Inhibitors of heat shock protein 70 (Hsp70) with enhanced metabolic stability reduce tau levels.

The molecular chaperone, Heat Shock Protein 70 (Hsp70), is an emerging drug target for neurodegenerative diseases, because of its ability to promote degradation of microtubule-associated protein tau (MAPT/tau). Recently, we reported YM-08 as a brain penetrant, allosteric Hsp70 inhibitor, which reduces tau levels. However, the benzothiazole moiety of YM-08 is vulnerable to metabolism by CYP3A4, limiting its further application as a chemical probe. In this manuscript, we designed and synthesized seventeen YM-08 derivatives by systematically introducing halogen atoms to the benzothiazole ring and shifting the position of the heteroatom in a distal pyridine. In microsome assays, we found that compound JG-23 has 12-fold better metabolic stability and it retained the ability to reduce tau levels in two cell-based models. These chemical probes of Hsp70 are expected to be useful tools for studying tau homeostasis.

A Phosphoramidate Strategy Enables Membrane Permeability of a Non-nucleotide Inhibitor of the Prolyl Isomerase Pin1.

The membrane permeability of nucleotide-based drugs, such as sofosbuvir (Sovaldi), requires installation of phosphate-caging groups. One strategy, termed "ProTide", masks the anionic phosphate through an N-linked amino ester and an O-linked aromatic phospho-ester, such that release of the active drug requires consecutive enzymatic liberation by an esterase and then a phosphoramidase, such as Hint1. Because Hint1 is known to be selective for nucleotides, it was not clear if the ProTide approach could be deployed for non-nucleotides. Here, we demonstrate that caging of a phosphate-containing inhibitor of the prolyl isomerase Pin1 increases its permeability. Moreover, this compound was processed by both esterase and phosphoramidase activity, releasing the active molecule to bind and inhibit Pin1 in cells. Thus, Hint1 appears to recognize a broader set of substrates than previously appreciated. It seems possible that other potent, but impermeable, phosphate-containing inhibitors might likewise benefit from this approach.

Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding.

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of α-synuclein, a dynamic protein linked to Parkinson's disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of α-synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to α-synuclein biology and pathobiology. We found that the conformation of α-synuclein previously shown to drive yeast toxicity-an extended, membrane-bound helix-is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress α-synuclein toxicity. Moreover, we find that sequence determinants of α-synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that α-synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.

Temperature-jump solution X-ray scattering reveals distinct motions in a dynamic enzyme.

Correlated motions of proteins are critical to function, but these features are difficult to resolve using traditional structure determination techniques. Time-resolved X-ray methods hold promise for addressing this challenge, but have relied on the exploitation of exotic protein photoactivity, and are therefore not generalizable. Temperature jumps, through thermal excitation of the solvent, have been utilized to study protein dynamics using spectroscopic techniques, but their implementation in X-ray scattering experiments has been limited. Here, we perform temperature-jump small- and wide-angle X-ray scattering measurements on a dynamic enzyme, cyclophilin A, demonstrating that these experiments are able to capture functional intramolecular protein dynamics on the microsecond timescale. We show that cyclophilin A displays rich dynamics following a temperature jump, and use the resulting time-resolved signal to assess the kinetics of conformational changes. Two relaxation processes are resolved: a fast process is related to surface loop motions, and a slower process is related to motions in the core of the protein that are critical for catalytic turnover.

Author Correction: Hsp70 and Hsp40 inhibit an inter-domain interaction necessary for transcriptional activity in the androgen receptor.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hsp70 and Hsp40 inhibit an inter-domain interaction necessary for transcriptional activity in the androgen receptor.

Molecular chaperones such as Hsp40 and Hsp70 hold the androgen receptor (AR) in an inactive conformation. They are released in the presence of androgens, enabling transactivation and causing the receptor to become aggregation-prone. Here we show that these molecular chaperones recognize a region of the AR N-terminal domain (NTD), including a FQNLF motif, that interacts with the AR ligand-binding domain (LBD) upon activation. This suggests that competition between molecular chaperones and the LBD for the FQNLF motif regulates AR activation. We also show that, while the free NTD oligomerizes, binding to Hsp70 increases its solubility. Stabilizing the NTD-Hsp70 interaction with small molecules reduces AR aggregation and promotes its degradation in cellular and mouse models of the neuromuscular disorder spinal bulbar muscular atrophy. These results help resolve the mechanisms by which molecular chaperones regulate the balance between AR aggregation, activation and quality control.

Extending chemical perturbations of the ubiquitin fitness landscape in a classroom setting reveals new constraints on sequence tolerance.

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.

Bicyclic [3.3.0]-Octahydrocyclopenta[c]pyrrolo Antagonists of Retinol Binding Protein 4: Potential Treatment of Atrophic Age-Related Macular Degeneration and Stargardt Disease.

Antagonists of retinol-binding protein 4 (RBP4) impede ocular uptake of serum all-trans retinol (1) and have been shown to reduce cytotoxic bisretinoid formation in the retinal pigment epithelium (RPE), which is associated with the pathogenesis of both dry age-related macular degeneration (AMD) and Stargardt disease. Thus, these agents show promise as a potential pharmacotherapy by which to stem further neurodegeneration and concomitant vision loss associated with geographic atrophy of the macula. We previously disclosed the discovery of a novel series of nonretinoid RBP4 antagonists, represented by bicyclic [3.3.0]-octahydrocyclopenta[c]pyrrolo analogue 4. We describe herein the utilization of a pyrimidine-4-carboxylic acid fragment as a suitable isostere for the anthranilic acid appendage of 4, which led to the discovery of standout antagonist 33. Analogue 33 possesses exquisite in vitro RBP4 binding affinity and favorable drug-like characteristics and was found to reduce circulating plasma RBP4 levels in vivo in a robust manner (>90%).

Design, synthesis, and evaluation of nonretinoid retinol binding protein 4 antagonists for the potential treatment of atrophic age-related macular degeneration and Stargardt disease.

Accumulation of lipofuscin in the retina is associated with pathogenesis of atrophic age-related macular degeneration and Stargardt disease. Lipofuscin bisretinoids (exemplified by N-retinylidene-N-retinylethanolamine) seem to mediate lipofuscin toxicity. Synthesis of lipofuscin bisretinoids depends on the influx of retinol from serum to the retina. Compounds antagonizing the retinol-dependent interaction of retinol-binding protein 4 (RBP4) with transthyretin in the serum would reduce serum RBP4 and retinol and inhibit bisretinoid formation. We recently showed that A1120 (3), a potent carboxylic acid based RBP4 antagonist, can significantly reduce lipofuscin bisretinoid formation in the retinas of Abca4(-/-) mice. As part of the NIH Blueprint Neurotherapeutics Network project we undertook the in vitro exploration to identify novel conformationally flexible and constrained RBP4 antagonists with improved potency and metabolic stability. We also demonstrate that upon acute and chronic dosing in rats, 43, a potent cyclopentyl fused pyrrolidine antagonist, reduced circulating plasma RBP4 protein levels by approximately 60%.