The elucidation of species-specific receptor pharmacology: a case study using subtype selective para- and meta-carborane estrogen receptor agonists.

Estrogen receptors are essential pharmacological targets for treating hormonal disorders and estrogen-dependent malignancies. Selective activation of estrogen receptor (ER) ß is hypothesized to provide therapeutic benefit with reduced risk of unwanted estrogenic side-effects associated with ERα activity. However, activating ERß without activating α is challenging due to the high sequence and structural homology between the receptor subtypes. We assessed the impact of structural modifications to the parent compound OSU-ERß-12 on receptor subtype binding selectivity using cell-free binding assays. Functional selectivity was evaluated by transactivation in HEK-293 cells overexpressing human or murine estrogen receptors. In vivo selectivity was examined through the uterotrophic effects of the analogs after oral administration in estrogen-naïve female mice. Furthermore, we evaluated the in vivo pharmacokinetics of the analogs following single dose IV and oral administration. Regarding selectivity, a single compound exhibited greater functional selectivity than OSU-ERß-12 for human ERß. However, like others in the meta-carborane series, its poor in vivo pharmacokinetics limit its suitability for further development. Surprisingly, and at odds with their pharmacokinetic and in vitro human activity data, most analogs potently induced uterotrophic effects in estrogen-naïve female mice. Further investigation of activity in HEK293 cells expressing murine estrogen receptors revealed species-specific differences in the ER-subtype selectivity of these analogs. Our findings highlight species-specific receptor pharmacology and the challenges it poses to characterizing developmental therapeutics in preclinical species. Significance Statement This study investigates para- and meta-substituted carborane analogs targeting estrogen receptors, revealing the greater selectivity of carborane analogs for human ERß compared to the mouse homolog. These findings shed light on the intricacies of using preclinical species in drug development to predict human pharmacology. The report also provides insights for the refinement and optimization of carborane analogs as potential therapeutic agents for estrogen-related disease states.

PP2A is a therapeutically targetable driver of cell fate decisions via a c-Myc/p21 axis in human and murine acute myeloid leukemia.

Dysregulated cellular differentiation is a hallmark of acute leukemogenesis. Phosphatases are widely suppressed in cancers but have not been traditionally associated with differentiation. In this study, we found that the silencing of protein phosphatase 2A (PP2A) directly blocks differentiation in acute myeloid leukemia (AML). Gene expression and mass cytometric profiling revealed that PP2A activation modulates cell cycle and transcriptional regulators that program terminal myeloid differentiation. Using a novel pharmacological agent, OSU-2S, in parallel with genetic approaches, we discovered that PP2A enforced c-Myc and p21 dependent terminal differentiation, proliferation arrest, and apoptosis in AML. Finally, we demonstrated that PP2A activation decreased leukemia-initiating stem cells, increased leukemic blast maturation, and improved overall survival in murine Tet2-/-Flt3ITD/WT and human cell-line derived xenograft AML models in vivo. Our findings identify the PP2A/c-Myc/p21 axis as a critical regulator of the differentiation/proliferation switch in AML that can be therapeutically targeted in malignancies with dysregulated maturation fate.

Discovery of Nanomolar Inhibitors for Human Dihydroorotate Dehydrogenase Using Structure-Based Drug Discovery Methods.

We used a structure-based drug discovery approach to identify novel inhibitors of human dihydroorotate dehydrogenase (DHODH), which is a therapeutic target for treating cancer and autoimmune and inflammatory diseases. In the case of acute myeloid leukemia, no previously discovered DHODH inhibitors have yet succeeded in this clinical application. Thus, there remains a strong need for new inhibitors that could be used as alternatives to the current standard-of-care. Our goal was to identify novel inhibitors of DHODH. We implemented prefiltering steps to omit PAINS and Lipinski violators at the earliest stages of this project. This enriched compounds in the data set that had a higher potential of favorable oral druggability. Guided by Glide SP docking scores, we found 20 structurally unique compounds from the ChemBridge EXPRESS-pick library that inhibited DHODH with IC50, DHODH values between 91 nM and 2.7 µM. Ten of these compounds reduced MOLM-13 cell viability with IC50, MOLM-13 values between 2.3 and 50.6 µM. Compound 16 (IC50, DHODH = 91 nM) inhibited DHODH more potently than the known DHODH inhibitor, teriflunomide (IC50, DHODH = 130 nM), during biochemical characterizations and presented a promising scaffold for future hit-to-lead optimization efforts. Compound 17 (IC50, MOLM-13 = 2.3 µM) was most successful at reducing survival in MOLM-13 cell lines compared with our other hits. The discovered compounds represent excellent starting points for the development and optimization of novel DHODH inhibitors.

Benchmarking a universal smoking machine adaptor for tobacco product testing.

SIGNIFICANCE: Characterisation of tobacco product emissions is an important step in assessing their impact on public health. Accurate and repeatable emissions data require that a leak-tight seal be made between the smoking or vaping machine and the mouth-end of the tobacco product being tested. This requirement is challenging because of the variety of tobacco product mouth-end geometries being puffed on by consumers today. We developed and tested a prototype universal smoking machine adaptor (USMA) that interfaces with existing machines and reliably seals with a variety of tobacco product masses and geometries. METHODS: Emissions were machine-generated using the USMA and other available adaptors for a variety of electronic cigarettes (n=7 brands), cigars (n=4), cigarillos (n=2), a heated tobacco product, and a reference cigarette (1R6F), and mainstream total particulate matter (TPM) and nicotine were quantified. Data variability (precision, n≥10 replicates/brand) for all products and error (accuracy) from certified values (1R6F) were compared across adaptors. RESULTS: TPM and nicotine emissions generated using the USMA were accurate, precise and agreed with certified values for the 1R6F reference cigarette. Replicate data indicate that USMA repeatability across all tobacco products tested generally meets or exceeds that from the comparison adaptors and extant data. CONCLUSION: The USMA seals well with a variety of combustible tobacco products, e-cigarettes with differing geometries and plastic-tipped cigarillos. Variability for all measures was similar or smaller for the USMA compared with other adaptors.

Universal smoking machine adaptor for tobacco product testing.

SIGNIFICANCE: Historically, tobacco product emissions testing using smoking machines has largely focused on combustible products, such as cigarettes and cigars. However, the popularity of newer products, such as electronic cigarettes (e-cigarettes), has complicated emissions testing because the products' mouth-end geometries do not readily seal with existing smoking and vaping machines. The demand for emissions data on popularly used products has led to inefficient and non-standardised solutions, such as laboratories making their geometry-specific custom adaptors and/or employing flexible tubing, for each unique mouth-end geometry tested. A user-friendly, validated, universal smoking machine adaptor (USMA) is needed for testing the variety of tobacco products reflecting consumer use, including e-cigarettes, heated tobacco products, cigarettes, plastic-tipped cigarillos and cigars. METHODS: A prototype USMA that is compatible with existing smoking/vaping machines was designed and fabricated. The quality of the seal between the USMA and different tobacco products, including e-cigarettes, cigars and cigarillos, was evaluated by examining the leak rate. RESULTS: Unlike commercial, product-specific adaptors, the USMA seals well with a wide range of tobacco product mouth-end geometries and masses. This includes e-cigarettes with non-cylindrical mouth ends and cigarillos with cuboid-like plastic tips. USMA leak rates were lower than or equivalent to commercial, product-specific adaptors. CONCLUSION: This report provides initial evidence that the USMA seals reliably with a variety of tobacco product mouth-end geometries and can be used with existing linear smoking/vaping machines to potentially improve the precision, repeatability and reproducibility of machine smoke yield data. Accurate and reproducible emissions testing is critical for regulating tobacco products.

Potent, non-covalent reversible BTK inhibitors with 8-amino-imidazo[1,5-a]pyrazine core featuring 3-position bicyclic ring substitutes.

Bruton's tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition, and for the treatment of B cell related diseases. Many BTK inhibitors have been discovered for the treatment of cancer and rheumatoid arthritis, including a series of BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine we recently reported. The X-ray crystal structures of BTK with inhibitors were also published, which provided great help for the SAR design. Here we report our SAR work introducing ring constraints for the 3-position piperidine amides on the BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine. This modification improved the potency in BTK inhibitions, as well as the PK profile and the off-target selectivity. The dose-dependent efficacy of two BTK inhibitors was observed in the rat collagen induced arthritis (CIA) model.

Understanding systemic problems in providing mental health services to people with an intellectual disability and co-morbid mental disorders in Victoria.

OBJECTIVE: This paper explores the difficulties the services system in Victoria has in responding to people with intellectual disability (ID) and mental health problems and identifies the underlying assumptions that have led to these. These issues are discussed and where possible put into a Victorian context with the intention of informing service development in the area of Dual Disability (co-morbid mental disorders in people with intellectual disability). CONCLUSIONS: People with ID have high levels of mental health need. Generic services have difficulty responding to these needs due to a range of patient, professional and service system factors as well as some of the conceptual issues underpinning policy and legislation.

Pyrimidine depletion enhances targeted and immune therapy combinations in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a fatal disease characterized by the accumulation of undifferentiated myeloblasts, and agents that promote differentiation have been effective in this disease but are not curative. Dihydroorotate dehydrogenase inhibitors (DHODHi) have the ability to promote AML differentiation and target aberrant malignant myelopoiesis. We introduce HOSU-53, a DHODHi with significant monotherapy activity, which is further enhanced when combined with other standard-of-care therapeutics. We further discovered that DHODHi modulated surface expression of CD38 and CD47, prompting the evaluation of HOSU-53 combined with anti-CD38 and anti-CD47 therapies, where we identified a compelling curative potential in an aggressive AML model with CD47 targeting. Finally, we explored using plasma dihydroorotate (DHO) levels to monitor HOSU-53 safety and found that the level of DHO accumulation could predict HOSU-53 intolerability, suggesting the clinical use of plasma DHO to determine safe DHODHi doses. Collectively, our data support the clinical translation of HOSU-53 in AML, particularly to augment immune therapies. Potent DHODHi to date have been limited by their therapeutic index; however, we introduce pharmacodynamic monitoring to predict tolerability while preserving antitumor activity. We additionally suggest that DHODHi is effective at lower doses with select immune therapies, widening the therapeutic index.

Structure activity relationship studies of tricyclic bispyran sulfone γ-secretase inhibitors.

An investigation is detailed of the structure activity relationships (SAR) of two sulfone side chains of compound (-)-1a (SCH 900229), a potent, PS1-selective γ-secretase inhibitor and clinical candidate for the treatment of Alzheimer's disease. Specifically, 4-CF(3) and 4-Br substituted arylsulfone analogs, (-)-1b and (-)-1c, are equipotent to compound (-)-1a. On the right hand side chain, linker size and terminal substituents of the pendant sulfone group are also investigated.

Fused tricyclic mGluR1 antagonists for the treatment of neuropathic pain.

A series of fused tricyclic mGluR1 antagonists containing a pyridone ring were synthesized. In vitro, these antagonists were potent against both human and rat isozymes, as well as selective for inhibiting mGluR1 over mGluR5. When dosed orally, several examples were active in vivo in a rat SNL test.

Synthesis and SAR development of novel mGluR1 antagonists for the treatment of chronic pain.

High throughput screening identified the pyridothienopyrimidinone 1 as a ligand for the metabotropic glutamate receptor 1 (mGluR1=10 nM). Compound 1 has an excellent in vivo profile; however, it displays unfavorable pharmacokinetic issues and metabolic stability. Therefore, using 1 as a template, novel analogues (10i) were prepared. These analogues displayed improved oral exposure and activity in the Spinal Nerve Ligation (SNL) pain model.

A Novel Estrogen Receptor ß Agonist Diminishes Ovarian Cancer Stem Cells via Suppressing the Epithelial-to-Mesenchymal Transition.

Epithelial ovarian cancer is the most lethal malignancy of the female reproductive tract. A healthy ovary expresses both Estrogen Receptor α (ERα) and ß (ERß). Given that ERα is generally considered to promote cell survival and proliferation, thereby, enhancing tumor growth, while ERß shows a protective effect against the development and progression of tumors, the activation of ERß by its agonists could be therapeutically beneficial for ovarian cancer. Here, we demonstrate that the activation of ERß using a newly developed ERß agonist, OSU-ERb-12, can impede ovarian cancer cell expansion and tumor growth in an ERα-independent manner. More interestingly, we found that OSU-ERb-12 also reduces the cancer stem cell (CSC) population in ovarian cancer by compromising non-CSC-to-CSC conversion. Mechanistically, we revealed that OSU-ERb-12 decreased the expression of Snail, a master regulator of the epithelial-to-mesenchymal transition (EMT), which is associated with de novo CSC generation. Given that ERα can mediate EMT and facilitate maintenance of the CSC subpopulation and that OSU-ERb-12 can block the transactivity of ERα, we conclude that OSU-ERb-12 reduces the CSC subpopulation by inhibiting EMT in an ERα-dependent manner. Taken together, our data indicate that the ERß agonist OSU-ERb-12 could be used to hinder tumor progression and limit the CSC subpopulation with the potential to prevent tumor relapse and metastasis in patients with ovarian cancer.

Activity of Estrogen Receptor ß Agonists in Therapy-Resistant Estrogen Receptor-Positive Breast Cancer.

Background: Among women, breast cancer is the leading cause of cancer-related death worldwide. Estrogen receptor α-positive (ERα+) breast cancer accounts for 70% of all breast cancer subtypes. Although ERα+ breast cancer initially responds to estrogen deprivation or blockade, the emergence of resistance compels the use of more aggressive therapies. While ERα is a driver in ERα+ breast cancer, ERß plays an inhibitory role in several different cancer types. To date, the lack of highly selective ERß agonists without ERα activity has limited the exploration of ERß activation as a strategy for ERα+ breast cancer. Methods: We measured the expression levels of ESR1 and ESR2 genes in immortalized mammary epithelial cells and different breast cancer cell lines. The viability of ERα+ breast cancer cell lines upon treatments with specific ERß agonists, including OSU-ERb-12 and LY500307, was assessed. The specificity of the ERß agonists, OSU-ERb-12 and LY500307, was confirmed by reporter assays. The effects of ERß agonists on cell proliferation, cell cycle, apoptosis, colony formation, cell migration, and expression of tumor suppressor proteins were analyzed. The expression of ESR2 and genes containing ERE-AP1 composite response elements was examined in ERα+ human breast cancer samples to determine the correlation between ESR2 expression and overall survival and that of putative ESR2-regulated genes. Results: In this study, we demonstrate the efficacy of highly selective ERß agonists in ERα+ breast cancer cell lines and drug-resistant derivatives. ERß agonists blocked cell proliferation, migration, and colony formation and induced apoptosis and S and/or G2/M cell-cycle arrest of ERα+ breast cancer cell lines. Also, increases in the expression of the key tumor suppressors FOXO1 and FOXO3a were noted. Importantly, the strong synergy between ERß agonists and ERα antagonists suggested that the efficacy of ERß agonists is maximized by combination with ERα blockade. Lastly, ESR2 (ERß gene) expression was negatively correlated with ESR1 (ERα gene) and CCND1 RNA expression in human metastatic ERα+/HER2- breast cancer samples. Conclusion: Our results demonstrate that highly selective ERß agonists attenuate the viability of ERα+ breast cancer cell lines in vitro and suggest that this therapeutic strategy merits further evaluation for ERα+ breast cancer.

Structure-Activity Relationship of para-Carborane Selective Estrogen Receptor ß Agonists.

Selective agonism of the estrogen receptor (ER) subtypes, ERα and ERß, has historically been difficult to achieve due to the high degree of ligand-binding domain structural similarity. Multiple efforts have focused on the use of classical organic scaffolds to model 17ß-estradiol geometry in the design of ERß selective agonists, with several proceeding to various stages of clinical development. Carborane scaffolds offer many unique advantages including the potential for novel ligand/receptor interactions but remain relatively unexplored. We synthesized a series of para-carborane estrogen receptor agonists revealing an ERß selective structure-activity relationship. We report ERß agonists with low nanomolar potency, greater than 200-fold selectivity for ERß over ERα, limited off-target activity against other nuclear receptors, and only sparse CYP450 inhibition at very high micromolar concentrations. The pharmacological properties of our para-carborane ERß selective agonists measure favorably against clinically developed ERß agonists and support further evaluation of carborane-based selective estrogen receptor modulators.

Tetracyclic sulfones as potent gamma-secretase inhibitors: synthesis and structure-activity relationship studies.

Complex tetracyclic sulfones were designed as gamma-secretase inhibitors and a stereoselective synthesis was achieved. Gamma-secretase activity was seen predominately in the (-) enantiomeric series. Compounds such as 2a and 2b showed remarkable in vitro and in vivo potency.

Iminoheterocycles as gamma-secretase modulators.

The synthesis of a novel series of iminoheterocycles and their structure-activity relationship (SAR) as modulators of gamma-secretase activity will be detailed. Encouraging SAR generated from a monocyclic core led to a structurally unique bicyclic core. Selected compounds exhibit good potency as gamma-secretase modulators, excellent rat pharmacokinetics, and lowering of Abeta42 levels in various in vivo models.

T-type calcium channel blockers: spiro-piperidine azetidines and azetidinones-optimization, design and synthesis.

A series of spiro-azetidines and azetidinones has been evaluated as novel blockers of the T-type calcium channel (Ca(V)3.2) which is a new therapeutic target for the potential treatment of both inflammatory and neuropathic pain. Confirmation and optimization of the potency, selectivity and DMPK properties of leads will be described.

Design and synthesis of tricyclic sulfones as gamma-secretase inhibitors with greatly reduced Notch toxicity.

A novel series of tricyclic gamma-secretase inhibitors was designed and synthesized via a conformational analysis of literature compounds. The preliminary results have shown that compounds in this new series have much improved in vitro potency and in vivo profiles. More importantly, they have greatly reduced Notch related toxicity that was associated with previous gamma-secretase inhibitors.

Pharmacokinetics and Tolerability of the Novel Non-immunosuppressive Fingolimod Derivative, OSU-2S, in Dogs and Comparisons with Data in Mice and Rats.

In this study, we characterized the pharmacokinetics of OSU-2S, a fingolimod-derived, non-immunosuppressive phosphatase activator, in mice, rats, and dogs, as well as tolerability and food effects in dogs. Across all species tested, plasma protein binding for OSU-2S was > 99.5%, and metabolic stability and hepatic intrinsic clearance were in the moderate range. OSU-2S did not significantly modulate CYP enzyme activity up until 50 µM, and Caco-2 data suggested low permeability with active efflux at 2 µM. Apparent oral bioavailability in mice was 16% and 69% at 10 and 50 mg/kg, respectively. In rats, bioavailability was 24%, 35%, and 28% at 10, 30, and 100 mg/kg, respectively, while brain/plasma ratio was 36 at 6-h post-dose at 30 mg/kg. In dogs, OSU-2S was well tolerated with oral capsule bioavailability of 27.5%. Plasma OSU-2S exposures increased proportionally over a 2.5-20 mg/kg dose range. After 4 weeks of 3 times weekly, oral administration (20 mg/kg), plasma AUClast (26.1 µM*h), and Cmax (0.899 µM) were nearly 2-fold greater than those after 1 week of dosing, and no food effects were observed. The elimination half-life (29.7 h), clearance (22.9 mL/min/kg), and plasma concentrations of repeated oral doses support a 3-times weekly dosing schedule in dogs. No significant CBC, serum biochemical, or histopathological changes were observed. OSU-2S has favorable oral PK properties similar to fingolimod in rodents and dogs and is well tolerated in healthy animals. This work supports establishing trials of OSU-2S efficacy in dogs with spontaneous tumors to guide its clinical development as a cancer therapeutic for human patients.

Arrowtail RNA for Ligand Display on Ginger Exosome-like Nanovesicles to Systemic Deliver siRNA for Cancer Suppression.

Exosomes have shown increasing potential as delivery vesicles for therapy, but challenges like cost/yield, drug payload, and targeting specificity still exist. Plant derived exosome-like nanoparticles have been reported as a promising substitution and exhibit biocompatibility through oral, intranasal administration; however, systemic delivery of siRNA by exosome-like nanoparticles directly isolated from plants has not been reported. Recently, we reported the control of RNA orientation to decorate human derived exosome with cell targeting ligands for specific delivery of siRNA to tumors. Here, we expand to the application of arrowtail RNA nanoparticles for displaying ligands on ginger derived exosome-like nanovesicles (GDENs) for siRNA delivery and tumor inhibition through IV administration. Cushion ultracentrifugation coupled with equilibrium density gradient ultracentrifugation were used for purifying GDENs that displayed size, density, and morphology similar to human derived exosomes. Folic acid (FA), as a ligand, was displayed on the surface of GDENs for targeted delivery of survivin siRNA to KB cancer models. In vitro gene knockdown efficacy by FA-3WJ/GDENs/siRNA complex was comparable to transfection. We observed inhibition of tumor growth on a xenograft model by intravenous administration, which reveals the potential of GDENs as an economic delivery system for siRNA.