Application of the model-informed drug development paradigm to datopotamab deruxtecan dose selection for late-stage development.

To replace the conventional maximum tolerated dose (MTD) approach, a paradigm for dose optimization and dose selection that relies on model-informed drug development (MIDD) approaches has been proposed in oncology. Here, we report our application of an MIDD approach during phase I to inform dose selection for the late-stage development of datopotamab deruxtecan (Dato-DXd). Dato-DXd is a TROP2-directed antibody-drug conjugate being developed for advanced/metastatic non-small cell lung cancer (NSCLC) and other tumors. Data on pharmacokinetics (PKs), efficacy, and safety in NSCLC were collected in the TROPION-PanTumor01 phase I dose-expansion and -escalation study over a wide dose range of 0.27-10 mg/kg administered every 3 weeks. Population PK and exposure-response analyses were performed iteratively at three data cutoffs to inform dose selection. The 6 mg/kg dose was identified as the optimal dose by the second data cutoff analysis and confirmed by the subsequent third data cutoff analysis. The 6 mg/kg dose was more tolerable (i.e., lower rates of interstitial lung disease, stomatitis, and mucosal inflammation) than the MTD (8 mg/kg) and was more efficacious than 4 mg/kg (simulated mean objective response rate: 23.8% vs. 18.6%; mean hazard ratio of progression-free survival: 0.74) - a candidate dose studied just below 6 mg/kg. Therefore, the 6 mg/kg dose was judged to afford the optimal benefit-risk balance. This case study demonstrated the utility of an MIDD approach for dose optimization and dose selection.

Telomere-to-telomere genome assembly of matsutake (Tricholoma matsutake).

Here, we report the first telomere-to-telomere genome assembly of matsutake (Tricholoma matsutake), which consists of 13 sequences (spanning 161.0 Mb) and a 76 kb circular mitochondrial genome. All the 13 sequences were supported with telomeric repeats at the ends. GC-rich regions are located at the middle of the sequences and are enriched with long interspersed nuclear elements (LINEs). Repetitive sequences including long-terminal repeats (LTRs) and LINEs occupy 71.6% of the genome. A total of 21,887 potential protein-coding genes were predicted. The genomic data reported in this study served not only matsutake gene sequences but also genome structures and intergenic sequences. The information gained would be a great reference for exploring the genetics, genomics, and evolutionary study of matsutake in the future, and ultimately facilitate the conservation of this vulnerable genetic resource.

Population Pharmacokinetics of Patritumab Deruxtecan in Patients With Solid Tumors.

Patritumab deruxtecan is an antibody-drug conjugate consisting of a fully human monoclonal antibody against human epidermal growth factor receptor 3 (HER3) attached to a topoisomerase I inhibitor payload via a tetrapeptide-based cleavable linker. As part of the pharmacometric analysis informing dose selection for later-stage development, population pharmacokinetics (PK) analysis of patritumab deruxtecan was conducted with pooled serum PK data from patients with HER3-expressing solid tumors (from 3 phase 1/2 studies in breast, lung, and colorectal cancer; N = 425) treated over the dose range of 1.6 to 8.0 mg/kg intravenously every 3 weeks. Population PK modeling for deruxtecan (DXd)-conjugated antibody (representing patritumab deruxtecan) and unconjugated MAAA-1181a (DXd, payload) was carried out sequentially. DXd-conjugated antibody PK was described using a 2-compartment model with parallel linear and nonlinear clearance. Unconjugated DXd PK was described using a 1-compartment model with linear clearance and release of DXd as a first-order, time-dependent function of the level of DXd-conjugated antibody in the central compartment. Preliminary covariate evaluation was conducted for prespecified covariates of pharmacological plausibility and clinical interest. The final model retained weight (on linear clearance and central volume) and albumin level, sex, and tumor type (on linear clearance) for DXd-conjugated antibody, and weight (on release rate constant) and hepatic function (on clearance) for unconjugated DXd. Effects of these covariates on the exposure metrics were generally mild and did not require dose adjustment for subpopulations in subsequent development. Further PK characterization for patritumab deruxtecan will evolve with emerging data.

Comparative analysis of the relationship between translation efficiency and sequence features of endogenous proteins in multiple organisms.

The translation efficiency of protein genes is known to be affected by sequence features. Previous studies have found that various sequence features based on codon usage and mRNA secondary structure contribute to translation efficiency. However, most studies have focused on a specific organism, usually a model organism such as Escherichia coli or Saccharomyces cerevisiae. Here, we investigate whether the relationship between translation efficiency and sequence features is conserved among multiple organisms using publicly available ribosome profiling data and RNA-Seq data. We analyze nine organisms from various taxa: Staphylococcus aureus, five species of Streptomyces, two strains of E. coli, and S. cerevisiae. We reveal that the relationship between translation efficiency and sequence features differs across organisms, partly reflecting their taxonomy. The codon adaptation index shows high correlation in all analyzed organisms. Our study provides an insight into the diversity and commonality of sequence determinants of protein expression in these organisms.

A phase I study of pexidartinib, a colony-stimulating factor 1 receptor inhibitor, in Asian patients with advanced solid tumors.

Background Pexidartinib, a novel, orally administered small-molecule tyrosine kinase inhibitor, has strong selectivity against colony-stimulating factor 1 receptor. This phase I, nonrandomized, open-label multiple-dose study evaluated pexidartinib safety and efficacy in Asian patients with symptomatic, advanced solid tumors. Materials and Methods Patients received pexidartinib: cohort 1, 600 mg/d; cohort 2, 1000 mg/d for 2 weeks, then 800 mg/d. Primary objectives assessed pexidartinib safety and tolerability, and determined the recommended phase 2 dose; secondary objectives evaluated efficacy and pharmacokinetic profile. Results All 11 patients (6 males, 5 females; median age 64, range 23-82; cohort 1 n = 3; cohort 2 n = 8) experienced at least one treatment-emergent adverse event; 5 experienced at least one grade ≥ 3 adverse event, most commonly (18%) for each of the following: increased aspartate aminotransferase, blood alkaline phosphatase, gamma-glutamyl transferase, and anemia. Recommended phase 2 dose was 1000 mg/d for 2 weeks and 800 mg/d thereafter. Pexidartinib exposure, area under the plasma concentration-time curve from zero to 8 h (AUC0-8h), and maximum observed plasma concentration (Cmax) increased on days 1 and 15 with increasing pexidartinib doses, and time at Cmax (Tmax) was consistent throughout all doses. Pexidartinib exposure and plasma levels of adiponectin and colony-stimulating factor 1 increased following multiple daily pexidartinib administrations. One patient (13%) with tenosynovial giant cell tumor showed objective tumor response. Conclusions This was the first study to evaluate pexidartinib in Asian patients with advanced solid tumors. Pexidartinib was safe and tolerable in this population at the recommended phase 2 dose previously determined for Western patients (funded by Daiichi Sankyo; clinicaltrials.gov number, NCT02734433).

Developing a codon optimization method for improved expression of recombinant proteins in actinobacteria.

Codon optimization by synonymous substitution is widely used for recombinant protein expression. Recent studies have investigated sequence features for codon optimization based on large-scale expression analyses. However, these studies have been limited to common host organisms such as Escherichia coli. Here, we develop a codon optimization method for Rhodococcus erythropolis, a gram-positive GC-rich actinobacterium attracting attention as an alternative host organism. We evaluate the recombinant protein expression of 204 genes in R. erythropolis with the same plasmid vector. The statistical analysis of these expression data reveals that the mRNA folding energy at 5' regions as well as the codon frequency are important sequence features for codon optimization. Intriguingly, other sequence features such as the codon repetition rate show a different tendency from the previous study on E. coli. We optimize the coding sequences of 12 genes regarding these sequence features, and confirm that 9 of them (75%) achieve increased expression levels compared with wild-type sequences. Especially, for 5 genes whose expression levels for wild-type sequences are small or not detectable, all of them are improved by optimized sequences. These results demonstrate the effectiveness of our codon optimization method in R. erythropolis, and possibly in other actinobacteria.

Complete Genome Sequence of the Nonheterocystous Cyanobacterium Pseudanabaena sp. ABRG5-3.

We report here the complete sequences of the main genome (4.8 Mb) and seven plasmids of the semifilamentous, nonheterocystous cyanobacterium Pseudanabaena sp. ABRG5-3, a strain isolated from a pond in Japan. These data are expected to enhance our understanding of the Pseudanabaena subclade near the root of cyanobacterial diversity.

Pharmacokinetics and Safety of a Single Oral Dose of Mirogabalin in Japanese Subjects With Varying Degrees of Renal Impairment.

Mirogabalin (DS-5565) is a novel preferentially selective α2 δ-1 ligand being developed for the treatment of diabetic peripheral neuropathic pain and postherpetic neuralgia. The current multicenter open-label study determined the effect of varying degrees of renal impairment on the pharmacokinetics and safety of a single dose of mirogabalin 5 mg in Japanese subjects. A total of 30 subjects (6 subjects per renal function category [normal, mild, moderate, or severe impairment; and end-stage renal disease (ESRD)]) were enrolled and completed the study. The AUClast increased with severity of renal impairment; the geometric least-squares mean ratios of AUClast compared with subjects with normal renal function were 1.3, 1.9, 3.6, and 5.3 for patients with mild, moderate, and severe impairment and ESRD, respectively. In accordance with this AUClast increase, apparent total body clearance (CL/F), renal clearance (CLr), and the cumulative percentage of mirogabalin dose excreted into urine all decreased with severity of renal impairment. There were no deaths and no severe treatment-related adverse events (TEAEs), serious TEAEs, or TEAEs resulting in study discontinuation. Mirogabalin was well tolerated in Japanese subjects with normal renal function and those with mild to severe renal impairment. It was also tolerated in subjects with ESRD but with a higher incidence of TEAEs. The most frequently reported TEAEs were dizziness (ESRD, n = 3), somnolence (ESRD, n = 2), and vomiting (ESRD, n = 2). Based on these data, a mirogabalin dose adjustment will be considered in Japanese subjects with moderate to severe renal impairment and those with ESRD.

Single-Pixel Densitometry Revealed the Presence of Peptidoglycan in the Intermembrane Space of the Moss Chloroplast Envelope in Conventional Electron Micrographs.

Chloroplasts are believed to be descendants of ancestral cyanobacteria that have a peptidoglycan layer between the outer and the inner membranes. In particular, cyanelles having peptidoglycan in Cyanophora paradoxa are considered as evidence for the endosymbiotic origin of chloroplasts. The moss Physcomitrella patens has a complete set of genes involved in the synthesis of peptidoglycan, but a peptidoglycan layer has not been observed by conventional electron microscopy to date. Recently, a new metabolic labeling technique using a fluorescent probe was applied to visualize putative peptidoglycan surrounding the chloroplasts. The exact localization of the peptidoglycan, however, has not been clearly identified. Here we examined conventional electron micrographs of two types of moss materials (mutants or ampicillin-treated plants), one presumably having peptidoglycan and the other presumably lacking peptidoglycan, and analyzed in detail, by single-pixel densitometry, the electron density of the chloroplast envelope membranes and the intermembrane space. Statistical analysis showed that the relative electron density within the intermembrane space with respect to that of the envelope membranes was significantly higher in the materials presumably having peptidoglycan than in the materials presumably devoid of peptidoglycan. We consider this difference as bona fide evidence for the presence of peptidoglycan between the outer and the inner envelope membranes in the wild-type chloroplasts of the moss, although its density is lower than that in bacteria and cyanelles. We will also discuss this low-density peptidoglycan in the light of the phylogenetic origin of peptidoglycan biosynthesis enzymes.

A phase 1 study comparing the proposed biosimilar BS-503a with bevacizumab in healthy male volunteers.

This is a randomized, double-blind, single-dose, parallel group phase 1 study to assess pharmacokinetic similarity, safety, and tolerability of BS-503a, a proposed bevacizumab biosimilar. A total of 114 male healthy subjects were randomized (1:1) to receive a single 3 mg/kg intravenous dose of either BS-503a or bevacizumab (Avastin®). Pharmacokinetic (PK) blood samples were collected up to Day 78, and serum drug concentrations were measured using a validated enzyme-linked immunosorbent assay. Pharmacokinetic similarity was evaluated using area under the serum concentration-time curve from zero to infinity (AUC inf) as a primary PK parameter, and maximum serum concentration (Cmax) and area under the serum concentration-time curve from zero to the last measurable time (AUC last) as secondary PK parameters. The 90% confidence intervals (CIs) of geometric mean ratio of AUC inf ranged 0.980-1.105, which met the predefined criteria of 0.80-1.25. The 90% CIs of geometric mean ratios for Cmax and AUC last were 1.009-1.125 and 0.982-1.096, respectively, falling into the same criteria. At least one drug-related treatment emergent adverse event occurred in 18 and 21 subjects treated with BS-503a and bevacizumab, respectively. The most common adverse events were headache, epistaxis, and rhinorrhea. Most adverse events were mild or moderate; however, one drug-related serious adverse event of duodenal ulcer perforation was reported by a subject 47 days after treatment of BS-503a. In conclusion, BS-503a was demonstrated to have highly similar PK to bevacizumab and adverse events observed were consistent with those observed for bevacizumab.

Draft Genome Sequences of Four Species of Chlamydomonas Containing Phosphatidylcholine.

Phosphatidylcholine (PC) is one of the essential phospholipids for most eukaryotes. Although the model green alga Chlamydomonas reinhardtii lacks PC, four species containing PC were found in the genus Chlamydomonas Here, we report the draft genome sequences of the four species of Chlamydomonas containing PC.

Sequencing and analysis of the complete organellar genomes of Parmales, a closely related group to Bacillariophyta (diatoms).

We sequenced the complete plastid and mitochondrial genomes of the unicellular marine phytoplankton Triparma laevis, belonging to the order Parmales (Heterokonta). The cells of Parmales are surrounded by silicified cell walls, similar to Bacillariophyta (diatoms). T. laevis was recognized as a sister group of Bacillariophyta using a molecular phylogenetic analysis based on SSU rDNA and rbcL sequences. Bacillariophyta are the most successful group of phytoplankton in the modern ocean, but the origin and early evolution of them have not been clearly established. Detailed molecular analyses of T. laevis may increase our understanding of the evolutionary relationships among Parmales and Bacillariophyta. The gene contents of the plastid and mitochondrial genomes are similar between T. laevis and Bacillariophyta. The gene order of the plastid genome is also similar to Bacillariophyta, whereas the gene order of the mitochondrial genome is not conserved in Bacillariophyta, but the structure is more compact than Bacillariophyta. Phylogenetic analyses, using plastid-encoded concatenated amino acid datasets and mitochondria-encoded concatenated amino acid datasets suggest that T. laevis is a sister group of Bacillariophyta. These results suggest that the characteristics of the organellar genomes of T. laevis are similar and conserve ancestral characteristics more than Bacillariophyta.

Characterization of three putative xylulose 5-phosphate/fructose 6-phosphate phosphoketolases in the cyanobacterium Anabaena sp. PCC 7120.

Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) is a key enzyme in the central carbohydrate metabolism in heterofermentative bacteria, in which enzymatic property of Xfps is well characterized. This is not the case in other microbes. The cyanobacterium Anabaena sp. PCC 7120 possesses three putative genes encoding Xfp, all1483, all2567, and alr1850. We purified three putative Xfps as recombinant proteins. The results of gel filtration indicated that these proteins form homomultimer complex. All1483 and All2567 showed phosphoketolase activity, whereas Alr1850 did not show the activity. Kinetic analyses demonstrated that substrates, fructose 6-phosphate and inorganic phosphate, are cooperatively bound to enzymes positively and negatively, respectively.

Cellular Dynamics Drives the Emergence of Supracellular Structure in the Cyanobacterium, Phormidium sp. KS.

Motile filamentous cyanobacteria, such as Oscillatoria, Phormidium and Arthrospira, are ubiquitous in terrestrial and aquatic environments. As noted by Nägeli in 1860, many of them form complex three-dimensional or two-dimensional structures, such as biofilm, weed-like thalli, bundles of filaments and spirals, which we call supracellular structures. In all of these structures, individual filaments incessantly move back and forth. The structures are, therefore, macroscopic, dynamic structures that are continuously changing their microscopic arrangement of filaments. In the present study, we analyzed quantitatively the movement of individual filaments of Phormidium sp. KS grown on agar plates. Junctional pores, which have been proposed to drive cell movement by mucilage/slime secretion, were found to align on both sides of each septum. The velocity of movement was highest just after the reversal of direction and, then, attenuated exponentially to a final value before the next reversal of direction. This kinetics is compatible with the "slime gun" model. A higher agar concentration restricts the movement more severely and, thus, resulted in more spiral formation. The spiral is a robust form compatible with non-homogeneous movements of different parts of a long filament. We propose a model of spiral formation based on the microscopic movement of filaments.

Molecular phylogeny and intricate evolutionary history of the three isofunctional enzymes involved in the oxidation of protoporphyrinogen IX.

Tetrapyrroles such as heme and chlorophyll are essential for biological processes, including oxygenation, respiration, and photosynthesis. In the tetrapyrrole biosynthesis pathway, protoporphyrinogen IX oxidase (Protox) catalyzes the formation of protoporphyrin IX, the last common intermediate for the biosynthesis of heme and chlorophyll. Three nonhomologous isofunctional enzymes, HemG, HemJ, and HemY, for Protox have been identified. To reveal the distribution and evolution of the three Protox enzymes, we identified homologs of each along with other heme biosynthetic enzymes by whole-genome clustering across three domains of life. Most organisms possess only one of the three Protox types, with some exceptions. Detailed phylogenetic analysis revealed that HemG is mostly limited to γ-Proteobacteria whereas HemJ may have originated within α-Proteobacteria and transferred to other Proteobacteria and Cyanobacteria. In contrast, HemY is ubiquitous in prokaryotes and is the only Protox in eukaryotes, so this type may be the ancestral Protox. Land plants have a unique HemY homolog that is also shared by Chloroflexus species, in addition to the main HemY homolog originating from Cyanobacteria. Meanwhile, organisms missing any Protox can be classified into two groups; those lacking most heme synthetic genes, which necessarily depend on external heme supply, and those lacking only genes involved in the conversion of uroporphyrinogen III into heme, which would use a precorrin2-dependent alternative pathway. However, hemN encoding coproporphyrinogen IX oxidase was frequently found in organisms lacking Protox enzyme, which suggests a unique role of this gene other than in heme biosynthesis.

Klebsormidium flaccidum genome reveals primary factors for plant terrestrial adaptation.

The colonization of land by plants was a key event in the evolution of life. Here we report the draft genome sequence of the filamentous terrestrial alga Klebsormidium flaccidum (Division Charophyta, Order Klebsormidiales) to elucidate the early transition step from aquatic algae to land plants. Comparison of the genome sequence with that of other algae and land plants demonstrate that K. flaccidum acquired many genes specific to land plants. We demonstrate that K. flaccidum indeed produces several plant hormones and homologues of some of the signalling intermediates required for hormone actions in higher plants. The K. flaccidum genome also encodes a primitive system to protect against the harmful effects of high-intensity light. The presence of these plant-related systems in K. flaccidum suggests that, during evolution, this alga acquired the fundamental machinery required for adaptation to terrestrial environments.

Analysis of the complete plastid genome of the unicellular red alga Porphyridium purpureum.

We determined the complete nucleotide sequence of the plastid genome of the unicellular marine red alga Porphyridium purpureum strain NIES 2140, belonging to the unsequenced class Porphyridiophyceae. The genome is a circular DNA composed of 217,694 bp with the GC content of 30.3%. Twenty-nine of the 224 protein-coding genes contain one or multiple intron(s). A group I intron was found in the rpl28 gene, whereas the other introns were group II introns. The P. purpureum plastid genome has one non-coding RNA (ncRNA) gene, 29 tRNA genes and two nonidentical ribosomal RNA operons. One rRNA operon has a tRNA(Ala)(UGC) gene between the rrs and the rrl genes, whereas another has a tRNA(Ile)(GAU) gene. Phylogenetic analyses suggest that the plastids of Heterokontophyta, Cryptophyta and Haptophyta originated from the subphylum Rhodophytina. The order of the genes in the ribosomal protein cluster of the P. purpureum plastid genome differs from that of other Rhodophyta and Chromalveolata. These results suggest that a large-scale rearrangement occurred in the plastid genome of P. purpureum after its separation from other Rhodophyta.

Localization and phylogenetic analysis of enzymes related to organellar genome replication in the unicellular rhodophyte Cyanidioschyzon merolae.

Plants and algae possess plastids and mitochondria harboring their own genomes, which are replicated by the apparatus consisting of DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, and primer removal enzyme. In the higher plant Arabidopsis thaliana, organellar replication-related enzymes (OREs) are similar in plastids and mitochondria because many of them are dually targeted to plastids and mitochondria. In the red algae, there is a report about a DNA replicase, plant/protist organellar DNA polymerase, which is localized to both plastids and mitochondria. However, other OREs remain unclear in algae. Here, we identified OREs possibly localized to organelles in the unicellular rhodophyte Cyanidioschyzon merolae. We then examined intracellular localization of green fluorescent protein-fusion proteins of these enzymes in C. merolae, whose cell has a single plastid and a single mitochondrion and is suitable for localization analysis, demonstrating that the plastid and the mitochondrion contain markedly different components of replication machinery. Phylogenetic analyses revealed that the organelle replication apparatus was composed of enzymes of various different origins, such as proteobacterial, cyanobacterial, and eukaryotic, in both red algae and green plants. Especially in the red alga, many enzymes of cyanobacterial origin remained. Finally, on the basis of the results of localization and phylogenetic analyses, we propose a model on the succession of OREs in eukaryotes.

The all0458/lti46.2 gene encodes a low temperature-induced Dps protein homologue in the cyanobacteria Anabaena sp. PCC 7120 and Anabaena variabilis M3.

DNA-binding proteins from starved cells (Dps), which are encoded by many bacterial genomes, protect genomic DNA via non-specific DNA binding, as well as inhibition of free radical formation by chelating Fe(II). In the filamentous cyanobacterium Anabaena, the second gene (lti46.2) in the low temperature-induced gene operon lti46 in strain M3 was found to encode a homologue of Dps, but for a long time this gene remained poorly characterized. A gene cluster, all0459-all0458-all0457, was found later to be 100% identical to the lti46 gene cluster in a closely related strain, PCC 7120. In the present study, we detected ferroxidase activity of the Lti46.2/All0458 protein, which formed a dodecamer, as found in other Dps proteins. In addition, three homologues of all0458 were found in strain PCC 7120, namely, all1173, alr3808 and all4145. We analysed expression of the lti46 or all0459-8-7 gene cluster in both strains, M3 and PCC 7120, and confirmed its induction by low temperature. We found that the All0458-GFP fusion protein and the All1173-GFP fusion protein were localized to the nucleoids. In the all0458 null mutant, the transcript of the alr3808 gene accumulated. These results suggest that there might be complex cooperation of various members of the dps family in protecting the genome from environmental stresses such as changing temperature.

Statistics of N-terminal alignment as a guide for refining prokaryotic gene annotation.

Identification of a correct N-terminus of a protein is an important step in genome annotation. However, we sometimes encounter incorrectly annotated N-termini in genomic databases. We analyzed statistics of surplus or missing N-terminal amino acid residues in tentatively translated coding sequence of cyanobacterial database entries, and found that, on average, about 8-9% of the aligned proteins have a putative incorrect N-terminus, although the percentage was dependent on the database entry. In an attempt to find more plausible N-termini for these proteins, we were able to estimate a better-aligning N-terminus in 90% of the cases. TTG was found as a putative initiation codon in most cases of recessed N-termini. This statistical approach, applicable to any group of prokaryotes, will help identify a plausible translation initiation site for each protein-coding gene in newly sequenced genomes, and also is a method of refining the N-terminus of proteins in already published genomes.