High Throughput Combinatorial Formatting of PcrV Nanobodies for Efficient Potency Improvement.

Improving potencies through concomitant blockage of multiple epitopes and avid binding by fusing multiple (different) monovalent Nanobody building blocks via linker sequences into one multivalent polypeptide chain is an elegant alternative to affinity maturation. We explored a large and random formatting library of bivalent (combinations of two identical) and biparatopic (combinations of two different) Nanobodies for functional blockade of Pseudomonas aeruginosa PcrV. PcrV is an essential part of the P. aeruginosa type III secretion system (T3SS), and its oligomeric nature allows for multiple complex binding and blocking options. The library screening yielded a large number of promising biparatopic lead candidates, revealing significant (and non-trivial) preferences in terms of Nanobody building block and epitope bin combinations and orientations. Excellent potencies were confirmed upon further characterization in two different P. aeruginosa T3SS-mediated cytotoxicity assays. Three biparatopic Nanobodies were evaluated in a lethal mouse P. aeruginosa challenge pneumonia model, conferring 100% survival upon prophylactic administration and reducing lung P. aeruginosa burden by up to 2 logs. At very low doses, they protected the mice from P. aeruginosa infection-related changes in lung histology, myeloperoxidase production, and lung weight. Importantly, the most potent Nanobody still conferred protection after therapeutic administration up to 24 h post-infection. The concept of screening such formatting libraries for potency improvement is applicable to other targets and biological therapeutic platforms.

Dose- and Time-dependency of the Toxicity and Pharmacokinetic Profiles of Bedaquiline and Its N-desmethyl Metabolite in Dogs.

Bedaquiline (BDQ) is an antibiotic to treat pulmonary multidrug-resistant tuberculosis (MDR-TB). Studies up to 39 weeks were conducted orally in dogs to assess the toxicity and pharmacokinetics of BDQ and its N-desmethyl metabolite (D-BDQ). Phospholipidosis (PLD) seen in the monocytic phagocytic system was considered an adaptive change. Skeletal muscle, heart, stomach, liver, and pancreas toxicities with D-BDQ as the main contributor were associated with a less-than-dose-proportional increase in plasma exposure and an overproportional tissue uptake of BDQ and D-BDQ at high-dose levels. Tissue concentrations of BDQ and D-BDQ slowly decreased after lowering the dose, contributing to the recovery of the pathological findings. Treatment was better tolerated at mid-dose levels, characterized by a dose-proportional increase in plasma and tissue exposures. Treatment at a low dose, reaching exposures approximating therapeutic exposures, was without adverse effects and not associated with PLD. There was no evidence of delayed toxicities after treatment cessation. Intermittent dosing was better tolerated at high doses. Since MDR-TB patients are dosed within the linear plasma exposure range and plasma levels of BDQ and D-BDQ are similar or lower than in dogs, PLD and adverse findings related to tissue accumulation that occurred at high doses in dogs are unlikely to occur in humans.

Correlated positions in protein evolution and engineering.

Statistical analysis of a protein multiple sequence alignment can reveal groups of positions that undergo interdependent mutations throughout evolution. At these so-called correlated positions, only certain combinations of amino acids appear to be viable for maintaining proper folding, stability, catalytic activity or specificity. Therefore, it is often speculated that they could be interesting guides for semi-rational protein engineering purposes. Because they are a fingerprint from protein evolution, their analysis may provide valuable insight into a protein's structure or function and furthermore, they may also be suitable target positions for mutagenesis. Unfortunately, little is currently known about the properties of these correlation networks and how they should be used in practice. This review summarises the recent findings, opportunities and pitfalls of the concept.

Synthesis and SAR assessment of novel Tubathian analogs in the pursuit of potent and selective HDAC6 inhibitors.

The synthesis of novel isoform-selective HDAC inhibitors is considered to be an important, emerging field in medicinal chemistry. In this paper, the preparation and assessment of thirteen selective HDAC6 inhibitors is disclosed, elaborating on a previously developed thiaheterocyclic Tubathian series. All compounds were evaluated in vitro for their ability to inhibit HDAC6, and a selection of five potent compounds was further screened toward all HDAC isoforms (HDAC1-11). The capability of these Tubathian analogs to inhibit α-tubulin deacetylation was assessed as well, and ADME/Tox data were collected. This thorough SAR evaluation revealed that the oxidized, para-substituted hydroxamic acids can be recognized as valuable lead structures in the pursuit of novel potent and selective HDAC6 inhibitors.

Creating Space for Large Acceptors: Rational Biocatalyst Design for Resveratrol Glycosylation in an Aqueous System.

Polyphenols display a number of interesting properties but their low solubility limits practical applications. In that respect, glycosylation offers a solution for which sucrose phosphorylase has been proposed as a cost-effective biocatalyst. However, its activity on alternative acceptor substrates is too low for synthetic purposes and typically requires the addition of organic (co-)solvents. Here, we describe the engineering of the enzyme from Thermoanaerobacterium thermosaccharolyticum to enable glycosylation of resveratrol as test case. Based on docking and modeling studies, an active-site loop was predicted to hinder binding. Indeed, the unbolted loop variant R134A showed useful affinity for resveratrol (K(m)=185 mM) and could be used for the quantitative production of resveratrol 3-α-glucoside in an aqueous system. Improved activity was also shown for other acceptors, introducing variant R134A as promising new biocatalyst for glycosylation reactions on bulky phenolic acceptors.

Engineering the carbohydrate-binding site of Epa1p from Candida glabrata: generation of adhesin mutants with different carbohydrate specificity.

The N-terminal domain of the Epa1p adhesin from Candida glabrata (N-Epa1p) is a calcium-dependent lectin, which confers the opportunistic yeast the ability to adhere to human epithelial cells. This lectin domain is able to interact with galactosides and, more precisely, with glycan molecules containing the Galß-1,3-GalNAc disaccharide, also known as the T-antigen. Based on the crystallographic structure of the N-Epa1p domain and the role of the variable loop CBL2 in glycan binding, saturation mutagenesis on some residues of the CBL2 loop was used to increase the binding affinity of N-Epa1p for fibronectin, which was selected as a model of a human glycoprotein. Two adhesin mutants, E227A and Y228W, with improved binding features were obtained. More importantly, a glycan array screening revealed that single-point mutations in the CBL2 could produce significant changes in the carbohydrate specificity of the protein. In particular, lectin molecules were generated with a high affinity for sulfated glycans, which may find an application as molecular probes for the identification of 6-sulfogalactose containing glycans and glycoconjugates.

The quest for a thermostable sucrose phosphorylase reveals sucrose 6'-phosphate phosphorylase as a novel specificity.

Sucrose phosphorylase is a promising biocatalyst for the glycosylation of a wide range of compounds, but its industrial application has been hampered by the low thermostability of known representatives. Hence, in this study, the putative sucrose phosphorylase from the thermophile Thermoanaerobacterium thermosaccharolyticum was recombinantly expressed and fully characterised. The enzyme showed significant activity on sucrose (optimum at 55 °C), and with a melting temperature of 79 °C and a half-life of 60 h at the industrially relevant temperature of 60 °C, it is far more stable than known sucrose phosphorylases. Substrate screening and detailed kinetic characterisation revealed however a preference for sucrose 6'-phosphate over sucrose. The enzyme can thus be considered as a sucrose 6'-phosphate phosphorylase, a specificity not yet reported to date. Homology modelling and mutagenesis pointed out particular residues (Arg134 and His344) accounting for the difference in specificity. Moreover, phylogenetic and sequence analysis suggest that glycoside hydrolase 13 subfamily 18 might harbour even more specificities. In addition, the second gene residing in the same operon as sucrose 6'-phosphate phosphorylase was identified as well, and found to be a phosphofructokinase. The concerted action of both these enzymes implies a new pathway for the breakdown of sucrose, in which the reaction products end up at different stages of the glycolysis.

Therapeutic siRNA Loaded to RISC as Single and Double Strands Requires an Appropriate Quantitative Assay for RISC PK Assessment.

In recent years, therapeutic siRNA projects are booming in the biotech and pharmaceutical industries. As these drugs act by silencing the target gene expression, a critical step is the binding of antisense strands of siRNA to RNA-induced silencing complex (RISC) and then degrading their target mRNA. However, data that we recently obtained suggest that double-stranded siRNA can also load to RISC. This brings a new understanding of the mechanism of RISC loading which may have a potential impact on how quantification of RISC loaded siRNA should be performed. By combining RNA immune precipitation and probe-based hybridization LC-fluorescence approach, we have developed a novel assay that can accurately quantify the RISC-bound antisense strand, irrespective of which form (double-stranded or single-stranded) is loaded on RISC. In addition, this novel assay can discriminate between the 5'-phosphorylated antisense (5'p-AS) and the nonphosphorylated forms, therefore specifically quantifying the RISC bound 5'p-AS. In comparison, stem-loop qPCR assay does not provide discrimination and accurate quantification when the oligonucleotide analyte exists as a mixture of double and single-stranded forms. Taking together, RISC loading assay with probe-hybridization LC-fluorescence technique would be a more accurate and specific quantitative approach for RISC-associated pharmacokinetic assessment.

Recommendations and feedback from the European Bioanalysis Forum Workshop: 1 year into ICH M10 - keeping our finger on the pulse.

The ICH M10 guideline on bioanalytical method validation and sample analysis is being adopted since 2023. However, and inevitably, some paragraphs or requirements remain ambiguous and are open for different interpretations. In support of a harmonized interpretation by the industry and health authorities, the European Bioanalysis Forum organized a workshop on 14 November 2023 in Barcelona, Spain, to discuss unclear and/or ambiguous paragraphs which were identified by the European Bioanalysis Forum community and delegates of the workshop prior to the workshop. This manuscript reports back from the workshop with recommendations and aims at continuing an open scientific discussion within the industry and with regulators in support of a science-driven guideline for the bioanalytical community and in line with the ICH mission - that is, achieve greater harmonization worldwide to ensure that safe, effective and high-quality medicines are developed and registered in the most resource-efficient manner.

Consensus engineering of sucrose phosphorylase: the outcome reflects the sequence input.

Consensus engineering, which is replacing amino acids by the most frequently occurring one at their positions in a multiple sequence alignment (MSA), is a known strategy to increase the stability of a protein. The application of this concept to the entire sequence of an enzyme, however, has been tried only a few times mainly because of the problems determining the consensus in highly variable regions. We show that this problem can be solved by replacing such problematic regions by the corresponding sequence of the natural homologue closest to the consensus. When one or a few sub-families are overrepresented in the MSA the consensus sequence is a biased representation of the sequence space. We examine the influence of this bias by constructing three consensus sequences using different MSAs of sucrose phosphorylase (SP). Each consensus enzyme contained about 70 mutations compared to its closest natural homologue and folded correctly and displayed activity on sucrose. Correlation analysis revealed that the family's co-evolution network was kept intact, which is one of the main advantages of full-length consensus design. The consensus enzymes displayed an "average" thermostability, that is, one that is higher than some but not all known representatives. We cautiously present practical rules for the design of consensus sequences, but warn that the measure of success depends on which natural enzyme is used as point of comparison.

Synthesis of 2-aminomethyl-4-phenyl-1-azabicyclo[2.2.1]heptanes via LiAlH4-induced reductive cyclization of 2-(4-chloro-2-cyano-2-phenylbutyl)aziridines and evaluation of their antimalarial activity.

2-(4-Chloro-2-cyano-2-phenylbutyl)aziridines were employed for the one-step stereoselective construction of both endo- and exo-2-aminomethyl-4-phenyl-1-azabicyclo[2.2.1]heptanes as new azaheterobicyclic scaffolds via a double LiAlH(4)-induced reductive cyclization protocol. Antiplasmodial assessment of these 1-azabicyclo[2.2.1]heptanes revealed moderate to good activities in the micromolar range, with the exo-isomers being the most promising structures. Furthermore, the proposed mode of action was supported by ligand docking studies, pointing to a strong binding interaction with the enzyme plasmepsin II.

Conference Report from the European Bioanalysis Forum Workshop: toward harmonized implementation of the ICH M10 guideline.

In this report, the European Bioanalysis Forum shares the proposals for harmonized implementation of the ICH M10 guideline on bioanalytical method validation and study sample analysis from the ICH M10 workshop. The focus of the discussions was to understand new, changed or still ambiguous regulatory expectations in the guideline, as identified in feedback from the pre-workshop surveys or during the workshop. The proposals from the workshop aim at stimulating and helping a harmonized implementation of the guideline, and using our community as a sounding board during and after implementation to highlight areas of misalignment and to create a platform for continued sharing with the regulatory authorities in an effort to contribute to industry and regulators developing similar interpretations on guideline expectations.

Multimodal biomarker discovery for active Onchocerca volvulus infection.

The neglected tropical disease onchocerciasis, or river blindness, is caused by infection with the filarial nematode Onchocerca volvulus. Current estimates indicate that 17 million people are infected worldwide, the majority of them living in Africa. Today there are no non-invasive tests available that can detect ongoing infection, and that can be used for effective monitoring of elimination programs. In addition, to enable pharmacodynamic studies with novel macrofilaricide drug candidates, surrogate endpoints and efficacy biomarkers are needed but are non-existent. We describe the use of a multimodal untargeted mass spectrometry-based approach (metabolomics and lipidomics) to identify onchocerciasis-associated metabolites in urine and plasma, and of specific lipid features in plasma of infected individuals (O. volvulus infected cases: 68 individuals with palpable nodules; lymphatic filariasis cases: 8 individuals; non-endemic controls: 20 individuals). This work resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine (CCG) as biomarker for O. volvulus. During the targeted validation study, metabolite-specific cutoffs were determined (inosine: 34.2 ng/ml; hypoxanthine: 1380 ng/ml; CCG: 29.7 ng/ml) and sensitivity and specificity profiles were established. Subsequent evaluation of these biomarkers in a non-endemic population from a different geographical region invalidated the urine metabolite CCG as biomarker for O. volvulus. The plasma metabolites inosine and hypoxanthine were confirmed as biomarkers for filarial infection. With the availability of targeted LC-MS procedures, the full potential of these 2 biomarkers in macrofilaricide clinical trials, MDA efficacy surveys, and epidemiological transmission studies can be investigated.

Capillary microsampling in clinical studies: opportunities and challenges in two case studies.

Aim: Capillary microsampling of 15 µl whole blood from fingersticks or heelsticks was used to collect pharmacokinetic (PK) samples from pediatric subjects in two projects. Results: In a mebendazole multisite study in Ethiopia and Rwanda in subjects between 1 and 16 years old, complete PK profiles (7 timepoints) could be obtained, although some of the fingerstick samples were contaminated by the dosing formulation. In a multisite study with a respiratory syncytial virus drug in children between 1 and 24 months old, sparse PK sampling was done (2 samples). All samples were successfully analyzed even though some capillaries were not properly filled. Conclusion: CMS shows potential for PK sampling in pediatrics but may need further optimization.

European Bioanalysis Forum feedback on draft ICH M10 guideline on bioanalytical method validation during the Step 2b public consultation period.

Once released, the ICH M10 Guideline on bioanalytical method validation will become one of the most important milestones in the history of regulated bioanalysis, closing a chapter on intense discussions among the industry and health authorities started in Crystal City in 2001. In this manuscript, the European Bioanalysis Forum community reports back on their feedback on the ICH M10 draft guideline gathered during the public consultation period. The comments given are intended to contribute to a guideline that combines several decades of experience and current scientific vision. They should provide future generations of bioanalytical scientist a regulatory framework so their bioanalytical work can contribute to safe, effective and high-quality medicines, which can be developed and registered in the most resource-efficient manner.

Pharmacokinetics of carisbamate (RWJ-333369) in healthy Japanese and Western subjects.

PURPOSE: To compare the pharmacokinetics of carisbamate (RWJ-333369) in healthy Japanese and Western adults, and to comparatively assess carisbamate safety and tolerability between the two populations. METHODS: An open-label study was conducted in 24 Japanese and 24 Caucasian healthy subjects. Subjects received a single oral dose of 250 mg carisbamate on day 1 followed by a 3-day washout period; twice-daily dosing of 250 mg carisbamate on days 5-8; subsequently, 500 mg on days 9-12 and a single dose of 500 mg on day 13. Plasma samples were collected for a pharmacokinetic analysis on days 1, 8, and 13. Plasma and urine samples were analyzed for carisbamate and its urinary metabolites by liquid-chromatography-mass-spectrometry. RESULTS: Following a single dose, carisbamate Cmax and area under the curve (AUC) geometric mean ratios were 16.4% and 28.8% higher in Japanese than in Caucasians, respectively; these differences were statistically significant and their 90% confidence intervals (CIs) fell outside of the 80-125% limits, which are considered not to be of clinical significance. With dose-body weight normalization, Cmax and AUC were similar in Japanese and Caucasian subjects and the 90% CIs were within the 80-125% boundaries. Carisbamate was well tolerated, and its mean oral clearance and half-life were similar in both groups, ranging from 35.1-41.4 ml/h/kg and 11.5-12.8 h. DISCUSSION: Carisbamate plasma exposure (AUC) and C(max) in Japanese subjects is approximately 20-25% higher than in Caucasians due to a higher mg/kg dose. After body weight normalization, carisbamate pharmacokinetics was similar between Japanese and Caucasian subjects following single and multiple dosing, and showed the same dose proportionality.

Systematic internal standard variability and issue resolution: two case studies.

Two case studies are presented of validated assays where the internal standard showed high variability, and there was a clear response difference between study samples and standards and quality controls. In the first case a co-eluting peak boosted the stable isotope labeled internal standard response in samples from hepatically impaired subjects. In the second case the blank plasma matrix suppressed the structural analog internal standard response. For both assays the issue could be resolved by adapting the chromatographic conditions and re-validating the assay (case 1) or by diluting the study samples with blank plasma (case 2).

LC-MS quantification of oligonucleotides in biological matrices with SPE or hybridization extraction.

Aim: Quantitative LC-MS analysis of oligonucleotides (OGNs) in biological matrices is needed to support candidate selection of new therapeutic OGNs. Methodology & results: A set of 20 single stranded antisense oligonucleotides (ASO) and five siRNAs were extracted from plasma and tissue homogenates. Anion Exchange (AEX) SPE was selected as generic extraction approach, resulting in recoveries from plasma >70%. Extraction from tissue homogenates showed often more variation and lower recoveries. A proof of concept of a novel tailored hybridization extraction is demonstrated for two 16-mer reference OGNs. Conclusion: Two methods for extraction of OGNs were investigated and applied for quantitative analysis. The AEX-SPE is considered a more generic approach preferred when multiple compounds are evaluated. Hybridization extraction has great potential but critical reagents per analyte are needed.

Comparison of toxicokinetic parameters of a drug and two metabolites following traditional and capillary microsampling in rat.

Aim: Following the request of a regulatory authority, a rat study was conducted to compare pharmacokinetic parameters from traditional large volume sampling and capillary microsampling. Materials & methods: Rats were dosed with a proprietary compound in three dose groups and blood samples were collected via capillary microsampling (32 µl), immediately followed by traditional large volume sampling (300 µl) up to 24 h postdose. Resulting plasma samples were analyzed for parent drug and two metabolites. AUCs were compared between sampling techniques. Results: There was no statistical difference between AUCs from traditional and microsampling across different doses and analytes. Conclusion: Toxicokinetic parameters generated from plasma collected as a capillary microsample or traditional large volume sample are highly comparable.