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, internalization 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 normalized 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 labeled 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 characterizing the ADME properties of both the ADC and its payload, to enable better translation from animals to human for ADC development.

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.

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.

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.

Transcriptional control of carbohydrate catabolism by the CcpA protein in the ruminal bacterium Streptococcus bovis.

As a potent, pleiotropic regulatory protein in Gram-positive bacteria, catabolite control protein A (CcpA) mediates the transcriptional control of carbohydrate metabolism in Streptococcus bovis, a lactate-producing bacterium that plays an essential role in rumen acidosis in dairy cows. Although the rumen uptake of carbohydrates is multi-substrate, the focus of S. bovis research thus far has been on the glucose. With the aid of gene deletion, whole-genome sequencing, and transcriptomics, we have unraveled the role of CcpA in carbohydrate metabolism, on the one hand, and acidosis, on the other, and we show that the S. bovis strain S1 encodes "Carbohydrate-Active Enzymes" and that ccpA deletion slows the organism's growth rate and modulates the organic acid fermentation pathways toward lower lactate, higher formate, and acetate in the maltose and cellobiose. Furthermore, this study revealed the different regulatory functions of the CcpA protein in rumen metabolism and acidosis.IMPORTANCEThis study is important as it illustrates the varying regulatory role of the Streptococcus bovis catabolite control protein A protein in carbohydrate metabolism and the onset of acidosis in dairy cattle.

Interplay of emerging and established technologies drives innovation in natural product antibiotic discovery.

A continued rise of antibiotic resistance and shortages of effective antibiotics necessitate the discovery and development of new antibiotics with novel modes of action (MoAs) against resistant pathogens. While natural products remain the best resource for antibiotic discovery, their exploration faces many challenges, including (i) unknown MoAs, (ii) high rediscovery rates, (iii) tedious isolation and structure elucidation, and (iv) insufficient production for further development. We have identified recent innovations in screening methods, microbiology, bioinformatics, and metabolomics technologies, as well as natural product-inspired synthesis and synthetic biology, that have contributed to new natural product antibiotics in the past two years. We highlight their interplay as the key element for successful applications, driving future opportunities to increase the pool of natural product-based antibacterial antibiotics.

The many facets of sulfur incorporation in natural product biosynthesis.

Sulfur-containing natural products (S-containing NPs) exhibit diverse chemical structures and biosynthetic machineries. Unraveling the intricate chemistry of S-incorporation requires innovative and multidisciplinary approaches. In this review, we surveyed the landscape of S-containing NP biosynthetic machineries, classified the S-incorporation chemistry into four distinct classes, and highlighted each of the four classes with representative examples from recent studies. All highlighted chemistry has been correlated to the genes encoding the biosynthetic machineries of the S-containing NPs, which open new opportunities to discover S-containing NPs through genome mining. These examples should inspire the community to explore uncharted territories in NP research, promoting further advancements in both novel S-containing NP discovery and S-incorporation chemistry.

Utilizing PK and PD Biomarkers to Guide the First-in-Human Starting Dose Selection of MTBT1466A: A Novel Humanized Monoclonal Anti-TGFß3 Antibody for the Treatment of Fibrotic Diseases.

MTBT1466A is a high-affinity TGFß3-specific humanized IgG1 monoclonal antibody with reduced Fc effector function, currently under investigation in clinical trials as a potential anti-fibrotic therapy. Here, we characterized the pharmacokinetics (PK) and pharmacodynamics (PD) of MTBT1466A in mice and monkeys and predicted the PK/PD of MTBT1466A in humans to guide the selection of the first-in-human (FIH) starting dose. MTBT1466A demonstrated a typical IgG1-like biphasic PK profile in monkeys, and the predicted human clearance of 2.69 mL/day/kg and t1/2 of 20.4 days are consistent with those expected for a human IgG1 antibody. In a mouse model of bleomycin-induced lung fibrosis, changes in expression of TGFß3-related genes, serpine1, fibronectin-1, and collagen 1A1 were used as PD biomarkers to determine the minimum pharmacologically active dose of 1 mg/kg. Unlike in the fibrosis mouse model, evidence of target engagement in healthy monkeys was only observed at higher doses. Using a PKPD-guided approach, the recommended FIH dose of 50 mg, IV, provided exposures that were shown to be safe and well tolerated in healthy volunteers. MTBT1466A PK in healthy volunteers was predicted reasonably well using a PK model with allometric scaling of PK parameters from monkey data. Taken together, this work provides insights into the PK/PD behavior of MTBT1466A in preclinical species, and supports the translatability of the preclinical data into the clinic.

A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products.

The enediynes are structurally characterized by a 1,5-diyne-3-ene motif within a 9- or 10-membered enediyne core. The anthraquinone-fused enediynes (AFEs) are a subclass of 10-membered enediynes that contain an anthraquinone moiety fused to the enediyne core as exemplified by dynemicins and tiancimycins. A conserved iterative type I polyketide synthase (PKSE) is known to initiate the biosynthesis of all enediyne cores, and evidence has recently been reported to suggest that the anthraquinone moiety also originates from the PKSE product. However, the identity of the PKSE product that is converted to the enediyne core or anthraquinone moiety has not been established. Here, we report the utilization of recombinant E. coli coexpressing various combinations of genes that encode a PKSE and a thioesterase (TE) from either 9- or 10-membered enediyne biosynthetic gene clusters to chemically complement ΔPKSE mutant strains of the producers of dynemicins and tiancimycins. Additionally, 13C-labeling experiments were performed to track the fate of the PKSE/TE product in the ΔPKSE mutants. These studies reveal that 1,3,5,7,9,11,13-pentadecaheptaene is the nascent, discrete product of the PKSE/TE that is converted to the enediyne core. Furthermore, a second molecule of 1,3,5,7,9,11,13-pentadecaheptaene is demonstrated to serve as the precursor of the anthraquinone moiety. The results establish a unified biosynthetic paradigm for AFEs, solidify an unprecedented biosynthetic logic for aromatic polyketides, and have implications for the biosynthesis of not only AFEs but all enediynes.

Neogrisemycin, a Trisulfide-Bridged Angucycline, Produced upon Expressing the Thioangucycline Biosynthetic Gene Cluster in Streptomyces albus J1074.

Neogrisemycin (1) was isolated from recombinant Streptomyces albus J1074 strain SB4061 expressing an engineered thioangucycline (TAC) biosynthetic gene cluster (BGC). The structure and absolute configuration of 1 were established by a combination of mass spectrometry, nuclear magnetic resonance, and single-crystal X-ray diffraction analyses. Like the TACs, 1 was also proposed to derive non-enzymatically from the common epoxide (8), the nascent product encoded by the tac BGC, mediated by endogenous hydrogen trisulfide.

Protocol for 3D screening of lung cancer spheroids using natural products.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and accounts for ∼84% of all lung cancer cases. NSCLC remains one of the leading causes of cancer-associated death, with a 5-year survival rate less than 25%. This type of cancer begins with healthy cells that change and start growing out of control, leading to the formation of lesions or tumors. Understanding the dynamics of how the tumor microenvironment promotes cancer initiation and progression that leads to cancer metastasis is crucial to help identify new molecular therapies. 3D primary cell tumor models have received renewed recognition due to their ability to better mimic the complexity of in vivo tumors and as a potential bridge between traditional 2D culture and in vivo studies. Vast improvements in 3D cell culture technologies make them much more cost effective and efficient largely because of the use of a cell-repellent surfaces and a novel angle plate adaptor technology. To exploit this technology, we accessed the Natural Products Library (NPL) at UF Scripps, which consists of crude extracts, partially purified fractions, and pure natural products (NPs). NPs generally are not very well represented in most drug discovery libraries and thus provide new insights to discover leads that could potentially emerge as novel molecular therapies. Herein we describe how we combined these technologies for 3D screening in 1536 well format using a panel of ten NSCLC cells lines (5 wild type and 5 mutant) against ∼1280 selected members of the NPL. After further evaluation, the selected active hits were prioritized to be screened against all 10 NSCLC cell lines as concentration response curves to determine the efficacy and selectivity of the compounds between wild type and mutant 3D cell models. Here, we demonstrate the methods needed for automated 3D screening using microbial NPs, exemplified by crude extracts, partially purified fractions, and pure NPs, that may lead to future use targeting human cancer.

Application of a Biocatalytic Strategy for the Preparation of Tiancimycin-Based Antibody-Drug Conjugates Revealing Key Insights into Structure-Activity Relationships.

Antibody-drug conjugates (ADCs) are cancer chemotherapeutics that utilize a monoclonal antibody (mAb)-based delivery system, a cytotoxic payload, and a chemical linker. ADC payloads must be strategically functionalized to allow linker attachment without perturbing the potency required for ADC efficacy. We previously developed a biocatalytic system for the precise functionalization of tiancimycin (TNM)-based payloads. The TNMs are anthraquinone-fused enediynes (AFEs) and have yet to be translated into the clinic. Herein, we report the translation of biocatalytically functionalized TNMs into ADCs in combination with the dual-variable domain (DVD)-mAb platform. The DVD enables both site-specific conjugation and a plug-and-play modularity for antigen-targeting specificity. We evaluated three linker chemistries in terms of TNM-based ADC potency and antigen selectivity, demonstrating a trade-off between potency and selectivity. This represents the first application of AFE-based payloads to DVDs for ADC development, a workflow that is generalizable to further advance AFE-based ADCs for multiple cancer types.

Intramolecular C-C Bond Formation Links Anthraquinone and Enediyne Scaffolds in Tiancimycin Biosynthesis.

First discovered in 1989, the anthraquinone-fused enediynes are a class of DNA-cleaving bacterial natural products composed of a DNA-intercalating anthraquinone moiety and a 10-membered enediyne warhead. However, until recently, there has been a lack of genetically amenable hosts and sequenced biosynthetic gene clusters available for solving the biosynthetic questions surrounding these molecules. Herein, we have identified and biochemically and structurally characterized TnmK1, a member of the α/ß-hydrolase fold superfamily responsible for the C-C bond formation linking the anthraquinone moiety and enediyne core together in tiancimycin (TNM) biosynthesis. In doing so, two intermediates, TNM H and TNM I, in anthraquinone-fused enediyne biosynthesis, containing an unprecedented cryptic C16 aldehyde group, were identified. This aldehyde plays a key role in the TnmK1-catalyzed C-C bond formation via a Michael addition, representing the first example of this chemistry for the α/ß-hydrolase fold superfamily. Additionally, TNM I shows sub-nanomolar cytotoxicity against selected cancer cell lines, indicating a new mechanism of action compared to previously known anthraquinone-fused enediynes. Together, the findings from this study are expected to impact enzymology, natural product biosynthesis, and future efforts at enediyne discovery and drug development.

A homogeneous high-DAR antibody-drug conjugate platform combining THIOMAB antibodies and XTEN polypeptides.

The antibody-drug conjugate (ADC) is a well-validated modality for the cell-specific delivery of small molecules with impact expanding rapidly beyond their originally-intended purpose of treating cancer. However, antibody-mediated delivery (AMD) remains inefficient, limiting its applicability to targeting highly potent payloads to cells with high antigen expression. Maximizing the number of payloads delivered per antibody is one key way in which delivery efficiency can be improved, although this has been challenging to carry out; with few exceptions, increasing the drug-to-antibody ratio (DAR) above ∼4 typically destroys the biophysical properties and in vivo efficacy for ADCs. Herein, we describe the development of a novel bioconjugation platform combining cysteine-engineered (THIOMAB) antibodies and recombinant XTEN polypeptides for the unprecedented generation of homogeneous, stable "TXCs" with DAR of up to 18. Across three different bioactive payloads, we demonstrated improved AMD to tumors and Staphylococcus aureus bacteria for high-DAR TXCs relative to conventional low-DAR ADCs.

Rational Approach to Identify RNA Targets of Natural Products Enables Identification of Nocathiacin as an Inhibitor of an Oncogenic RNA.

The discovery of biofunctional natural products (NPs) has relied on the phenotypic screening of extracts and subsequent laborious work to dereplicate active NPs and define cellular targets. Herein, NPs present as crude extracts, partially purified fractions, and pure compounds were screened directly against molecular target libraries of RNA structural motifs in a library-versus-library fashion. We identified 21 hits with affinity for RNA, including one pure NP, nocathiacin I (NOC-I). The resultant data set of NOC-I-RNA fold interactions was mapped to the human transcriptome to define potential bioactive interactions. Interestingly, one of NOC-I's most preferred RNA folds is present in the nuclease processing site in the oncogenic, noncoding microRNA-18a, which NOC-I binds with low micromolar affinity. This affinity for the RNA translates into the selective inhibition of its nuclease processing in vitro and in prostate cancer cells, in which NOC-I also triggers apoptosis. In principle, adaptation of this combination of experimental and predictive approaches to dereplicate NPs from the other hits (extracts and partially purified fractions) could fundamentally transform the current paradigm and accelerate the discovery of NPs that bind RNA and their simultaneous correlation to biological targets.