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.

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.

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.