Predicted Biological Activity of Purchasable Chemical Space.

Whereas 400 million distinct compounds are now purchasable within the span of a few weeks, the biological activities of most are unknown. To facilitate access to new chemistry for biology, we have combined the Similarity Ensemble Approach (SEA) with the maximum Tanimoto similarity to the nearest bioactive to predict activity for every commercially available molecule in ZINC. This method, which we label SEA+TC, outperforms both SEA and a naïve-Bayesian classifier via predictive performance on a 5-fold cross-validation of ChEMBL's bioactivity data set (version 21). Using this method, predictions for over 40% of compounds (>160 million) have either high significance (pSEA ≥ 40), high similarity (ECFP4MaxTc ≥ 0.4), or both, for one or more of 1382 targets well described by ligands in the literature. Using a further 1347 less-well-described targets, we predict activities for an additional 11 million compounds. To gauge whether these predictions are sensible, we investigate 75 predictions for 50 drugs lacking a binding affinity annotation in ChEMBL. The 535 million predictions for over 171 million compounds at 2629 targets are linked to purchasing information and evidence to support each prediction and are freely available via https://zinc15.docking.org and https://files.docking.org .

Evolutionarily Conserved Roles for Blood-Brain Barrier Xenobiotic Transporters in Endogenous Steroid Partitioning and Behavior.

Central nervous system (CNS) chemical protection depends upon discrete control of small-molecule access by the blood-brain barrier (BBB). Curiously, some drugs cause CNS side-effects despite negligible transit past the BBB. To investigate this phenomenon, we asked whether the highly BBB-enriched drug efflux transporter MDR1 has dual functions in controlling drug and endogenous molecule CNS homeostasis. If this is true, then brain-impermeable drugs could induce behavioral changes by affecting brain levels of endogenous molecules. Using computational, genetic, and pharmacologic approaches across diverse organisms, we demonstrate that BBB-localized efflux transporters are critical for regulating brain levels of endogenous steroids and steroid-regulated behaviors (sleep in Drosophila and anxiety in mice). Furthermore, we show that MDR1-interacting drugs are associated with anxiety-related behaviors in humans. We propose a general mechanism for common behavioral side effects of prescription drugs: pharmacologically challenging BBB efflux transporters disrupts brain levels of endogenous substrates and implicates the BBB in behavioral regulation.

Determination of ubiquitin fitness landscapes under different chemical stresses in a classroom setting.

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.

Activation of neurokinin 3 receptors stimulates GnRH release in a location-dependent but kisspeptin-independent manner in adult mice.

GnRH neurons form the final common pathway for the central control of reproduction. GnRH release occurs from terminals in the external layer of the median eminence (ME) for neuroendocrine control of the pituitary, and near GnRH-GnRH fiber appositions within the preoptic area (POA). Whether or not control of GnRH secretion by neuromodulators is different in these 2 areas is unknown. Mutations in neurokinin B (NKB) or the neurokinin-3 receptor (NK3R) are linked to hypogonadotropic hypogonadism in humans, suggesting that NKB may regulate GnRH secretion. Using fast scan cyclic voltammetry through carbon-fiber microelectrodes, we examined real-time GnRH release in response to the NK3R agonist senktide in the ME and POA. Coronal brain slices were acutely prepared from adult gonad-intact GnRH-green fluorescent protein male mice, and carbon-fiber microelectrodes were placed either within green fluorescent protein-positive terminal fields of the ME or near GnRH-GnRH fiber appositions in the POA. Senktide induced GnRH release consistently in the ME but not the POA, indicating that GnRH release is differentially regulated by NKB in a location-dependent manner. Senktide also induced GnRH secretion in the ME of kisspeptin-knockout (Kiss1 knockout) mice. Interestingly, release amplitude was lower compared with wild-type mice. These data indicate regulation of GnRH release by NK3R agonists is site specific and suggest that kisspeptin is not a required mediator between NK3R activation and GnRH secretion in the ME. This information will be useful for informing future models of afferent regulation of GnRH release.

Orexin a suppresses gonadotropin-releasing hormone (GnRH) neuron activity in the mouse.

GnRH neurons are critical for the central regulation of fertility, integrating steroidal, metabolic and other cues. GnRH neurons appear to lack receptors for many of these cues, suggesting involvement of afferent systems to convey information. Orexin A (orexin) is of interest in this regard as a neuromodulator that up-regulates metabolic activity, increases wakefulness, and affects GnRH/LH release. We examined the electrophysiological response of GnRH neurons to orexin application and how this response changes with estradiol and time of day in a defined animal model. Mice were either ovariectomized (OVX) or OVX and implanted with estradiol capsules (OVX+E). GnRH neurons from OVX+E mice exhibit low firing rates in the morning, due to estradiol-negative feedback, and high firing rates in the evening, due to positive feedback. Orexin inhibited activity of GnRH neurons from OVX mice independent of time of day. In GnRH neurons from OVX+E mice, orexin was inhibitory during the evening, suggesting orexin inhibition is not altered by estradiol. No effect of orexin was observed in OVX+E morning recordings, due to low basal GnRH activity. Inhibitory effects of orexin were mediated by the type 1 orexin receptor, but antagonism of this receptor did not increase GnRH neuron activity during estradiol-negative feedback. Spike pattern analysis revealed orexin increases interevent interval by reducing the number of single spikes and bursts. Orexin reduced spikes/burst and burst duration but did not affect intraburst interval. This suggests orexin may reduce overall firing rate by suppressing spike initiation and burst maintenance in GnRH neurons.