A novel approach to quantitate biodistribution and transduction of adeno-associated virus gene therapy using radiolabeled AAV vectors in mice.

An understanding of recombinant adeno-associated virus (AAV) biodistribution profiles is an important element of a preclinical development program. Here, we have developed a radiolabeling strategy utilizing the co-delivery of 125I (non-residualizing) and 111In (residualizing) radionuclide-conjugated AAVs to provide a detailed distribution quantification at tissue level delineating between the cellular internalized AAV (degraded, 111In-125I) and AAV remaining in the extracellular matrix (intact, 125I). This labeling method has been successfully applied to AAV9 and AAV-PHP.eB as tool molecules without altering the physical properties and biological activities of the AAVs. Upon labeling with either of the radioactive probes, these molecules were systemically injected into C57BL/6 mice. The biodistribution results indicate that AAVs, with a fast distribution profile, were mainly located in the extracellular matrix of highly perfused organs such as liver and spleen at early time points, leading to a difference between capsid quantification and vector genome quantification. The results suggest that the 125I-AAV/111In-AAV co-delivery approach offers a robust and efficient analytical strategy to investigate the detailed tissue distribution of AAV vectors, including both vector genome and protein capsids. This novel method has the potential to be applied to capsid optimization, selection, and lead candidate development.

Enediyne natural product biosynthesis unified by a diiodotetrayne intermediate.

Enediyne natural products are renowned for their potent cytotoxicities but the biosynthesis of their defining 1,5-diyne-3-ene core moiety remains largely enigmatic. Since the discovery of the enediyne polyketide synthase cassette in 2002, genome sequencing has revealed thousands of distinct enediyne biosynthetic gene clusters, each harboring the conserved enediyne polyketide synthase cassette. Here we report that (1) the products of this cassette are an iodoheptaene, a diiodotetrayne and two pentaynes; (2) the diiodotetrayne represents a common biosynthetic intermediate for all known enediynes; and (3) cryptic iodination can be exploited to increase enediyne titers. These findings establish a unified biosynthetic pathway for the enediynes, set the stage to further advance enediyne core biosynthesis and enable fundamental breakthroughs in chemistry, enzymology and translational applications of enediyne natural products.

Monomethyl auristatin E (MMAE), a payload for multiple antibody drug conjugates (ADCs), demonstrates differential red blood cell partitioning across human and animal species.

Background: Monomethyl auristatin E (MMAE) has been used as a payload for several Food and Drug Administration (FDA) approved antibody-drug conjugates (ADCs). It is known that MMAE is released from the ADC following binding, internalisation and proteolytic degradation in target tissues. A striking discrepancy in systemic MMAE levels has been observed across species with 50-fold higher MMAE levels in human than that in rodents when normalised by ADC dose with unknown mechanism.Hypothesis and purpose: Multiple factors could affect systemic MMAE levels such as production and elimination of unconjugated MMAE following ADC dosing. In this study, we have explored whether MMAE displays differential red blood cell (RBC) partitioning across species that may contribute to the different MMAE levels seen between human and animals.Experiments: To determine MMAE RBC partitioning, tritium labelled MMAE ([3H]-MMAE) was incubated in whole blood from mice, rats, monkeys and humans in vitro, then RBC partitioning was determined and compared across species. To test whether MMAE released from the ADC would show any difference in RBC partitioning, pinatuzumab vedotin or polatuzumab vedotin was administered to mice, rats, and monkeys. MMAE levels were measured in both blood and plasma, and the ratios of MMAE levels were calculated as blood-to-plasma ratio (in vivo RBC partitioning).Results: Our in vitro data showed that unconjugated MMAE has a species-dependent RBC partitioning with strong RBC partitioning in mouse, rat, followed by monkey blood, whereas minimal RBC partitioning was seen in human blood. Incubation of 2 nM of MMAE in mouse blood resulted in a blood-to-plasma ratio of 11.8 ± 0.291, followed by rat, monkey, and human at 2.36 ± 0.0825, 1.57 ± 0.0250, and 0.976 ± 0.0620, respectively. MMAE RBC partitioning is also concentration-dependent, with an inverse relationship between RBC partitioning and MMAE concentration (higher RBC partitioning at lower concentration). In vivo dosing of pinatuzumab vedotin in mouse displayed systemic MMAE at about a 5-fold higher blood concentration compared to plasma concentration once MMAE reached a pseudo-equilibrium, while systemic MMAE from blood and plasma concentration showed a 1.65-fold difference in rat.Implication and conclusion: These data demonstrated that MMAE has a distinct RBC partitioning across different species, which may contribute to, at least in part, to the differential in the systemic MMAE levels observed in vivo between preclinical and clinical studies. These findings highlight the importance of fully characterising the ADME properties of both the ADC and its payload, to enable better translation from animals to human for ADC development.

Clinical pharmacology strategies to accelerate the development of polatuzumab vedotin and summary of key findings.

The favorable benefit-risk profile of polatuzumab vedotin, as demonstrated in a pivotal Phase Ib/II randomized study (GO29365; NCT02257567), coupled with the need for effective therapies in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL), prompted the need to accelerate polatuzumab vedotin development. An integrated, fit-for-purpose clinical pharmacology package was designed to support regulatory approval. To address key clinical pharmacology questions without dedicated clinical pharmacology studies, we leveraged non-clinical and clinical data for polatuzumab vedotin, published clinical data for brentuximab vedotin, a similar antibody-drug conjugate, and physiologically based pharmacokinetic and population pharmacokinetic modeling approaches. We review strategies and model-informed outcomes that contributed to regulatory approval of polatuzumab vedotin plus bendamustine and rituximab in R/R DLBCL. These strategies made polatuzumab vedotin available to patients earlier than previously possible; depending on the strength of available data and the regulatory/competitive environment, they may also prove useful in accelerating the development of other agents.

Discrete Acyltransferases and Thioesterases in Iso-Migrastatin and Lactimidomycin Biosynthesis.

Iso-Migrastatin (iso-MGS) and lactimidomycin (LTM) are glutarimide-containing polyketide natural products (NPs) that are biosynthesized by homologous acyltransferase (AT)-less type I polyketide synthase (PKS) assembly lines. The biological activities of iso-MGS and LTM have inspired numerous efforts to generate analogues via genetic manipulation of their biosynthetic machinery in both native producers and model heterologous hosts. A detailed understanding of the MGS and LTM AT-less type I PKSs would serve to inspire future engineering efforts while advancing the fundamental knowledge of AT-less type I PKS enzymology. The mgs and ltm biosynthetic gene clusters (BGCs) encode for two discrete ATs of the architecture AT-enoylreductase (AT-ER) and AT-type II thioesterase (AT-TE). Herein, we report the functional characterization of the mgsB and ltmB and the mgsH and ltmH gene products, revealing that MgsB and LtmB function as type II thioesterases (TEs) and MgsH and LtmH are the dedicated trans-ATs for the MGS and LTM AT-less type I PKSs. In vivo and in vitro experiments demonstrated that MgsB was devoid of any AT activity, despite the presence of the conserved catalytic triad of canonical ATs. Cross-complementation experiments demonstrated that MgsH and LtmH are functionally interchangeable between the MGS and LTM AT-less type I PKSs. This work sets the stage for future mechanistic studies of AT-less type I PKSs and efforts to engineer the MGS and LTM AT-less type I PKS assembly lines for novel glutarimide-containing polyketides.

Biochemical and Structural Studies of the Carminomycin 4-O-Methyltransferase DnrK.

Structural and functional studies of the carminomycin 4-O-methyltransferase DnrK are described, with an emphasis on interrogating the acceptor substrate scope of DnrK. Specifically, the evaluation of 100 structurally and functionally diverse natural products and natural product mimetics revealed an array of pharmacophores as productive DnrK substrates. Representative newly identified DnrK substrates from this study included anthracyclines, angucyclines, anthraquinone-fused enediynes, flavonoids, pyranonaphthoquinones, and polyketides. The ligand-bound structure of DnrK bound to a non-native fluorescent hydroxycoumarin acceptor, 4-methylumbelliferone, along with corresponding DnrK kinetic parameters for 4-methylumbelliferone and native acceptor carminomycin are also reported for the first time. The demonstrated unique permissivity of DnrK highlights the potential for DnrK as a new tool in future biocatalytic and/or strain engineering applications. In addition, the comparative bioactivity assessment (cancer cell line cytotoxicity, 4E-BP1 phosphorylation, and axolotl embryo tail regeneration) of a select set of DnrK substrates/products highlights the ability of anthracycline 4-O-methylation to dictate diverse functional outcomes.

The Natural Products Discovery Center: Release of the First 8490 Sequenced Strains for Exploring Actinobacteria Biosynthetic Diversity.

Actinobacteria, the bacterial phylum most renowned for natural product discovery, has been established as a valuable source for drug discovery and biotechnology but is underrepresented within accessible genome and strain collections. Herein, we introduce the Natural Products Discovery Center (NPDC), featuring 122,449 strains assembled over eight decades, the genomes of the first 8490 NPDC strains (7142 Actinobacteria), and the online NPDC Portal making both strains and genomes publicly available. A comparative survey of RefSeq and NPDC Actinobacteria highlights the taxonomic and biosynthetic diversity within the NPDC collection, including three new genera, hundreds of new species, and ~7000 new gene cluster families. Selected examples demonstrate how the NPDC Portal's strain metadata, genomes, and biosynthetic gene clusters can be leveraged using genome mining approaches. Our findings underscore the ongoing significance of Actinobacteria in natural product discovery, and the NPDC serves as an unparalleled resource for both Actinobacteria strains and genomes.

Development of platensimycin, platencin, and platensilin overproducers by biosynthetic pathway engineering and fermentation medium optimization.

The platensimycin (PTM), platencin (PTN), and platensilin (PTL) family of natural products continues to inspire the discovery of new chemistry, enzymology, and medicine. Engineered production of this emerging family of natural products, however, remains laborious due to the lack of practical systems to manipulate their biosynthesis in the native-producing Streptomyces platensis species. Here we report solving this technology gap by implementing a CRISPR-Cas9 system in S. platensis CB00739 to develop an expedient method to manipulate the PTM, PTN, and PTL biosynthetic machinery in vivo. We showcase the utility of this technology by constructing designer recombinant strains S. platensis SB12051, SB12052, and SB12053, which, upon fermentation in the optimized PTM-MS medium, produced PTM, PTN, and PTL with the highest titers at 836 mg L-1, 791 mg L-1, and 40 mg L-1, respectively. Comparative analysis of these resultant recombinant strains also revealed distinct chemistries, catalyzed by PtmT1 and PtmT3, two diterpene synthases that nature has evolved for PTM, PTN, and PTL biosynthesis. The ΔptmR1/ΔptmT1/ΔptmT3 triple mutant strain S. platensis SB12054 could be envisaged as a platform strain to engineer diterpenoid biosynthesis by introducing varying ent-copalyl diphosphate-acting diterpene synthases, taking advantage of its clean metabolite background, ability to support diterpene biosynthesis in high titers, and the promiscuous tailoring biosynthetic machinery. ONE-SENTENCE SUMMARY: Implementation of a CRISPR-Cas9 system in Streptomyces platensis CB00739 enabled the construction of a suite of designer recombinant strains for the overproduction of platensimycin, platencin, and platensilin, discovery of new diterpene synthase chemistries, and development of platform strains for future diterpenoid biosynthesis engineering.

Cofactorless oxygenases guide anthraquinone-fused enediyne biosynthesis.

The anthraquinone-fused enediynes (AFEs) combine an anthraquinone moiety and a ten-membered enediyne core capable of generating a cytotoxic diradical species. AFE cyclization is triggered by opening the F-ring epoxide, which is also the site of the most structural diversity. Previous studies of tiancimycin A, a heavily modified AFE, have revealed a cryptic aldehyde blocking installation of the epoxide, and no unassigned oxidases could be predicted within the tnm biosynthetic gene cluster. Here we identify two consecutively acting cofactorless oxygenases derived from methyltransferase and α/ß-hydrolase protein folds, TnmJ and TnmK2, respectively, that are responsible for F-ring tailoring in tiancimycin biosynthesis by comparative genomics. Further biochemical and structural characterizations reveal that the electron-rich AFE anthraquinone moiety assists in catalyzing deformylation, epoxidation and oxidative ring cleavage without exogenous cofactors. These enzymes therefore fill important knowledge gaps for the biosynthesis of this class of molecules and the underappreciated family of cofactorless oxygenases.

Unveiling the Quantitative Relationships between Electron Distribution and Steric Hindrance of Organic Amines and Their Reaction Rates with Carbonyl Sulfur: A Theoretical Calculation Investigation.

The removal of carbonyl sulfide (COS) commonly contained in natural gas is of great significance but still very challenging via a widely employed absorption process due to its low reactivity and solubility in various commercial solvents. Artificial intelligence (AI) is playing an increasingly important role in the exploration of desulfurization solvents. However, practically feasible AI models still lack a thorough understanding of the reaction mechanisms. Machine learning (ML) models established on chemical mechanisms exhibit enhanced chemical interpretability and prediction performance. In this study, we constructed a series of solvent molecules with varying functional groups, including linear aliphatic amines, cyclic aliphatic amines, and aromatic amines and proposed a three-step reaction pathway to dissect the effects of charge and steric hindrance of different substituents on their reaction rates with COS. Chemical descriptors, based on electrostatic potential (ESP), average local ionization energy (ALIE) theory, Hirshfeld charges, and Fukui functions, were used to correlate and predict the electrophilic reactivity of amine groups with COS. Substituents influence the reaction rate by changing the attraction interaction of amine groups to COS molecules and the electron rearrangement in the electrophilic reaction. Furthermore, they have more pronounced steric effects on the reaction rate in the linear amines. The descriptors N_ALIE and q(N) were found to be crucial in predicting the reactivity of amine groups with COS. Present study provides a comprehensive understanding of the reaction mechanisms of COS with amine compounds, offers specific chemical principles for the development of chemistry-driven ML models, sheds light on other types of electrophilic reactions occurring on amine and phosphine groups, and guides the development of chemical solvents in gas absorption processes.