An FDA/CDER perspective on nonclinical testing strategies: Classical toxicology approaches and new approach methodologies (NAMs).

Nonclinical testing of human pharmaceuticals is conducted to assess the safety of compounds to be studied in human clinical trials and for marketing of new drugs. Although there is no exact number and type of nonclinical studies required for safety assessments, as there is inherent flexibility for each new compound, the traditional approach is outlined in various FDA and ICH guidance documents and involves a combination of in vitro assays and whole animal testing methods. Recent advances in science have led to the emergence of numerous new approach methodologies (NAMs) for nonclinical testing that are currently being used in various aspects of drug development. Traditional nonclinical testing methods can predict clinical outcomes, although improvements in these methods that can increase predictivity of clinical outcomes are encouraged and needed. This paper discusses FDA/CDER's view on the opportunities and challenges of using NAMs in drug development especially for regulatory purposes, and also includes examples where NAMs are currently being used in nonclinical safety assessments and where they may supplement and/or enhance current testing methods. FDA/CDER also encourages communication with stakeholders regarding NAMs and is committed to exploring the use of NAMs to improve regulatory efficiency and potentially expedite drug development.

Fixed-target protein serial microcrystallography with an x-ray free electron laser.

We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

Femtosecond X-ray diffraction from two-dimensional protein crystals.

X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Šresolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.

Laboratory-based cryogenic soft x-ray tomography with correlative cryo-light and electron microscopy.

Here we present a novel laboratory-based cryogenic soft X-ray microscope for whole cell tomography of frozen hydrated samples. We demonstrate the capabilities of this compact cryogenic microscope by visualizing internal subcellular structures of Saccharomyces cerevisiae cells. The microscope is shown to achieve better than 50 nm half-pitch spatial resolution with a Siemens star test sample. For whole biological cells, the microscope can image specimens up to 5 µm thick. Structures as small as 90 nm can be detected in tomographic reconstructions following a low cumulative radiation dose of only 7.2 MGy. Furthermore, the design of the specimen chamber utilizes a standard sample support that permits multimodal correlative imaging of the exact same unstained yeast cell via cryo-fluorescence light microscopy, cryo-soft X-ray microscopy, and cryo-transmission electron microscopy. This completely laboratory-based cryogenic soft X-ray microscope will enable greater access to three-dimensional ultrastructure determination of biological whole cells without chemical fixation or physical sectioning.

The bipolar assembly domain of the mitotic motor kinesin-5.

An outstanding unresolved question is how does the mitotic spindle utilize microtubules and mitotic motors to coordinate accurate chromosome segregation during mitosis? This process depends upon the mitotic motor, kinesin-5, whose unique bipolar architecture, with pairs of motor domains lying at opposite ends of a central rod, allows it to crosslink microtubules within the mitotic spindle and to coordinate their relative sliding during spindle assembly, maintenance and elongation. The structural basis of kinesin-5's bipolarity is, however, unknown, as protein asymmetry has so far precluded its crystallization. Here we use electron microscopy of single molecules of kinesin-5 and its subfragments, combined with hydrodynamic analysis plus mass spectrometry, circular dichroism and site-directed spin label electron paramagnetic resonance spectroscopy, to show how a staggered antiparallel coiled-coil 'BASS' (bipolar assembly) domain directs the assembly of four kinesin-5 polypeptides into bipolar minifilaments.

Low-cost cryo-light microscopy stage fabrication for correlated light/electron microscopy.

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

A dynamic role for the Ah receptor in cell signaling? Insights from a diverse group of Ah receptor interacting proteins.

The aryl hydrocarbon (Ah) receptor (AhR) is a member of the basic helix-loop-helix PER-ARNT-SIM (PAS) transcription factor family. Consistent with the notion that PAS proteins are biological sensors, AhR binding to Ah toxicants induces or represses transcription of a wide range of genes and results in a cascade of toxic responses. However, an endogenous role for AhR in development and homeostasis is supported by (1) the discovery of low affinity, endogenous ligands; (2) studies demonstrating a role for the receptor in development of liver and vascular systems, that were established using mice lacking AhR expression; and (3) the presence of functional dioxin-responsive elements in promoter regions of genes involved in cellular growth and differentiation. A large body of recent literature has implicated AhR in multiple signal transduction pathways. AhR is known to interact with signaling pathways that are mediated by estrogen receptor and other hormone receptors, hypoxia, nuclear factor kappaB, and retinoblastoma protein. In addition, AhR complexes may affect cellular signaling through interactions with various other regulatory and signaling proteins, including PAS heterodimerization partners (ARNT), chaperone and immunophilin-like proteins (e.g. HSP90, XAP2/ARA9/AIP, p23), protein kinases and phosphatases (e.g. tyrosine kinases, casein kinase 2, protein kinase C), and coactivators (e.g. SRC-1, RIP 140, CBP/p300). Here we summarize the types of molecular cross talk that have been identified between AhR and cell signaling pathways.

Characterization of the phosphorylation status of the hepatitis B virus X-associated protein 2.

The cytosolic Ah receptor (AhR) heterocomplex consists of one molecule of the AhR, a 90-kDa heat shock protein (Hsp90) dimer, and one molecule of the hepatitis B virus X-associated protein 2 (XAP2). Serine residues 43,53,131-2, and 329 on XAP2-FLAG were identified as putative phosphorylation sites using site-directed mutagenesis followed by two-dimensional phosphopeptide mapping analysis. Protein kinase CK2 (CK2) was identified as the 45-kDa kinase from COS 1 cell or liver extracts that was responsible for phosphorylation of serine 43 in the XAP2 peptide 39-57. Loss of phosphorylation at any or all of the serine residues did not significantly affect the ability of XAP2-FLAG to bind to the murine AhR in rabbit reticulocyte lysate or Hsp90 in COS-1 cells. Furthermore, all of these serine mutants were able to sequester murine AhR-YFP into the cytoplasm as well as wild-type XAP2. YFP-XAP2 S53A was unable to enter the nucleus, indicating a potential role of phosphorylation in nuclear translocation of XAP2.

Sex-specific vitellogenin production in immature rainbow trout.

Vitellogenin (Vg) was measured in sexually immature rainbow trout (Oncorhynchus mykiss) exposed to 17ß-estradiol (E2 ) in the diet. Mixed-sex populations of trout aged 3, 6, 12, or 18 months were maintained separately and fed E2 at 0.05 or 2.5 mg/kg for 7 d. Females fed E2 at 0.05 mg/kg consistently produced three- to fourfold greater amounts of Vg than similarly aged males. Age- and sex-matched fish fed E2 at 2.5 mg/kg produced equivalent amounts of Vg. Sex differences in Vg production were apparent only at a dose of E2 (0.05 mg/kg) that results in submaximal Vg induction. Our results document the importance of considering the sex of juvenile fish when using Vg production as a marker of xenoestrogen exposure.