-BDB-TcAMP, but no protection was noted with ADP or ATP, which are not hydrolyzed by the enzyme. This compound, 8-BDB-TcAMP, and similar affinity reagents should prove useful in delineating amino acids in the active site of PDE2.

Cancer cells display metabolic properties distinct from normal cells and are therefore believed to be dependent on key metabolic enzymes. The effectiveness of targeting metabolism for cancer therapy has been largely restricted due to metabolic adaptability. We postulate that tumors with metabolic vulnerabilities will be limited in such adaptation, thus providing a unique opportunity for therapeutic intervention. Hereditary leiomyomatosis renal cell carcinoma (HLRCC) is a metabolically vulnerable cancer, due to mutations in a key TCA cycle enzyme - fumarate hydratase (FH), thus rendering them deficient in oxidative phosphorylation (OXPHOS). We performed a loss-of-function genetic screen in a FH-null HLRCC model (UOK262 cells) and identified 6-phosphogluconate dehydrogenase (6PGD) - a rate-limiting enzyme in the non-oxidative arm of pentose phosphate pathway - as a critical regulator of cell growth. Inhibition of 6PGD using either dox-inducible shRNA or a small molecule inhibitor dramatically blocks the growth of UOK262 cells, and in contrast, has no effect on 6PGD-deficient NB1 cells. The growth inhibition from knockdown is fully rescued by expressing shRNA-resistant 6PGD. Conversely, ectopic expression of 6PGD in NB1 cells results in a gain of function phenotype. Importantly, in vivo, 6PGD knockdown causes robust regression of established UOK262 xenografts. Mechanistically, 6PGD inhibition causes the accumulation of 6-phosphogluconate, blocks glycolysis, and induces ROS production. Furthermore, in tumors with intact TCA cycle, 6PGD inhibition shows synergism with OXPHOS Complex I inhibitor IACS-10759. Together, our data suggest that 6PGD is a bona fide oncology target and provide a strong rationale for developing small molecule inhibitors that could be used to treat OXPHOS-deficient tumors as single agent, or combined with inhibitors of OXPHOS. Citation Format: Yuting Sun, Madhavi Bandi, Timothy Lofton, Melinda Smith, Christopher Bristow, Norma Rogers, Chang Edward, Mary Geck Do, Yongying Jiang, Pietro Morlacchi, Florian Muller, Faika Mseeh, Barbara Czako, Wylie Palmer, Carlo Toniatti, Philip Jones, Giulio Draetta, Timothy Heffernan, Joseph Marszalek. Functional genomics reveals dependency on 6-phosphogluconate dehydrogenase in OXPHOS-deficient tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1023.

Sodium-glucose cotransporter 2 (SGLT2) is the major, and SGLT1 the minor, transporter responsible for renal glucose reabsorption. Increasing urinary glucose excretion (UGE) by selectively inhibiting SGLT2 improves glycemic control in diabetic patients. We generated Sglt1 and Sglt2 knockout (KO) mice, Sglt1/Sglt2 double-KO (DKO) mice, and wild-type (WT) littermates to study their relative glycemic control and to determine contributions of SGLT1 and SGLT2 to UGE. Relative to WTs, Sglt2 KOs had improved oral glucose tolerance and were resistant to streptozotocin-induced diabetes. Sglt1 KOs fed glucose-free high-fat diet (G-free HFD) had improved oral glucose tolerance accompanied by delayed intestinal glucose absorption and increased circulating glucagon-like peptide-1 (GLP-1), but had normal intraperitoneal glucose tolerance. On G-free HFD, Sglt2 KOs had 30%, Sglt1 KOs 2%, and WTs

The human skeleton is affected by mutations in Low-density lipoprotein Receptor-related Protein 5 (LRP5). To understand how LRP5 influences bone properties, we generated mice with inducible Lrp5 mutations that cause high bone mass and low bone mass phenotypes in humans. We conditionally-induced Lrp5 mutations in osteocytes and found that bone properties in these mice were comparable to bone properties in mice with inherited mutations. We also conditionally-induced an Lrp5 mutation in cells that contribute to the appendicular skeleton, and not to the axial skeleton, and we observed bone properties were altered in the limbs, and not in the spine. These data indicate that Lrp5 signaling functions locally and suggest increasing LRP5 signaling in mature bone cells as a strategy to treat human low bone mass disorders, such as osteoporosis.

The prevalence of diabetes throughout the world continues to increase and has become a major health issue. Recently there have been several reports of inhibitors directed toward the sodium-dependent glucose cotransporter 2 (SGLT2) as a method of maintaining glucose homeostasis in diabetic patients. Herein we report the discovery of the novel O-xyloside 7c that inhibits SGLT2 in vitro and urinary glucose reabsorption in vivo.

MTH1 is a protein that sanitizes oxidized dNTPs in the cell. It preferentially hydrolyzes 8-oxo-dGTP and 2-OH-dATP to their corresponding monophosphates and thereby prevents the incorporation of oxidized nucleotides into DNA or RNA. The functional result is a reduction in down-stream mutations, or DNA damage, thus preventing cell death. Recent publications suggest that MTH1 is a non-essential enzyme in normal cells, but is required for the survival of cancer cells as a consequence of being subjected to high levels of oxidative stress; hence its relevance as an oncology target. Gad et al., 2014 The interesting target rationale combined with a perceived high chemical tractability of the target resulting from availability of x-ray crystallographic information for the protein, led to a decision to undertake, in parallel, target validation, assay development and drug discovery. This effort resulted in the structure-based design of potent, cell active MTH1 small molecule inhibitors. This poster will describe the discovery and optimization of these inhibitors, and their use to evaluate the potential of MTH1 as an oncology target. This work was complemented by parallel genetic studies. Pharmacodynamic evaluation of target engagement using proximal biomarkers will be presented, as will phenotypic responses across a range of cancer cell lines. Citation Format: Elisabetta Leo, Alessia Petrocchi, Jennifer Bardenhagen, Maria Alimova, Xi Shi, Connor Parker, Naphtali Reyna, Matthew Hamilton, Edward Felix, Andrzej Mazan, Christian Dillon, Faika Mseeh, Joseph R. Marszalek, Carlo Toniatti, Giulio Draetta, Phil Jones, Richard T. Lewis. Identification of potent, cell active MTH1 inhibitors and their use in target validation studies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B48.

Melatonin mediates its physiological effects through activation of high affinity G protein-coupled receptors. The vertebrate MT1, MT2 and Mel1c melatonin receptors are molecularly and pharmacologically distinct. Three molecular models of melatonin recognition for the MT1 and/or Mel1c melatonin receptors have been proposed. To determine if these models applied to the MT2 melatonin receptor, we mutated seven conserved residues to alanine in the hMT2 melatonin receptor and expressed the receptors in HEK-293 cells. Competition of melatonin for 2-[ 125 I ]-iodomelatonin binding revealed that mutation of Asn 16 in TM4 or His 7 in TM5 of the hMT2 melatonin receptor significantly decreased the binding affinity for melatonin when compared with wild-type. In addition, competition of 4P-ADOT, N-acetyltryptamine, luzindole, and 5-methoxytryptophol for 2-[ 125 I ]-iodomelatonin binding suggested Asn 16 in TM4 may facilitate binding of the 5-methoxy group of the melatonin molecule to the hMT2 melatonin receptor. Trp 13 or Phe 6 in TM6 while not critical for melatonin binding, may interact with aromatic regions of luzindole and 4P-ADOT. Mutation of Ser 8 or Ser 12 in TM3, or Ser 6 in TM7 did not affect the affinity of melatonin for competition with 2-[ 125 I ]-iodomelatonin to the hMT2 melatonin receptor, although equivalent serines (Ser 8 and Ser 12 in TM3) were reported to be critical for melatonin binding to the hMT1 melatonin receptor. Thus these results are the first to identify residues within the transmembrane regions of the hMT2 melatonin receptor critical for melatonin binding, highlighting potential structural differences between the MT1 and MT2 melatonin receptor binding pockets.