Mouse sperm energy restriction and recovery (SER) revealed novel metabolic pathways.

Mammalian sperm must undergo capacitation to become fertilization-competent. While working on mice, we recently developed a new methodology for treating sperm in vitro, which results in higher rates of fertilization and embryo development after in vitro fertilization. Sperm incubated in media devoid of nutrients lose motility, although they remain viable. Upon re-adding energy substrates, sperm resume motility and become capacitated with improved functionality. Here, we explore how sperm energy restriction and recovery (SER) treatment affects sperm metabolism and capacitation-associated signaling. Using extracellular flux analysis and metabolite profiling and tracing via nuclear magnetic resonance (NMR) and mass spectrometry (MS), we found that the levels of many metabolites were altered during the starvation phase of SER. Of particular interest, two metabolites, AMP and L-carnitine, were significantly increased in energy-restricted sperm. Upon re-addition of glucose and initiation of capacitation, most metabolite levels recovered and closely mimic the levels observed in capacitating sperm that have not undergone starvation. In both control and SER-treated sperm, incubation under capacitating conditions upregulated glycolysis and oxidative phosphorylation. However, ATP levels were diminished, presumably reflecting the increased energy consumption during capacitation. Flux data following the fate of 13C glucose indicate that, similar to other cells with high glucose consumption rates, pyruvate is converted into 13C-lactate and, with lower efficiency, into 13C-acetate, which are then released into the incubation media. Furthermore, our metabolic flux data show that exogenously supplied glucose is converted into citrate, providing evidence that in sperm cells, as in somatic cells, glycolytic products can be converted into Krebs cycle metabolites.

A cryptic K48 ubiquitin chain binding site on UCH37 is required for its role in proteasomal degradation.

Degradation by the 26 S proteasome is an intricately regulated process fine tuned by the precise nature of ubiquitin modifications attached to a protein substrate. By debranching ubiquitin chains composed of K48 linkages, the proteasome-associated ubiquitin C-terminal hydrolase UCHL5/UCH37 serves as a positive regulator of protein degradation. How UCH37 achieves specificity for K48 chains is unclear. Here, we use a combination of hydrogen-deuterium mass spectrometry, chemical crosslinking, small-angle X-ray scattering, nuclear magnetic resonance (NMR), molecular docking, and targeted mutagenesis to uncover a cryptic K48 ubiquitin (Ub) chain-specific binding site on the opposite face of UCH37 relative to the canonical S1 (cS1) ubiquitin-binding site. Biochemical assays demonstrate the K48 chain-specific binding site is required for chain debranching and proteasome-mediated degradation of proteins modified with branched chains. Using quantitative proteomics, translation shutoff experiments, and linkage-specific affinity tools, we then identify specific proteins whose degradation depends on the debranching activity of UCH37. Our findings suggest that UCH37 and potentially other DUBs could use more than one S1 site to perform different biochemical functions.

Modulation of Amyloid-ß42 Conformation by Small Molecules Through Nonspecific Binding.

Aggregation of amyloid-ß (Aß) peptides is a crucial step in the progression of Alzheimer's disease (AD). Identifying aggregation inhibitors against AD has been a great challenge. We report an atomistic simulation study of the inhibition mechanism of two small molecules, homotaurine and scyllo-inositol, which are AD drug candidates currently under investigation. We show that both small molecules promote a conformational change of the Aß42 monomer toward a more collapsed phase through a nonspecific binding mechanism. This finding provides atomistic-level insights into designing potential drug candidates for future AD treatments.

"Virtual fragment linking": an approach to identify potent binders from low affinity fragment hits.

In this work we explore the possibilities of using fragment-based screening data to prioritize compounds from a full HTS library, a method we call virtual fragment linking (VFL). The ability of VFL to identify compounds of nanomolar potency based on micromolar fragment binding data was tested on 75 target classes from the WOMBAT database and succeeded in 57 cases. Further, the method was demonstrated for seven drug targets from in-house screening programs that performed both FBS of 8800 fragments and screens of the full library. VFL captured between 28% and 67% of the hits (IC 50 < 10microM) in the top 5% of the ranked library for four of the targets (enrichment between 5-fold and 13-fold). Our findings lead us to conclude that proper coverage of chemical space by the fragment library is crucial for the VFL methodology to be successful in prioritizing HTS libraries from fragment-based screening data.

Application of NMR screening in drug discovery.

The application of NMR screening in drug discovery has recently attained heightened importance throughout the pharmaceutical industry. NMR screening can be applied at various points in a drug discovery program, ranging from very early in the program, when new targets can be screened long before an HTS enzymatic assay is developed, to later in the program, as in the case where no useful hits have been detected by HTS using biological assays. The binders determined in primary NMR screens are used to guide secondary screens, which can be either completely NMR driven or use NMR in combination with other biophysical techniques. In this review we briefly discuss the methods and techniques used in NMR screening. Then, we describe in detail the NMR screening strategies and their applications to specific targets, including successful examples from actual drug design programs at our own and other pharmaceutical companies.

Applications of SHAPES screening in drug discovery.

The SHAPES strategy combines nuclear magnetic resonance (NMR) screening of a library of small drug-like molecules with a variety of complementary methods, such as virtual screening, high throughput enzymatic assays, combinatorial chemistry, X-ray crystallography, and molecular modeling, in a directed search for new medicinal chemistry leads. In the past few years, the SHAPES strategy has found widespread utility in pharmaceutical research. To illustrate a variety of different implementations of the method, we will focus in this review on recent applications of the SHAPES strategy in several drug discovery programs at Vertex Pharmaceuticals.

Conformational refinement of hydroxamate-based histone deacetylase inhibitors and exploration of 3-piperidin-3-ylindole analogues of dacinostat (LAQ824).

Inspired by natural product HDAC inhibitors, we prepared a series of conformationally restrained HDAC inhibitors based on the hydroxamic acid dacinostat (LAQ824, 7). Several scaffolds with improved biochemical and cellular potency, as well as attenuated hERG inhibition, were identified, suggesting that the introduction of molecular rigidity is a viable strategy to enhance HDAC binding and mitigate hERG liability. Further SAR studies around a 3-piperidin-3-ylindole moiety resulted in the discovery of compound 30, for which a unique conformation was speculated to contribute to overcoming increased lipophilicity and attenuating hERG binding. Separation of racemate 30 afforded 32, the R enantiomer, which demonstrated improved potency in both enzyme and cellular assays compared to dacinostat.

Ultrahigh-resolution (1)H-(13)C HSQC spectra of metabolite mixtures using nonlinear sampling and forward maximum entropy reconstruction.

To obtain a comprehensive assessment of metabolite levels from extracts of leukocytes, we have recorded ultrahigh-resolution 1H-13C HSQC NMR spectra of cell extracts, which exhibit spectral signatures of numerous small molecules. However, conventional acquisition of such spectra is time-consuming and hampers measurements on multiple samples, which would be needed for statistical analysis of metabolite concentrations. Here we show that the measurement time can be dramatically reduced without loss of spectral quality when using nonlinear sampling (NLS) and a new high-fidelity forward maximum-entropy (FM) reconstruction algorithm. This FM reconstruction conserves all measured time-domain data points and guesses the missing data points by an iterative process. This consists of discrete Fourier transformation of the sparse time-domain data set, computation of the spectral entropy, determination of a multidimensional entropy gradient, and calculation of new values for the missing time-domain data points with a conjugate gradient approach. Since this procedure does not alter measured data points, it reproduces signal intensities with high fidelity and does not suffer from a dynamic range problem. As an example we measured a natural abundance 1H-13C HSQC spectrum of metabolites from granulocyte cell extracts. We show that a high-resolution 1H-13C HSQC spectrum with 4k complex increments recorded linearly within 3.7 days can be reconstructed from one-seventh of the increments with nearly identical spectral appearance, indistinguishable signal intensities, and comparable or even lower root-mean-square (rms) and peak noise patterns measured in signal-free areas. Thus, this approach allows recording of ultrahigh resolution 1H-13C HSQC spectra in a fraction of the time needed for recording linearly sampled spectra.

Leveraging structural approaches: applications of NMR-based screening and X-ray crystallography for inhibitor design.

In the last several years, NMR strategies in drug discovery have evolved from a primarily structural focus to a set of technologies that are non-structural in nature but that have a much greater impact on the identification and optimization of real drug leads. NMR-based screening methods, such as the SHAPES strategy, help rapidly identify good starting points for drug design in a relatively high throughput implementation. The SHAPES method uses simple NMR techniques to detect binding of a limited, but diverse library of low molecular weight, soluble compounds to a potential drug target. SHAPES library compounds are derived largely from molecular frameworks most commonly found in known therapeutic agents. The NMR experiments used in these protocols are based on the well-known NMR techniques, and may be applied to targets with no limitation on molecular weight and no requirement for isotope labeling. Following screening, SHAPES hits may be used to guide virtual screening, synthesis of combinatorial libraries, and bias the first compounds that undergo high throughput screening. Integration of the SHAPES strategy with iterative X-ray crystallographic structure determination can be very useful in deriving an initial structural pharmacophore model and achieving significant in vitro potency in a short time frame. Here, examples are provided of how the combination of NMR SHAPES screening, virtual screening, molecular modeling and X-ray crystallography has led to novel drug scaffolds in several drug discovery programs: JNK3 MAP kinase and the fatty acid binding protein, aP2.