A unified total synthesis route to 18-trideuterated and/or 19-trideuterated testosterone, androstenedione and progesterone.

A unified total synthesis route has been used to prepare 18- and 19-trideuterated testosterone, androstenedione and progesterone. The 18-trideuterated steroid synthetic method starts with the synthesis of 2-(methyl-d3)-1,3-cyclopentanedione from CD3I and 1,3-cyclopentanedione and is subsequently converted into the Hajos-Parrish ketone for synthesis of these trideuterated steroids. The 19-trideuterated steroid synthesis proceeds through non-deuterated Hajos-Parrish ketone with incorporation of the 19-methyl-d3 group from CD3I at a later stage of the same synthetic route. Utilization of CD3I at both the initial and later stages of the synthesis provides a route to 18,19-hexadeuterated steroids. The deuterated steroids are useful for studies of steroid biosynthesis and metabolism.

Neuroactive steroid effects on autophagy in a human embryonic kidney 293 (HEK) cell model.

Neuropsychiatric and neurodegenerative disorders are correlated with cellular stress. Macroautophagy (autophagy) may represent an important protective pathway to maintain cellular homeostasis and functionality, as it targets cytoplasmic components to lysosomes for degradation and recycling. Given recent evidence that some novel psychiatric treatments, such as the neuroactive steroid (NAS) allopregnanolone (AlloP, brexanolone), may induce autophagy, we stably transfected human embryonic kidney 293 (HEK) cells with a ratiometric fluorescent probe to assay NAS effects on autophagy. We hypothesized that NAS may modulate autophagy in part by the ability of uncharged NAS to readily permeate membranes. Microscopy revealed a weak effect of AlloP on autophagic flux compared with the positive control treatment of Torin1. In high-throughput microplate experiments, we found that autophagy induction was more robust in early passages of HEK cells. Despite limiting studies to early passages for maximum sensitivity, a range of NAS structures failed to reliably induce autophagy or interact with Torin1 or starvation effects. To probe NAS in a system where AlloP effects have been shown previously, we surveyed astrocytes and again saw minimal autophagy induction by AlloP. Combined with other published results, our results suggest that NAS may modulate autophagy in a cell-specific or context-specific manner. Although there is merit to cell lines as a screening tool, future studies may require assaying NAS in cells from brain regions involved in neuropsychiatric disorders.

Endogenous and fluorescent sterols reveal the molecular basis for ligand selectivity of human sterol transporters.

Sterol transport proteins (STPs) play a pivotal role in cholesterol homeostasis and therefore are essential for healthy human physiology. Despite recent advances in dissecting functions of STPs in the human cell, there is still a significant knowledge gap regarding their specific biological functions and a lack of suitable selective probes for their study. Here, we profile fluorescent steroid-based probes across ten STPs, uncovering substantial differences in their selectivity, aiding the retrospective and prospective interpretation of biological results generated with those probes. These results guided the establishment of an STP screening panel combining diverse biophysical assays, enabling the evaluation of 41 steroid-based natural products and derivatives. Combining this with a thorough structural analysis revealed the molecular basis for STP specific selectivity profiles, leading to the uncovering of several new potent and selective Aster-B inhibitors, and supporting the role of this protein in steroidogenesis.

Direct measurements of neurosteroid binding to specific sites on GABAA receptors.

BACKGROUND AND PURPOSE: Neurosteroids are allosteric modulators of GABAA currents, acting through several functional binding sites although their affinity and specificity for each site are unknown. The goal of this study was to measure steady-state binding affinities of various neurosteroids for specific sites on the GABAA receptor. EXPERIMENTAL APPROACH: Two methods were developed to measure neurosteroid binding affinity: (1) quenching of specific tryptophan residues in neurosteroid binding sites by the neurosteroid 17-methylketone group, and (2) FRET between MQ290 (an intrinsically fluorescent neurosteroid) and tryptophan residues in the binding sites. The assays were developed using ELIC-α1GABAAR, a chimeric receptor containing transmembrane domains of the α1-GABAA receptor. Tryptophan mutagenesis was used to identify specific interactions. KEY RESULTS: Allopregnanolone (3α-OH neurosteroid) was shown to bind at intersubunit and intrasubunit sites with equal affinity, whereas epi-allopregnanolone (3ß-OH neurosteroid) binds at the intrasubunit site. MQ290 formed a strong FRET pair with W246, acting as a site-specific probe for the intersubunit site. The affinity and site-specificity of several neurosteroid agonists and inverse agonists was measured using the MQ290 binding assay. The FRET assay distinguishes between competitive and allosteric inhibition of MQ290 binding and demonstrated an allosteric interaction between the two neurosteroid binding sites. CONCLUSIONS AND IMPLICATIONS: The affinity and specificity of neurosteroid binding to two sites in the ELIC-α1GABAAR were directly measured and an allosteric interaction between the sites was revealed. Adaptation of the MQ290 FRET assay to a plate-reader format will enable screening for high affinity agonists and antagonists for neurosteroid binding sites.

Synthetic routes to trifluoromethylphenyl diazirine photolabeling reagents containing an alkyne substituent (TPDYNE) for chemical biology applications.

The trifluoromethylphenyl diazirine (TPD) group is widely used in photoaffinity labeling studies. The TPDYNE group (TPD with an additional alkyne substituent on the phenyl ring) enables the use of click chemistry in conjunction with photoaffinity labeling and expands the utility of the TPD group. New methods for preparing previously known as well as new TPDYNE reagents are reported. Additional methods for preparation of a TPDYNE precursor from which the TPDYNE group can be generated once the precursor is attached to the molecule of interest are also described. Procedures for attaching the TPDYNE or TPDYNE precursor to carboxyl, amino, hydroxyl and alkyne groups are demonstrated using steroids as examples.