16th GCC Closed Forum: ICH M10 implementation; NGS, qPCR/dPCR, flow cytometry validation; tissue biomarkers; IS response; immunogenicity harmonization; bioanalytical industry status.

The 16th GCC Closed Forum was held in Orlando, FL, USA, on 23 June 2023. Representatives from international bioanalytical Contract Research Organizations were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at the meeting included: IS response, flow cytometry, changes to the bioanalytical industry, NGS assays, biomarker assay for tissues, dPCR validation, immunogenicity harmonization and ICH M10 implementation. Conclusions and consensus from discussions of these topics are included in this article.

Development and validation of a model gene therapy biodistribution assay for AVGN7 using digital droplet polymerase chain reaction.

Biodistribution assays are integral to gene therapy commercialization and have traditionally used real-time qPCR. Droplet digital PCR (ddPCR), however, has distinct advantages including higher sensitivity and absolute quantification but is underused because of lacking regulatory guidance and meaningful examples in the literature. We report a fit-for-purpose model process to validate a good laboratory practice (GLP)-compliant ddPCR assay for AVGN7, a Smad7 gene therapeutic for muscle wasting. Duplexed primer/probe sets for Smad7 and mouse TATA-box binding protein were optimized using gBlock DNA over a dynamic range of 10-80,000 copies/reaction in 250 ng mouse gDNA. Linearized plasmid and mouse gDNA were used for validation, which determined precision, accuracy, ruggedness/robustness, selectivity, recovery, specificity, dilution linearity, and stability. Inter-run precision and accuracy met previously established criteria with bias between -5% and 15%, coefficient of variation (CV) less than 19%, and total error within 8%-35%. The limit of detection was 2.5 copies/reaction, linearity was confirmed at 40-80,000 copies/reaction, specificity was demonstrated by single droplet populations and assay stability was demonstrated for benchtop, refrigerated storage, and repeated freeze-thaw cycles. The procedural road map provided exceeds recently established standards. It is also relevant to many IND-enabling processes, as validated ddPCR assays can be used in biodistribution studies and with vector titering and manufacturing quality control.

Discovery of 6-Phenylhexanamide Derivatives as Potent Stereoselective Mitofusin Activators for the Treatment of Mitochondrial Diseases.

Mutations in the mitochondrial fusion protein mitofusin (MFN) 2 cause the chronic neurodegenerative condition Charcot-Marie-Tooth disease type 2A (CMT2A), for which there is currently no treatment. Small-molecule activators of MFN1 and MFN2 enhance mitochondrial fusion and offer promise as therapy for this condition, but prototype compounds have poor pharmacokinetic properties. Herein, we describe a rational design of a series of 6-phenylhexanamide derivatives whose pharmacokinetic optimization yielded a 4-hydroxycyclohexyl analogue, 13, with the potency, selectivity, and oral bioavailability of a preclinical candidate. Studies of 13 cis- and trans-4-hydroxycyclohexyl isostereomers unexpectedly revealed functionality and protein engagement exclusively for the trans form, 13B. Preclinical absorption, distribution, metabolism, and excretion (ADME) and in vivo target engagement studies of 13B support further development of 6-phenylhexanamide derivatives as therapeutic agents for human CMT2A.

New insights on critical reagent optimization for antidrug antibody assays.

Aim: Conjugated critical reagents are a pillar of ligand binding analysis. Although good practices for characterization have already been discussed, little is known about how assays are affected by specific conjugation parameters. Results: Here we developed, characterized and screened a toolset of bioconjugates that provided new insights about the optimization of conjugated critical reagent attributes. Biotinylated and sulfo-tagged trastuzumab were utilized as capture and detection antibodies, respectively, in an antidrug antibody (ADA) assay. The optimal conjugation window was identified by functional assessment. Excess of unlabeled biotin, but not sulfo-tag, affected the assay performance. An increase in the assay baseline was observed when sulfo-tagged trastuzumab underwent increasing freeze-thaw cycles. Conclusion: Upfront systematic screening and characterization of conjugated critical reagent attributes benefit assay robustness.

MFN2 agonists reverse mitochondrial defects in preclinical models of Charcot-Marie-Tooth disease type 2A.

Mitofusins (MFNs) promote fusion-mediated mitochondrial content exchange and subcellular trafficking. Mutations in Mfn2 cause neurodegenerative Charcot-Marie-Tooth disease type 2A (CMT2A). We showed that MFN2 activity can be determined by Met376 and His380 interactions with Asp725 and Leu727 and controlled by PINK1 kinase-mediated phosphorylation of adjacent MFN2 Ser378 Small-molecule mimics of the peptide-peptide interface of MFN2 disrupted this interaction, allosterically activating MFN2 and promoting mitochondrial fusion. These first-in-class mitofusin agonists overcame dominant mitochondrial defects provoked in cultured neurons by CMT2A mutants MFN2 Arg94→Gln94 and MFN2 Thr105→Met105, as demonstrated by amelioration of mitochondrial dysmotility, fragmentation, depolarization, and clumping. A mitofusin agonist normalized axonal mitochondrial trafficking within sciatic nerves of MFN2 Thr105→Met105 mice, promising a therapeutic approach for CMT2A and other untreatable diseases of impaired neuronal mitochondrial dynamism and/or trafficking.

Cysteine desulfurase is regulated by phosphorylation of Nfs1 in yeast mitochondria.

The cysteine desulfurase Nfs1/Isd11 uses the amino acid cysteine as the substrate and its activity is absolutely required for contributing persulfide sulfur to the essential process of iron-sulfur (Fe-S) cluster assembly in mitochondria. Here we describe a novel regulatory process involving phosphorylation of Nfs1 in mitochondria. Phosphorylation enhanced cysteine desulfurase activity, while dephosphorylation decreased its activity. Nfs1 phosphopeptides were identified, and the corresponding phosphosite mutants showed impaired persulfide formation. Nfs1 pull down from mitochondria recovered an associated kinase activity, and Yck2, a kinase present in the pull down, was able to phosphorylate Nfs1 in vitro and stimulate cysteine desulfurase activity. Yck2 exhibited an eclipsed distribution in the mitochondrial matrix, although other cellular localizations have been previously described. Mitochondria lacking the Yck2 protein kinase (∆yck2) showed less phosphorylating activity for Nfs1. Compared with wild-type mitochondria, ∆yck2 mitochondria revealed slower persulfide formation on Nfs1 consistent with a role of Yck2 in regulating mitochondrial cysteine desulfurase activity. We propose that Nfs1 phosphorylation may provide a means of rapid adaptation to increased metabolic demand for sulfur and Fe-S clusters within mitochondria.

Liquid Chromatography-High Resolution Mass Spectrometry Analysis of Platelet Frataxin as a Protein Biomarker for the Rare Disease Friedreich's Ataxia.

Friedreich's ataxia (FA) is an autosomal recessive disease caused by an intronic GAA triplet expansion in the FXN gene, leading to reduced expression of the mitochondrial protein frataxin. FA is estimated to affect 1 in 50 000 with a mean age of death in the fourth decade of life. There are no approved treatments for FA, although experimental approaches, which involve up-regulation or replacement of frataxin protein, are being tested. Frataxin is undetectable in serum or plasma, and whole blood cannot be used because it is present in long-lived erythrocytes. Therefore, an assay was developed for analyzing frataxin in platelets, which have a half-life of 10 days. The assay is based on stable isotope dilution immunopurification two-dimensional nano-ultra high performance liquid chromatography/parallel reaction monitoring/mass spectrometry. The lower limit of quantification was 0.078 pg frataxin/µg protein, and the assay had 100% sensitivity and specificity for discriminating between controls and FA cases. The mean levels of control and FA platelet frataxin were 9.4 ± 2.6 and 2.4 ± 0.6 pg/µg protein, respectively. The assay should make it possible to rigorously monitor the effects of therapeutic interventions on frataxin expression in this devastating disease.

Nfs1 cysteine desulfurase protein complexes and phosphorylation sites as assessed by mass spectrometry.

Fe-S clusters are cofactors that participate in diverse and essential biological processes. Mitochondria contain a complete machinery for Fe-S cluster assembly. Cysteine desulfurase (Nfs1) is required generation of a form of activated sulfur and is essential for the initial Fe-S cluster assembly step. Using mass-spectometry we identified proteins that were copurified with Nfs1 using a pull-down strategy, including a novel protein kinase. Furthermore, we were able to identify phosphorylation sites on the Nfs1 protein. These data and analyses support the research article "Cysteine desulfurase is regulated by phosphorylation of Nfs1 in yeast mitochondria" by Rocha et al. (in press) [1].

In vitro characterization of a novel Isu homologue from Drosophila melanogaster for de novo FeS-cluster formation.

FeS-clusters are utilized by numerous proteins within several biological pathways that are essential for life. In eukaryotes, the primary FeS-cluster production pathway is the mitochondrial iron-sulfur cluster (ISC) pathway. In Saccharomyces cerevisiae, de novo FeS-cluster formation is accomplished through coordinated assembly with the substrates iron and sulfur by the scaffold assembly protein "Isu1". Sulfur for cluster assembly is provided by cysteine desulfurase "Nfs1", a protein that works in union with its accessory protein "Isd11". Frataxin "Yfh1" helps direct cluster assembly by serving as a modulator of Nfs1 activity, by assisting in the delivery of sulfur and Fe(ii) to Isu1, or more likely through a combination of these and other possible roles. In vitro studies on the yeast ISC machinery have been limited, however, due to the inherent instability of recombinant Isu1. Isu1 is a molecule prone to degradation and aggregation. To circumvent Isu1 instability, we have replaced yeast Isu1 with the fly ortholog to stabilize our in vitro ISC assembly system and assist us in elucidating molecular details of the yeast ISC pathway. Our laboratory previously observed that recombinant frataxin from Drosophila melanogaster has remarkable stability compared to the yeast ortholog. Here we provide the first characterization of D. melanogaster Isu1 (fIscU) and demonstrate its ability to function within the yeast ISC machinery both in vivo and in vitro. Recombinant fIscU has physical properties similar to that of yeast Isu1. It functions as a stable dimer with similar Fe(ii) affinity and ability to form two 2Fe-2S clusters as the yeast dimer. The fIscU and yeast ISC proteins are compatible in vitro; addition of Yfh1 to Nfs1-Isd11 increases the rate of FeS-cluster formation on fIscU to a similar extent observed with Isu1. Finally, fIscU expressed in mitochondria of a yeast strain lacking Isu1 (and its paralog Isu2) is able to completely reverse the deletion phenotypes. These results demonstrate fIscU can functionally replace yeast Isu1 and it can serve as a powerful tool for exploring molecular details within the yeast ISC pathway.

Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope.

PolyGly is present in many proteins in various organisms. One example is found in a transmembrane ß-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.

Life without Fe-S clusters.

Fe-S clusters are critically important cofactors implicated in numerous cellular processes, including respiration, amino acid biosynthesis, cofactor biosynthesis, tRNA modification, DNA repair and regulation of gene expression. In the accompanying manuscript, Tanaka et al. show that reengineering of the isoprenoid biosynthetic pathway in E. coli (to bypass the usage of essential Fe-S cluster proteins by inserting the mevalonate pathway) can offset the indispensability of the Fe-S cluster biosynthetic systems. They show that the resulting Δisc Δsuf double mutants supplemented with mevalonate can grow slowly without detectable Fe-S cluster proteins. This result is astounding and raises interesting questions about what is essential and what is dispensable in the compendium of Fe-S cluster protein functions in this cell.

Phosphorylation of Arabidopsis transketolase at Ser428 provides a potential paradigm for the metabolic control of chloroplast carbon metabolism.

Calcium is an important second messenger in eukaryotic cells that regulates many different cellular processes. To elucidate calcium regulation in chloroplasts, we identified the targets of calcium-dependent phosphorylation within the stromal proteome. A 73 kDa protein was identified as one of the most dominant proteins undergoing phosphorylation in a calcium-dependent manner in the stromal extracts of both Arabidopsis and Pisum. It was identified as TKL (transketolase), an essential enzyme of both the Calvin-Benson-Bassham cycle and the oxidative pentose phosphate pathway. Calcium-dependent phosphorylation of both Arabidopsis isoforms (AtTKL1 and AtTKL2) could be confirmed in vitro using recombinant proteins. The phosphorylation is catalysed by a stroma-localized protein kinase, which cannot utilize GTP. Phosphorylation of AtTKL1, the dominant isoform in most tissues, occurs at a serine residue that is conserved in TKLs of vascular plants. By contrast, an aspartate residue is present in this position in cyanobacteria, algae and mosses. Characterization of a phosphomimetic mutant (S428D) indicated that Ser428 phosphorylation exerts significant effects on the enzyme's substrate saturation kinetics at specific physiological pH values. The results of the present study point to a role for TKL phosphorylation in the regulation of carbon allocation.

Identification of CP12 as a Novel Calcium-Binding Protein in Chloroplasts.

Calcium plays an important role in the regulation of several chloroplast processes. However, very little is still understood about the calcium fluxes or calcium-binding proteins present in plastids. Indeed, classical EF-hand containing calcium-binding proteins appears to be mostly absent from plastids. In the present study we analyzed the stroma fraction of Arabidopsis chloroplasts for the presence of novel calcium-binding proteins using 2D-PAGE separation followed by calcium overlay assay. A small acidic protein was identified by mass spectrometry analyses as the chloroplast protein CP12 and the ability of CP12 to bind calcium was confirmed with recombinant proteins. CP12 plays an important role in the regulation of the Calvin-Benson-Bassham Cycle participating in the assembly of a supramolecular complex between phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase, indicating that calcium signaling could play a role in regulating carbon fixation.

Chloroplast-localized protein kinases: a step forward towards a complete inventory.

In addition to redox regulation, protein phosphorylation has gained increasing importance as a regulatory principle in chloroplasts in recent years. However, only very few chloroplast-localized protein kinases have been identified to date. Protein phosphorylation regulates important chloroplast processes such as photosynthesis or transcription. In order to better understand chloroplast function, it is therefore crucial to obtain a complete picture of the chloroplast kinome, which is currently constrained by two effects: first, recent observations showed that the bioinformatics-based prediction of chloroplast-localized protein kinases from available sequence data is strongly biased; and, secondly, protein kinases are of very low abundance, which makes their identification by proteomics approaches extremely difficult. Therefore, the aim of this study was to obtain a complete list of chloroplast-localized protein kinases from different species. Evaluation of protein kinases which were either highly predicted to be chloroplast localized or have been identified in different chloroplast proteomic studies resulted in the confirmation of only three new kinases. Considering also all reports of experimentally verified chloroplast protein kinases to date, compelling evidence was found for a total set of 15 chloroplast-localized protein kinases in different species. This is in contrast to a much higher number that would be expected based on targeting prediction or on the general abundance of protein kinases in relation to the entire proteome. Moreover, it is shown that unusual protein kinases with differing ATP-binding sites or catalytic centres seem to occur frequently within the chloroplast kinome, thus making their identification by mass spectrometry-based approaches even more difficult due to a different annotation.

The role of calcium in chloroplasts--an intriguing and unresolved puzzle.

More than 70 years of studies have indicated that chloroplasts contain a significant amount of calcium, are a potential storage compartment for this ion, and might themselves be prone to calcium regulation. Many of these studies have been performed on the photosynthetic light reaction as well as CO(2) fixation via the Calvin-Benson-Bassham cycle, and they showed that calcium is required in several steps of these processes. Further studies have indicated that calcium is involved in other chloroplast functions that are not directly related to photosynthesis and that there is a calcium-dependent regulation similar to cytoplasmic calcium signal transduction. Nevertheless, the precise role that calcium has as a functional and regulatory component of chloroplast processes remains enigmatic. Calcium concentrations in different chloroplast subcompartments have been measured, but the extent and direction of intra-plastidal calcium fluxes or calcium transport into and from the cytosol are not yet very well understood. In this review we want to give an overview over the current knowledge on the relationship between chloroplasts and calcium and discuss questions that need to be addressed in future research.

Cross-talk between calcium signalling and protein phosphorylation at the thylakoid.

The role of protein phosphorylation for adjusting chloroplast functions to changing environmental needs is well established, whereas calcium signalling in the chloroplast is only recently becoming appreciated. The work presented here explores the potential cross-talk between calcium signalling and protein phosphorylation in chloroplasts and provides the first evidence for targets of calcium-dependent protein phosphorylation at the thylakoid membrane. Thylakoid proteins were screened for calcium-dependent phosphorylation by 2D gel electrophoresis combined with phospho-specific labelling and PsaN, CAS, and VAR1, among other proteins, were identified repeatedly by mass spectrometry. Subsequently their calcium-dependent phosphorylation was confirmed in kinase assays using the purified proteins and chloroplast extracts. This is the first report on the protein targets of calcium-dependent phosphorylation of thylakoid proteins and provides ground for further studies in this direction.

Arabidopsis calcium-binding mitochondrial carrier proteins as potential facilitators of mitochondrial ATP-import and plastid SAM-import.

Chloroplasts and mitochondria are central to crucial cellular processes in plants and contribute to a whole range of metabolic pathways. The use of calcium ions as a secondary messenger in and around organelles is increasingly appreciated as an important mediator of plant cell signaling, enabling plants to develop or to acclimatize to changing environmental conditions. Here, we have studied the four calcium-dependent mitochondrial carriers that are encoded in the Arabidopsis genome. An unknown substrate carrier, which was previously found to localize to chloroplasts, is proposed to present a calcium-dependent S-adenosyl methionine carrier. For three predicted ATP/phosphate carriers, we present experimental evidence that they can function as mitochondrial ATP-importers.