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Invadopodia are actin-based proteolytic membrane protrusions required for invasive behavior and tumor growth. In this study, we used our high-content screening assay to identify kinases whose activity affects invadopodia formation. Among the top hits selected for further analysis was TAO3, an STE20-like kinase of the GCK subfamily. TAO3 was overexpressed in many human cancers and regulated invadopodia formation in melanoma, breast, and bladder cancers. Furthermore, TAO3 catalytic activity facilitated melanoma growth in three-dimensional matrices and in vivo. A novel, potent catalytic inhibitor of TAO3 was developed that inhibited invadopodia formation and function as well as tumor cell extravasation and growth. Treatment with this inhibitor demonstrated that TAO3 activity is required for endosomal trafficking of TKS5α, an obligate invadopodia scaffold protein. A phosphoproteomics screen for TAO3 substrates revealed the dynein subunit protein LIC2 as a relevant substrate. Knockdown of LIC2 or expression of a phosphomimetic form promoted invadopodia formation. Thus, TAO3 is a new therapeutic target with a distinct mechanism of action.Significance:An unbiased screening approach identifies TAO3 as a regulator of invadopodia formation and function, supporting clinical development of this class of target.

Effect of SBI-581 on TKS5alpha vesicle trafficking

Effect of SBI-581 on Rab11 vesicle trafficking

Includes Sup. Figures 1-7, Tables 1-6 and Methods.

Effect of SU11333 on TKS5alpha vesicle trafficking

Trafficking of TKS5alpha vesicles

Description of additional methods and procedures used in the study. Also includes Supplemental References.

SBI-756 inhibits AKT, mTOR signaling and alters cell cycle.

Pharmacological properties of top four BI-69A11 analogs (S1); Blood chemistry following SBI-756 administration (S2); List of biotinylated-BI-69A11-bound proteins, identified by LC/MS/MS (S3); RPPA based analyses identify proteins that were significantly inhibited by SBI-756, compared to DMSO treatment at the 24 h time point (S4); Gene variants identified in SBI-756-resistant clones but not SBI-756-sensitive melanoma cell cultures (S5); The top 10 enriched canonical pathways identified using the Ingenuity Pathway Analysis of genes mutated in SBI-756 resistant melanoma (S6); Ingenuity Pathway Analysis of genes differentially expressed between SBI-756-sensitive and - resistant melanoma cells (S7).

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of the nicotinamide adenine dinucleotide (NAD+) salvage pathway. Because NAD+ plays a pivotal role in energy metabolism and boosting NAD+ has positive effects on metabolic regulation, activation of NAMPT is an attractive therapeutic approach for the treatment of various diseases, including type 2 diabetes and obesity. Herein we report the discovery of 1-(2-phenyl-1,3-benzoxazol-6-yl)-3-(pyridin-4-ylmethyl)urea 12c (DS68702229), which was identified as a potent NAMPT activator. Compound 12c activated NAMPT, increased cellular NAD+ levels, and exhibited an excellent pharmacokinetic profile in mice after oral administration. Oral administration of compound 12c to high-fat diet-induced obese mice decreased body weight. These observations indicate that compound 12c is a promising anti-obesity drug candidate.

SUMMARYThe NTSR1 neurotensin receptor (NTSR1) is a G protein coupled receptor (GPCR) found in the brain and peripheral tissues with neurotensin (NTS) being its endogenous peptide ligand. In the brain, NTS modulates dopamine neuronal activity, induces opioid-independent analgesia, and regulates food intake. Recent studies indicate that biasing NTSR1 toward β-Arrestin signaling can attenuate the actions of psychostimulants and other drugs of abuse. Here we provide the cryoEM structures of NTSR1 ternary complexes with heterotrimeric Gq and Go with and without the brain penetrant small molecule SBI-553. In functional studies, we discovered that SBI-553 displays complex allosteric actions exemplified by negative allosteric modulation for G proteins that are Gαsubunit selective and positive allosteric modulation and agonism for β-Arrestin translocation at NTSR1. Detailed structural analysis of the allosteric binding site illuminated the structural determinants for biased allosteric modulation of SBI-553 on NTSR1. These insights promise to both accelerate the structure-guided design of more effective NTSR1 therapeutics and provide insights into the complexities of GPCR allosteric modulation.

Obesity-associated insulin resistance plays a central role in the pathogenesis of type 2 diabetes. A promising approach to decrease insulin resistance in obesity is to inhibit the protein tyrosine phosphatases that negatively regulate insulin receptor signaling. The low-molecular-weight protein tyrosine phosphatase (LMPTP) acts as a critical promoter of insulin resistance in obesity by inhibiting phosphorylation of the liver insulin receptor activation motif. Here, we report development of a novel purine-based chemical series of LMPTP inhibitors. These compounds inhibit LMPTP with an uncompetitive mechanism and are highly selective for LMPTP over other protein tyrosine phosphatases. We also report the generation of a highly orally bioavailable purine-based analogue that reverses obesity-induced diabetes in mice.

Background The BCL2 inhibitor venetoclax has shown efficacy in several hematologic malignancies, with the greatest response rates in indolent blood cancers such as chronic lymphocytic leukaemia. There is a lower response rate to venetoclax monotherapy in diffuse large B-cell lymphoma (DLBCL). Methods We tested inhibitors of cap-dependent mRNA translation for the ability to sensitise DLBCL and mantle cell lymphoma (MCL) cells to apoptosis by venetoclax. We compared the mTOR kinase inhibitor (TOR-KI) MLN0128 with SBI-756, a compound targeting eukaryotic translation initiation factor 4G1 (eIF4G1), a scaffolding protein in the eIF4F complex. Results Treatment of DLBCL and MCL cells with SBI-756 synergised with venetoclax to induce apoptosis in vitro, and enhanced venetoclax efficacy in vivo. SBI-756 prevented eIF4E-eIF4G1 association and cap-dependent translation without affecting mTOR substrate phosphorylation. In TOR-KI-resistant DLBCL cells lacking eIF4E binding protein-1, SBI-756 still sensitised to venetoclax. SBI-756 selectively reduced translation of mRNAs encoding ribosomal proteins and translation factors, leading to a reduction in protein synthesis rates in sensitive cells. When normal lymphocytes were treated with SBI-756, only B cells had reduced viability, and this correlated with reduced protein synthesis. Conclusions Our data highlight a novel combination for treatment of aggressive lymphomas, and establishes its efficacy and selectivity using preclinical models.

Sepsis is a host response to systemic inflammation and infection that may lead to multi-organ dysfunction and eventual death. While acute brain dysfunction is common among all sepsis patients, chronic neurological impairment is prevalent among sepsis survivors. The brain microvasculature has emerged as a major determinant of sepsis-associated brain dysfunction, yet the mechanisms that underlie its associated neuroimmune perturbations and behavioral deficits are not well understood. An emerging body of data suggests that inhibition of tissue-nonspecific alkaline phosphatase (TNAP) enzyme activity in cerebral microvessels may be associated with changes in endothelial cell barrier integrity. The objective of this study was to elucidate the connection between alterations in cerebrovascular TNAP enzyme activity and brain microvascular dysfunction in late sepsis. We hypothesized that the disruption of TNAP enzymatic activity in cerebral microvessels would be coupled to the sustained loss of brain microvascular integrity, elevated neuroinflammatory responses, and behavioral deficits. Male mice were subjected to cecal ligation and puncture (CLP), a model of experimental sepsis, and assessed up to seven days post-sepsis. All mice were observed daily for sickness behavior and underwent behavioral testing. Our results showed a significant decrease in brain microvascular TNAP enzyme activity in the somatosensory cortex and spinal cord of septic mice but not in the CA1 and CA3 hippocampal regions. Furthermore, we showed that loss of cerebrovascular TNAP enzyme activity was coupled to a loss of claudin-5 and increased perivascular IgG infiltration in the somatosensory cortex. Analyses of whole brain myeloid and T-lymphoid cell populations also revealed a persistent elevation of infiltrating leukocytes, which included both neutrophil and monocyte myeloid derived suppressor cells (MDSCs). Regional analyses of the somatosensory cortex, hippocampus, and spinal cord revealed significant astrogliosis and microgliosis in the cortex and spinal cord of septic mice that was accompanied by significant microgliosis in the CA1 and CA3 hippocampal regions. Assessment of behavioral deficits revealed no changes in learning and memory or evoked locomotion. However, the hot plate test uncovered a novel anti-nociceptive phenotype in our septic mice, and we speculate that this phenotype may be a consequence of sustained GFAP astrogliosis and loss of TNAP activity in the somatosensory cortex and spinal cord of septic mice. Taken together, these results demonstrate that the loss of TNAP enzyme activity in cerebral microvessels during late sepsis is coupled to sustained neuroimmune dysfunction which may underlie, in part, the chronic neurological impairments observed in sepsis survivors.

In Plasmodium, the first two and rate-limiting enzymes of the pentose phosphate pathway, glucose 6-phosphate dehydrogenase (G6PD) and the 6-phosphogluconolactonase, are bifunctionally fused to a unique enzyme named GluPho, differing structurally and mechanistically from the respective human orthologs. Consistent with the enzyme’s essentiality for malaria parasite proliferation and propagation, human G6PD deficiency has immense impact on protection against severe malaria, making PfGluPho an attractive antimalarial drug target. Herein we report on the optimized lead compound N-(((2R,4S)-1-cyclobutyl-4-hydroxypyrrolidin-2-yl)methyl)-6-fluoro-4-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxamide (SBI-0797750), a potent and fully selective PfGluPho inhibitor with robust nanomolar activity against recombinant PfGluPho, PvG6PD, and P. falciparum blood-stage parasites. Mode-of-action studies have confirmed that SBI-0797750 disturbs the cytosolic glutathione-dependent redox potential, as well as the cytosolic and mitochondrial H(2)O(2) homeostasis of P. falciparum blood stages, at low nanomolar concentrations. Moreover, SBI-0797750 does not harm red blood cell (RBC) integrity and phagocytosis and thus does not promote anemia. SBI-0797750 is therefore a very promising antimalarial lead compound.

Zika virus (ZIKV) is a mosquito-borne pathogen classified by the World Health Organization (WHO) as a public health emergency of international concern in 2016, and it is still identified as a priority disease. Although most infected individuals are asymptomatic or show mild symptoms, a risk of neurologic complications is associated with infection in adults. Additionally, infection during pregnancy is directly linked to microcephaly and other congenital malformations. Since there are no currently available vaccines or approved therapeutics for this virus, there is a critical unmet need in developing treatments to prevent future ZIKV outbreaks. Towards this end, we performed a large-scale cell-based high-content screen of 51,520 chemical compounds to identify potential antiviral drug candidates. The compound (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) was found to inhibit replication of multiple ZIKV strains and in different cell systems. SBI-0090799 did not affect viral entry or RNA translation but suppressed RNA replication by preventing the formation of the membranous replication compartment. Selection of drug-resistant viruses identified single amino acid substitions in the N-terminal region of non-structural protein NS4A arguing this to be the likely drug target. These resistance mutations rescued viral RNA replication and restored the formation of the membranous replication compartment. This mechanism of action is similar to clinically-approved NS5A inhibitors for hepatitis C virus (HCV). Taken together, SBI-0090799 represents a promising lead candidate for the development of an antiviral treatment against ZIKV infection for the mitigation of severe complications and potential resurgent outbreaks of the virus. IMPORTANCE This study describes the elucidation of (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) as selective and potent inhibitor of Zika virus (ZIKV) replication using a high throughput screening approach. Mapping and resistance studies, supported by electron microscopy observations, indicate that the small molecule is functioning through inhibition of NS4A-mediated formation of ZIKV replication compartments in the endoplasmic reticulum (ER). Intriguingly, this defines a novel non-enzymatic target and chemical matter for the development of a new class of ZIKV antivirals. Moreover, chemical modulation affecting this non-structural protein mirrors the identification and development of hepatitis C virus (HCV) NS5A inhibitor daclatasvir and its derivatives, similarly interfering with the formation of the viral replication compartment and also targeting a protein with no enzymatic activity, which have been part of a curative strategy for HCV.

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitous enzyme present in many cells and tissues, including the central nervous system. Yet its functions at the brain-immune axis remain unclear. The goal of this study was to use a novel small molecular inhibitor of TNAP, SBI-425, to interrogate the function of TNAP in neuroimmune disorders. Following intraperitoneal (IP) administration of SBI-425, mass spectrometry analysis revealed that the SBI-425 does not cross the blood-brain barrier (BBB) in healthy mice. To elucidate the role of TNAP at the brain-immune axis, mice were subjected to experimental sepsis and received either vehicle or SBI-425 (25 mg/kg, IP) daily for 7 days. While SBI-425 administration did not affect clinical severity outcomes, we found that SBI-425 administration suppressed CD4 + Foxp3+ CD25− and CD8 + Foxp3+ CD25− splenocyte T-cell populations compared to controls. Further evaluation of SBI-425’s effects in the brain revealed that TNAP activity was suppressed in the brain parenchyma of SBI-425-treated mice compared to controls. When primary brain endothelial cells were treated with a proinflammatory stimulus the addition of SBI-425 treatment potentiated the loss of barrier function in BBB endothelial cells. To further demonstrate a protective role for TNAP at endothelial barriers within this axis, transgenic mice with a conditional overexpression of TNAP were subjected to experimental sepsis and found to have increased survival and decreased clinical severity scores compared to controls. Taken together, these results demonstrate a novel role for TNAP activity in shaping the dynamic interactions within the brain-immune axis.

Neurotensin receptor 1 (NTR1) is a G protein coupled receptor that is widely expressed throughout the central nervous system where it acts as a neuromodulator. Neurotensin receptors have been implicated in a wide variety of CNS disorders, but despite extensive efforts to develop small molecule ligands there are few reports of such compounds. Herein we describe the optimization of a quinazoline based lead to give 18 (SBI-553), a potent and brain penetrant NTR1 allosteric modulator.

Inhibition of ubiquitin ligases with small molecule remains a very challenging task, given the lack of catalytic activity of the target and the requirement of disruption of its interactions with other proteins. Siah1/2, which are E3 ubiquitin ligases, are implicated in melanoma and prostate cancer and represent high-value drug targets. We utilized three independent screening approaches in our efforts to identify small-molecule Siah1/2 inhibitors: Affinity Selection-Mass Spectrometry, a protein thermal shift-based assay and an in silico based screen. Inhibitors were assessed for their effect on viability of melanoma and prostate cancer cultures, colony formation, prolyl-hydroxylase-HIF1α signaling, expression of selected Siah2-related transcripts, and Siah2 ubiquitin ligase activity. Several analogs were further characterized, demonstrating improved efficacy. Combination of the top hits identified in the different assays demonstrated an additive effect, pointing to complementing mechanisms that underlie each of these Siah1/2 inhibitors.

Invadopodia are actin-based proteolytic membrane protrusions required for invasive behavior and tumor growth. In this study, we used our high-content screening assay to identify kinases whose activity affects invadopodia formation. Among the top hits selected for further analysis was TAO3, an STE20-like kinase of the GCK subfamily. TAO3 was overexpressed in many human cancers and regulated invadopodia formation in melanoma, breast, and bladder cancers. Furthermore, TAO3 catalytic activity facilitated melanoma growth in three-dimensional matrices and in vivo. A novel, potent catalytic inhibitor of TAO3 was developed that inhibited invadopodia formation and function as well as tumor cell extravasation and growth. Treatment with this inhibitor demonstrated that TAO3 activity is required for endosomal trafficking of TKS5α, an obligate invadopodia scaffold protein. A phosphoproteomics screen for TAO3 substrates revealed the dynein subunit protein LIC2 as a relevant substrate. Knockdown of LIC2 or expression of a phosphomimetic form promoted invadopodia formation. Thus, TAO3 is a new therapeutic target with a distinct mechanism of action. Significance: An unbiased screening approach identifies TAO3 as a regulator of invadopodia formation and function, supporting clinical development of this class of target.

Obesity-associated insulin resistance plays a central role in the pathogenesis of type 2 diabetes. A promising approach to decrease insulin resistance in obesity is to inhibit the protein tyrosine phosphatases that negatively regulate insulin receptor signaling. The low molecular weight protein tyrosine phosphatase (LMPTP), encoded by the ACP1 gene, acts as a critical regulator of insulin signaling by dephosphorylating the activation motif of the insulin receptor. Human genetics studies suggest that high LMPTP catalytic activity promotes diabetes and insulin resistance in obesity. We recently discovered that LMPTP is a critical promoter of insulin resistance in obesity through an action on the liver and that pharmacological inhibition of LMPTP reverses obesity-induced diabetes in mice. Here we report our progress in the development of a new chemical series of LMPTP inhibitors that is orally bioavailable and effective at treating disease in obese diabetic mice. Disclosure S.M. Stanford: None. M.A. Diaz: None. J.J. Zou: None. R.J. Ardecky: None. A. Pinkerton: None. N. Bottini: Research Support; Self; Eli Lilly and Company, Gilead Sciences, Inc., Kyowa Hakko Kirin Co., Ltd., Roche Pharma. Funding American Diabetes Association/Pathway to Stop Diabetes (1-15-INI-13 to S.M.S.); National Institutes of Health (R01DK106233)

Background and Aims Vascular media calcification is frequently seen in elderly and patients with chronic kidney disease (CKD), diabetes and osteoporosis. Pyrophosphate is a well-known calcification inhibitor that binds to nascent hydroxyapatite crystals and prevents further incorporation of inorganic phosphate into these crystals. However, the enzyme tissue-nonspecific alkaline phosphatase (TNAP), which is highly expressed in calcified arteries, degrades extracellular pyrophosphate into phosphate ions, by which pyrophosphate loses its ability to block vascular calcification. Here, we aimed to evaluate whether a TNAP inhibitor is able to prevent the development of arterial calcification in a rat model of warfarin-induced vascular calcification. Method To induce vascular calcification, rats received a diet containing 0.30% warfarin and 0.15% vitamin K1 throughout the entire study and were subjected to the following daily treatments: (i) vehicle (n=10) or (ii) 10 mg/kg/day TNAP-inhibitor (n=10) administered via an intraperitoneal catheter from start of the study until sacrifice at week 7. Calcium, phosphorus and parathyroid hormone (PTH) levels were determined in serum samples as these are important determinants of vascular calcification. As TNAP is also expressed in the liver, serum alanine aminotransferase (ALT) and aspartate (AST) levels were analyzed. At sacrifice, vascular calcification was evaluated by measurement of the total calcium content in the arteries and quantification of the area % calcification on Von Kossa stained sections of the aorta. The mRNA expression of osteo/chondrogenic marker genes (runx2, TNAP, SOX9, collagen 1 and collagen 2) was analyzed in the aorta by qPCR to verify whether vascular smooth muscle cells underwent reprogramming towards bone-like cells. Bone histomorphometry was performed on the left tibia to measure static and dynamic bone parameters as TNAP also regulates physiological bone mineralization. Results No differences in serum calcium, phosphorus and PTH levels was observed between both study groups. Warfarin exposure resulted in distinct calcification in the aorta and peripheral arteries. Daily dosing with the TNAP inhibitor (10 mg/kg/day) for 7 weeks significantly reduced vascular calcification as indicated by a significant decrease in calcium content in the aorta (vehicle 3.84±0.64 mg calcium/g wet tissue vs TNAP inhibitor 0.70±0.23 mg calcium/g wet tissue) and peripheral arteries and a distinct reduction in area % calcification on Von Kossa stained aortic sections as compared to vehicle condition. The inhibitory effects of SBI-425 on vascular calcification were without altering serum liver markers ALT and AST levels. Furthermore, TNAP-inhibitor SBI-425 did not modulate the mRNA expression of osteo/chondrogenic marker genes runx2, TNAP, SOX9, collagen 1 and 2. Dosing with SBI-425 resulted in decreased bone formation rate and mineral apposition rate, and increased osteoid maturation time and this without significant changes in osteoclast- and eroded perimeter. Conclusion Dosing with TNAP inhibitor SBI-425 significantly reduced the calcification in the aorta and peripheral arteries of a rat model of warfarin-induced vascular calcification and this without affecting liver function. However, suppression of TNAP activity should be limited in order to maintain adequate physiological bone mineralization.

Summary Small molecule neurotensin receptor 1 (NTSR1) agonists have been pursued for more than 40 years as potential therapeutics for psychiatric disorders, including drug addiction. Clinical development of NTSR1 agonists has, however, been precluded by their severe side effects. NTSR1, a G protein-coupled receptor (GPCR), signals through the canonical activation of G proteins and engages β-arrestins to mediate distinct cellular signaling events. Here, we characterize the allosteric NTSR1 modulator SBI-553. This small molecule not only acts as a β-arrestin-biased agonist but also extends profound β-arrestin bias to the endogenous ligand by selectively antagonizing G protein signaling. SBI-553 shows efficacy in animal models of psychostimulant abuse, including cocaine self-administration, without the side effects characteristic of balanced NTSR1 agonism. These findings indicate that NTSR1 G protein and β-arrestin activation produce discrete and separable physiological effects, thus providing a strategy to develop safer GPCR-targeting therapeutics with more directed pharmacological action.

Invadopodia are actin-based proteolytic membrane protrusions required for invasive behavior and tumor growth. We used our high-content screening assay to identify kinases impacting invadopodia formation. Among the top hits we selected TAO3, a STE20-like kinase of the GCK subfamily, for further analysis. TAO3 was over-expressed in many human cancers, and regulated invadopodia formation in melanoma, breast and bladder cancers. Furthermore, TAO3 catalytic activity facilitated melanoma growth in 3-dimensional matrices andin vivo. We developed potent catalytic inhibitors of TAO3 that inhibited invadopodia formation and function, and tumor cell extravasation and growth. Using these inhibitors, we determined that TAO3 activity was required for endosomal trafficking of TKS5α, an obligate invadopodia scaffold protein. A phosphoproteomics screen for TAO3 substrates revealed the dynein subunit protein LIC2 as a relevant substrate. Knockdown of LIC2 or expression of a phosphomimetic form promoted invadopodia formation. Thus, TAO3 is a new therapeutic target with a distinct mechanism of action.SIGNIFICANCETargeting tumor invasive behavior represents an understudied opportunity. We used an unbiased screening approach to identify kinases required for invadopodia formation and function. We validated TAO3, both genetically and with a novel inhibitor, and determined TAO3 function. Our data support clinical development of this class of target.

Tissue-nonspecific alkaline phosphatase (TNAP) is an ectoenzyme crucial for bone matrix mineralization via its ability to hydrolyze extracellular inorganic pyrophosphate (ePPi), a potent mineralization inhibitor, to phosphate (Pi). By the controlled hydrolysis of ePPi, TNAP maintains the correct ratio of Pi to ePPi and therefore enables normal skeletal and dental calcification. In other areas of the body low ePPi levels lead to the development of pathological soft-tissue calcification, which can progress to a number of disorders. TNAP inhibitors have been shown to prevent these processes via an increase of ePPi. Herein we describe the use of a whole blood assay to optimize a previously described series of TNAP inhibitors resulting in 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent, selective and oral bioavailable compound that robustly inhibits TNAP in vivo.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of the NAD+ salvage pathway. Since NAD+ plays a pivotal role in many biological processes including metabolism and aging, activation of NAMPT is an attractive therapeutic target for treatment of diverse array of diseases. Herein, we report the continued optimization of novel urea-containing derivatives which were identified as potent NAMPT activators. Early optimization of HTS hits afforded compound 12, with a triazolopyridine core, as a lead compound. CYP direct inhibition (DI) was identified as an issue of concern, and was resolved through modulation of lipophilicity to culminate in 1-[2-(1-methyl-1H-pyrazol-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-6-yl]-3-(pyridin-4-ylmethyl)urea (21), which showed potent NAMPT activity accompanied with attenuated CYP DI towards multiple CYP isoforms.

Here we present an innovative computational-based drug discovery strategy, coupled with machine-based learning and functional assessment, for the rational design of novel small molecule inhibitors of the lipogenic enzyme stearoyl-CoA desaturase 1 (SCD1). Our methods resulted in the discovery of several unique molecules, of which our lead compound SSI-4 demonstrates potent anti-tumor activity, with an excellent pharmacokinetic and toxicology profile. We improve upon key characteristics, including chemoinformatics and absorption/distribution/metabolism/excretion (ADME) toxicity, while driving the IC50 to 0.6 nM in some instances. This approach to drug design can be executed in smaller research settings, applied to a wealth of other targets, and paves a path forward for bringing small-batch based drug programs into the Clinic.

Hepatic cystogenesis in Polycystic Liver Disease (PLD) is associated with increased levels of cAMP in cholangiocytes lining liver cysts. TGR5, a G protein-coupled bile acid receptor, is linked to cAMP and expressed in cholangiocytes. Therefore, we hypothesized that TGR5 might contribute to disease progression. We examined expression of TGR5 and Gα proteins in cultured cholangiocytes and in livers of animal models and humans with PLD. In vitro, we assessed cholangiocyte proliferation, cAMP levels, and cyst growth in response to: (i) TGR5 agonists [taurolithocholic acid (TLCA), oleanolic acid (OA) and two synthetic compounds]; (ii) a novel TGR5 antagonist (SBI-115); and (iii) a combination of SBI-115 and pasireotide, a somatostatin receptor (SSTR) analog. In vivo, we examined hepatic cystogenesis in OA-treated PCK rats and after genetic elimination of TGR5 in double mutant TGR5-/-;Pkhd1del2/del2 mice. Compared to control, expression of TGR5 and Gαs (but not Gαi and Gαq) proteins was increased 2-3-fold in cystic cholangiocytes in vitro and in vivo. In vitro, TGR5 stimulation enhanced cAMP production, cell proliferation and cyst growth by ∼40%; these effects were abolished after TGR5 reduction by shRNA. OA increased cystogenesis in PCK rats by 35%; in contrast, hepatic cystic areas were decreased by 45% in TGR5-deficient TGR5-/-;Pkhd1del2/del2 mice. TGR5 expression and its co-localization with Gαs were increased ∼2-fold upon OA treatment. Levels of cAMP, cell proliferation and cyst growth in vitro were decreased by ∼30% in cystic cholangiocytes after treatment with SBI-115 alone and by ∼50% when SBI-115 was combined with pasireotide. Conclusion: TGR5 contributes to hepatic cystogenesis by increasing cAMP and enhancing cholangiocyte proliferation. Our data suggest that a TGR5 antagonist alone or concurrently with SSTR agonists represents novel therapeutic approaches in PLD. This article is protected by copyright. All rights reserved.

The recent re-emergence of Zika virus (ZIKV)1, a member of the Flaviviridae family, has become a global emergency. Currently, there are no effective methods of preventing or treating ZIKV infection, which causes severe neuroimmunopathology and is particularly harmful to the developing fetuses of infected pregnant women. However, the pathology induced by ZIKV is unique among flaviviruses, and knowledge of the biology of other family members cannot easily be extrapolated to ZIKV. Thus, structure-function studies of ZIKV proteins are urgently needed to facilitate the development of effective preventative and therapeutic agents. Like other flaviviruses, ZIKV expresses an NS2B-NS3 protease, which consists of the NS2B cofactor and the NS3 protease domain and is essential for cleavage of the ZIKV polyprotein precursor and generation of fully functional viral proteins. Here, we report the enzymatic characterization of ZIKV protease, and we identify structural scaffolds for allosteric small-molecule inhibitors of this protease. Molecular modeling of the protease-inhibitor complexes suggests that these compounds bind to the druggable cavity in the NS2B-NS3 protease interface and affect productive interactions of the protease domain with its cofactor. The most potent compound demonstrated efficient inhibition of ZIKV propagation in vitro in human fetal neural progenitor cells and in vivo in SJL mice. The inhibitory scaffolds could be further developed into valuable research reagents and, ultimately, provide a roadmap for the selection of efficient inhibitors of ZIKV infection.

The chemokine system plays an important role in mediating a proinflammatory microenvironment for tumor growth in hepatocellular carcinoma (HCC). The CXCR6 receptor and its natural ligand CXCL16 are expressed at high levels in HCC cell lines and tumor tissues and receptor expression correlates with increased neutrophils in these tissues contributing to poor prognosis in patients. Availability of pharmacologcal tools targeting the CXCR6/CXCL16 axis are needed to elucidate the mechanism whereby neutrophils are affected in the tumor environment. We report the discovery of a series of small molecules with an exo-[3.3.1]azabicyclononane core. Our lead compound 81 is a potent (EC50 = 40 nM) and selective orally bioavailable small molecule antagonist of human CXCR6 receptor signaling that significantly decreases tumor growth in a 30-day mouse xenograft model of HCC.

Pharmacological strategies that boost intracellular NAD+ are highly coveted for their therapeutic potential. One approach is activation of nicotinamide phosphoribosyltransferase (NAMPT) to increase production of nicotinamide mononucleotide (NMN), the predominant NAD+ precursor in mammalian cells. A high-throughput screen for NAMPT activators and hit-to-lead campaign yielded SBI-797812, a compound that is structurally similar to active-site directed NAMPT inhibitors and blocks binding of these inhibitors to NAMPT. SBI-797812 shifts the NAMPT reaction equilibrium towards NMN formation, increases NAMPT affinity for ATP, stabilizes phosphorylated NAMPT at His247, promotes consumption of the pyrophosphate by-product, and blunts feedback inhibition by NAD+. These effects of SBI-797812 turn NAMPT into a “super catalyst” that more efficiently generates NMN. Treatment of cultured cells with SBI-797812 increases intracellular NMN and NAD+. Dosing of mice with SBI-797812 elevates liver NAD+. Small molecule NAMPT activators such as SBI-797812 are a pioneering approach to raise intracellular NAD+ and realize its associated salutary effects. Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate determining step for NAD+ synthesis and is of interest as a drug target. Here the authors identify and characterize a small molecule NAMPT activator SBI-797812, elucidate its mode of action and show that it increases intracellular NMN and NAD+ levels in cultured cells and elevates liver NAD+ in mice.

Medial arterial calcification (MAC) is a major complication of chronic kidney disease (CKD) and an indicator of poor prognosis. Aortic overexpression of tissue-nonspecific alkaline phosphatase (TNAP) accelerates MAC formation. The present study aimed to assess whether a TNAP inhibitor, SBI-425, protects against MAC and improves survival probability in a CKD-mineral and bone disorder (MBD) mouse model. CKD-MBD mice were divided in three groups: vehicle, SBI-10, and SBI-30. They were fed a 0.2% adenine and 0.8% phosphorus diet from 14 to 20 weeks of age to induce CKD, followed by a high-phosphorus (0.2% adenine and 1.8% phosphorus) diet for another 6 weeks. At 14-20 weeks of age, mice in the SBI-10 and SBI-30 groups were given 10 and 30 mg/kg SBI-425 by gavage once a day, respectively, while vehicle-group mice were given distilled water as vehicle. Control mice were fed a standard chow (0.8% phosphorus) between the ages of 8 and 20 weeks. Computed tomography imaging, histology, and aortic tissue calcium content revealed that, compared to vehicle animals, SBI-425 nearly halted the formation of MAC. Mice in the control, SBI-10 and SBI-30 groups exhibited 100% survival, which was significantly better than vehicle-treated mice (57.1%). Aortic mRNA expression of Alpl, encoding TNAP, as well as plasma and aortic tissue TNAP activity, were suppressed by SBI-425 administration, whereas plasma pyrophosphate increased. We conclude that a TNAP inhibitor successfully protected the vasculature from MAC and improved survival rate in a mouse CKD-MBD model, without causing any adverse effects on normal skeletal formation and residual renal function. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

NAD+ is a crucial cellular factor that plays multifaceted roles in wide ranging biological processes. Low levels of NAD+ have been linked to numerous diseases including metabolic disorders, cardiovascular disease, neurodegeneration, and muscle wasting disorders. A novel strategy to boost NAD+ is to activate nicotinamide phosphoribosyltransferase (NAMPT), the putative rate-limiting step in the NAD+ salvage pathway. We previously showed that NAMPT activators increase NAD+ levels in vitro and in vivo. Herein we describe the optimization of our NAMPT activator prototype (SBI-0797812) leading to the identification of 1-(4-((4-chlorophenyl)sulfonyl)phenyl)-3-(oxazol-5-ylmethyl)urea (34) that showed far more potent NAMPT activation and improved oral bioavailability.

Arterial media calcification is frequently seen in elderly and patients with chronic kidney disease (CKD), diabetes and osteoporosis. Pyrophosphate is a well-known calcification inhibitor that binds to nascent hydroxyapatite crystals and prevents further incorporation of inorganic phosphate into these crystals. However, the enzyme tissue-nonspecific alkaline phosphatase (TNAP), which is expressed in calcified arteries, degrades extracellular pyrophosphate into phosphate ions, by which pyrophosphate loses its ability to block vascular calcification. Here, we aimed to evaluate whether pharmacological TNAP inhibition is able to prevent the development of arterial calcification in a rat model of warfarin-induced vascular calcification. To investigate the effect of the pharmacological TNAP inhibitor SBI-425 on vascular calcification and bone metabolism, a 0.30% warfarin rat model was used. Warfarin exposure resulted in distinct calcification in the aorta and peripheral arteries. Daily administration of the TNAP inhibitor SBI-425 (10 mg/kg/day) for 7 weeks significantly reduced vascular calcification as indicated by a significant decrease in calcium content in the aorta (vehicle 3.84 ± 0.64 mg calcium/g wet tissue vs TNAP inhibitor 0.70 ± 0.23 mg calcium/g wet tissue) and peripheral arteries and a distinct reduction in area % calcification on Von Kossa stained aortic sections as compared to vehicle. Administration of SBI-425 resulted in decreased bone formation rate and mineral apposition rate, and increased osteoid maturation time and this without significant changes in osteoclast- and eroded perimeter. Administration of TNAP inhibitor SBI-425 significantly reduced the calcification in the aorta and peripheral arteries of a rat model of warfarin-induced vascular calcification. However, suppression of TNAP activity should be limited in order to maintain adequate physiological bone mineralization.

Pharmacological strategies that boost intracellular NAD+ are highly coveted for their therapeutic potential. One approach is activation of nicotinamide phosphoribosyltransferase (NAMPT) to increase production of nicotinamide mononucleotide (NMN), the predominant NAD+ precursor in mammalian cells. A high-throughput screen for NAMPT activators and hit-to-lead campaign yielded SBI-797812, a compound that is structurally similar to active-site directed NAMPT inhibitors and blocks binding of these inhibitors to NAMPT. SBI-797812 shifts the NAMPT reaction equilibrium towards NMN formation, increases NAMPT affinity for ATP, stabilizes phosphorylated NAMPT at His247, promotes consumption of the pyrophosphate by-product, and blunts feedback inhibition by NAD+. These effects of SBI-797812 turn NAMPT into a “super catalyst” that more efficiently generates NMN. Treatment of cultured cells with SBI-797812 increases intracellular NMN and NAD+. Dosing of mice with SBI-797812 elevates liver NAD+. Small molecule NAMPT activators such as SBI-797812 are a pioneering approach to raise intracellular NAD+ and realize its associated salutary effects., Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate determining step for NAD+ synthesis and is of interest as a drug target. Here the authors identify and characterize a small molecule NAMPT activator SBI-797812, elucidate its mode of action and show that it increases intracellular NMN and NAD+ levels in cultured cells and elevates liver NAD+ in mice.

Background Since malaria parasites highly depend on ribose 5-phosphate for DNA and RNA synthesis and on NADPH as a source of reducing equivalents, the pentose phosphate pathway (PPP) is considered an excellent anti-malarial drug target. In Plasmodium, a bifunctional enzyme named glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase (GluPho) catalyzes the first two steps of the PPP. PfGluPho has been shown to be essential for the growth of blood stage Plasmodium falciparum parasites. Methods Plasmodium vivax glucose 6-phosphate dehydrogenase (PvG6PD) was cloned, recombinantly produced in Escherichia coli, purified, and characterized via enzyme kinetics and inhibitor studies. The effects of post-translational cysteine modifications were assessed via western blotting and enzyme activity assays. Genetically encoded probes were employed to study the effects of G6PD inhibitors on the cytosolic redox potential of Plasmodium. Results Here the recombinant production and characterization of PvG6PD, the C-terminal and NADPH-producing part of PvGluPho, is described. A comparison with PfG6PD (the NADPH-producing part of PfGluPho) indicates that the P. vivax enzyme has higher KM values for the substrate and cofactor. Like the P. falciparum enzyme, PvG6PD is hardly affected by S-glutathionylation and moderately by S-nitrosation. Since there are several naturally occurring variants of PfGluPho, the impact of these mutations on the kinetic properties of the enzyme was analysed. Notably, in contrast to many human G6PD variants, the mutations resulted in only minor changes in enzyme activity. Moreover, nanomolar IC50 values of several compounds were determined on P. vivax G6PD (including ellagic acid, flavellagic acid, and coruleoellagic acid), inhibitors that had been previously characterized on PfGluPho. ML304, a recently developed PfGluPho inhibitor, was verified to also be active on PvG6PD. Using genetically encoded probes, ML304 was confirmed to disturb the cytosolic glutathione-dependent redox potential of P. falciparum blood stage parasites. Finally, a new series of novel small molecules with the potential to inhibit the falciparum and vivax enzymes were synthesized, resulting in two compounds with nanomolar activity. Conclusion The characterization of PvG6PD makes this enzyme accessible to further drug discovery activities. In contrast to naturally occurring G6PD variants in the human host that can alter the kinetic properties of the enzyme and thus the redox homeostasis of the cells, the naturally occurring PfGluPho variants studied here are unlikely to have a major impact on the parasites’ redox homeostasis. Several classes of inhibitors have been successfully tested and are presently being followed up. Electronic supplementary material The online version of this article (10.1186/s12936-019-2651-z) contains supplementary material, which is available to authorized users.

Neovascularization is the pathological driver of blinding eye diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. The loss of vision resulting from these diseases significantly impacts the productivity and quality of life of patients, and represents a substantial burden on the health care system. Current standard of care includes biologics that target vascular endothelial growth factor (VEGF), a key mediator of neovascularization. While anti-VGEF therapies have been successful, up to 30% of patients are non-responsive. Therefore, there is a need for new therapeutic targets, and small molecule inhibitors of angiogenesis to complement existing treatments. Apelin and its receptor have recently been shown to play a key role in both developmental and pathological angiogenesis in the eye. Through a cell-based high-throughput screen, we identified 4-aminoquinoline antimalarial drugs as potent selective antagonists of APJ. The prototypical 4-aminoquinoline, amodiaquine was found to be a selective, non-competitive APJ antagonist that inhibited apelin signaling in a concentration-dependent manner. Additionally, amodiaquine suppressed both apelin-and VGEF-induced endothelial tube formation. Intravitreal amodaiquine significantly reduced choroidal neovascularization (CNV) lesion volume in the laser-induced CNV mouse model, and showed no signs of ocular toxicity at the highest doses tested. This work firmly establishes APJ as a novel, chemically tractable therapeutic target for the treatment of ocular neovascularization, and that amodiaquine is a potential candidate for repurposing and further toxicological, and pharmacokinetic evaluation in the clinic.

In search of antiparasitic agents, we here identify arylmethylamino steroids as potent compounds and characterize more than 60 derivatives. The lead compound 1o is fast acting and highly active against intraerythrocytic stages of chloroquine-sensitive and resistant Plasmodium falciparum parasites (IC

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the U.S. National Institutes of Health launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines, but also highlight the need to innovate the science of therapeutic discovery.

Nematodes parasitize ∼1/3 of humans worldwide, and effective treatment via administration of anthelmintics is threatened by growing resistance to current therapies. The nematode transcription factor SKN-1 is essential for development of embryos and upregulates the expression of genes that result in modification, conjugation, and export of xenobiotics, which can promote resistance. Distinct differences in regulation and DNA binding relative to mammalian Nrf2 make SKN-1 a promising and selective target for the development of anthelmintics with a novel mode of action that targets stress resistance and drug detoxification. We report 17 (ML358), a first in class small molecule inhibitor of the SKN-1 pathway. Compound 17 resulted from a vanillamine-derived hit identified by high throughput screening that was advanced through analog synthesis and structure-activity studies. Compound 17 is a potent (IC50 = 0.24 μM, Emax = 100%) and selective inhibitor of the SKN-1 pathway and sensitizes the model nematode C. elegans to oxidants and anthelmintics. Compound 17 is inactive against Nrf2, the homologous mammalian detoxification pathway, and is not toxic to C. elegans (LC50 > 64 μM) and Fa2N-4 immortalized human hepatocytes (LC50 > 5.0 μM). In addition, 17 exhibits good solubility, permeability, and chemical and metabolic stability in human and mouse liver microsomes. Therefore, 17 is a valuable probe to study regulation and function of SKN-1 in vivo. By selective targeting of the SKN-1 pathway, 17 could potentially lead to drug candidates that may be used as adjuvants to increase the efficacy and useful life of current anthelmintics.

Currently there are no efficacious therapies for patients with anaplastic thyroid carcinoma (ATC) that result in long-term disease stabilization or regression.We sought to identify pathways critical for ATC cell progression and viability in an effort to develop new therapeutic strategies. We investigated the effects of targeted inhibition of stearoyl-CoA desaturase 1 (SCD1), a constituent of fatty acid metabolism overexpressed in ATC.A gene array of ATC and normal thyroid tissue was performed to identify gene transcripts demonstrating altered expression in tumor samples. Effects of pharmacological and the genetic inhibition of SCD1 on tumor cell viability as well as cell signaling responses to therapy were evaluated in in vitro and in vivo models of this rare, lethal malignancy.The gene array analysis revealed consistent distortion of fatty acid metabolism and overexpression of SCD1 in ATC and well-differentiated thyroid carcinomas. SCD1 is critical for ATC cell survival and proliferation, the inhibition of which induced endoplasmic reticulum stress, activation of the unfolded protein response, and apoptosis. Combined suppression of endoplasmic reticulum-associated degradation, a prosurvival component of the unfolded protein response, using proteasome inhibitors resulted in a synergistic decrease in tumor cell proliferation and increased cell death.SCD1 is a novel oncogenic factor specifically required for tumor cell viability in ATC. Furthermore, the expression of SCD1 appears to be correlated with thyroid tumor aggressiveness and may serve as a prognostic biomarker. These findings substantiate SCD1 as a novel tumor-specific target for therapy in patients with ATC and should be further investigated in a clinical setting.