Antibody-drug conjugates (ADCs) are transforming targeted cancer therapy, yet conventional IgG-based formats are often limited by stochastic conjugation, yielding heterogeneous ADCs. Dual-variable domain IgG1 (DVD-IgG1) antibodies offer a modular solution by enabling precise, site-specific conjugation through the catalytic lysine and engineered arginine or cysteine residues, affording homogeneous ADCs. This review summarizes recent advances in DVD-IgG1-based ADCs, highlighting site-specific conjugation, compatibility with diverse linker chemistries, and incorporation of both established and experimental payloads. Together, these developments position DVD-IgG1s as a versatile platform to expand the chemical and biological design space of next-generation ADCs.

Abstract The rise of antimicrobial resistance warrants renewed attention to established but overlooked antibiotics such as nitroxoline (NTX). Here, we systematically dissect NTX’s mode of action and investigate the contribution of its first-pass metabolites, NTX-sulphate and NTX-glucuronide. We identified metallophore-mediated cellular iron deprivation as the principal antibacterial mechanism of NTX, characterized by induction of iron acquisition pathways and Fe–S cluster proteins, and concomitant loss of protein-bound iron. In contrast, NTX metabolites were biologically inactive and lacked metal-chelating properties. Ex vivo assays demonstrated that clinically relevant uropathogens, including Escherichia coli and Klebsiella pneumoniae, efficiently reconvert these metabolites into active NTX in human urine. Together, our findings establish a mechanistic framework linking NTX antibacterial activity, host detoxification, and pathogen-dependent metabolite reactivation, and providing a molecular explanation for NTX’s enduring therapeutic potential and favourable safety profile .

Prenylation improves the bioactivity and therapeutic properties of peptides from natural product and synthetic origin. Prenyltransferases catalyze peptide prenylation under mild conditions with often remarkable chemo- and regioselectivity. Representatives from structurally distinct prenyltransferase families involved in ribosomal peptide biosynthesis revealed a large potential for chemoenzymatic peptide functionalization with linear prenyl donors. In contrast, the prenyltransferase-terpene cyclase fusion enzyme NctPC installs a polycyclic isoprenoid moiety during biosynthesis of the ribosomal peptide natural product steromaze. However, the two characterized steromaze-like prenyltransferases require at least the presence of a macrocyclic (methyl) lanthionine ring as a prerequisite for prenylation. Here, we report the characterization of standalone NctPC homologs, the steromaze-like prenyltransferase NdnP and the terpene cyclase NdnC, further expanding the chemical space of ribosomal peptides with cyclic isoprenoid moieties. NdnP is highly promiscuous for the geranylation of peptides containing C-terminal tryptophan from ribosomal and synthetic origin without being leader- or modification-dependent. The terpene cyclase NdnC utilizes the geranylated tryptophan to yield a tetracyclic amino acid at the peptide C-terminus. The promiscuity of both enzymes for ribosomal and synthetic peptides makes them promising biocatalysts for lipopeptide drug development creating so far enzymatically inaccessible peptide terpenoid hybrids.

Acute respiratory diseases in humans can be caused by various viral pathogens such as respiratory syncytial virus (RSV), human coronavirus 229E (hCoV-229E), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To prevent severe cases by an early treatment, one effective strategy is to inhibit viral infection at the entry stage of the replication cycle. However, there is a lack of efficient, FDA-approved small-molecule drugs targeting these pathogens. Previously, we identified two dual RSV/hCoV-229E small-molecule inhibitors with activity in the single-digit micromolar range. In this study, we focused on a structure-guided optimization approach of the more promising prototype addressing activity, cell viability, selectivity, solubility and metabolic stability. We present valuable insights into the structure-activity relationship (SAR), and report the discovery of a sub-micromolar RSV entry inhibitor, a dual RSV/CoV-229E inhibitor and a highly potent compound against hCoV-229E.

Antibody-drug conjugates (ADCs) are advanced cancer therapeutics that combine an antibody-based delivery system with a cytotoxic payload through chemical linkers. Anthraquinone-fused enediyne (AFE) natural products, such as tiancimycins (TNMs), are emerging payload candidates with exceptional potency and a validated DNA-damaging mechanism of action. In this study, we describe the translation of semisynthetically functionalized TNMs, incorporating various linker chemistries, into ADCs featuring dual variable domain monoclonal immunoglobulin G1 antibodies (DVD IgG1s). DVD IgG1s enable site-specific conjugation and modular antigen-targeting specificity. As a proof-of-principle, keto-TNM A-anti-CD79b DVD IgG1-based ADCs were constructed on the basis of the significance of CD79b as a clinical target, with only one FDA-approved ADC available. Inspired by the structure-activity relationship data, a chemical strategy to synthesize keto-TNM A was developed, which exhibited the highest potency among the TNMs we have examined to date. Keto-TNM A was elaborated to a panel of anti-CD79b DVD IgG1-based ADCs featuring varying linker chemistries. The optimized ADC exhibited potent and selective activity across multiple CD79b-expressing cell lines and, most significantly, patient-derived primary chronic lymphocytic leukemia cells. This study provides an efficient approach to access functionalized AFEs at scale and speed and exploit their utility as payloads for ADCs targeting CD79b and beyond as the next generation immunotherapies.

The rise of multidrug-resistant pathogens, such as Staphylococcus aureus and Mycobacterium tuberculosis, underscores an urgent need for therapeutic innovation. The antibiotic development pipeline targeting these bacteria is critically limited, with most discovered candidates exhibiting structurally similar features of prominent chemical entities and with well-established molecular targets or binding modes. The myxobacterial α-pyrone antibiotics, myxopyronins, represent a highly promising compound class due to their ability to inhibit RNA polymerase by binding to the "switch region", a distinct binding site to that of standard-of-care antibiotics. Mutasynthesis, leveraging engineered microorganisms and tailored precursors, provides a viable alternative to total synthesis for generating novel derivatives. This study utilized a heterologous expression system in Myxococcus xanthus DK1622 to generate analogs. Two carrier protein domain mutants were engineered to facilitate mutasynthesis-based production of structurally diverse derivatives. A trifluoromethyl-modified analog, once accessible only through total synthesis but now obtained via mutasynthesis, exhibits potent antimicrobial activity against Gram-positive pathogens including Mycobacterium tuberculosis and favorable in vitro absorption, distribution, metabolism, excretion and toxicity properties. These findings highlight a promising pathway for developing optimized α-pyrone antibiotics to address the global antimicrobial-resistance crisis.

The ubiquitous opportunistic pathogen Pseudomonas aeruginosa is responsible for severe infections and notoriously known for acquiring antimicrobial resistance. Inhibiting the bacterium’s extracellular elastase, LasB – a zinc-dependent protease – presents a promising strategy to mitigate its virulence. Within this medicinal chemistry–driven hit-to-lead optimization campaign, a new series of highly potent dipeptidic phosphonates is designed and synthesized following a structure–based drug discovery approach. In vitro and in vivo evaluation reveal beneficial pharmacokinetic profiles, excellent selectivity over human off-targets and good tolerability in murine toxicity studies. Ultimately, the scaffold presented herein demonstrates promising in vivo efficacy in a murine Pseudomonas aeruginosa keratitis model in combination with the antibiotic meropenem.

Based on an X-ray structure of cyclomarin bound to ClpC1, a new conformationally fixed, bicyclic cyclomarin derivative is synthesized in an effort to enhance antituberculosis activity. The synthesis of the linear heptapeptide and the two macrolactamizations proceed smoothly. Only the very last synthetic step, the cleavage of a benzyl ether, provides a low yield. Despite the successful synthesis, the resulting bicyclic compound shows reduced activity compared to cyclomarin.

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.

Acute respiratory diseases in humans can be caused by various viral pathogens such as respiratory syncytial virus (RSV), human coronavirus 229E (hCoV-229E), and severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2). To prevent severe cases by an early treatment, one effective strategy is to inhibit viral infection at the entry stage of the replication cycle. However, there is a lack of efficient, FDA-approved small molecule drugs targeting these pathogens. Previously, we identified two dual RSV/hCoV-229E small molecule inhibitors with activity in the single-digit micromolar range. In this study, we focused on optimizing the more promising starting point using a multiparametric hit optimization approach. Here, we present the results, including valuable insights into the structure activity relationship (SAR), and report the discovery of a submicromolar RSV entry inhibitor and a highly potent compound against hCoV-229E.

Die Art und Weise, mit welcher die Natur hochkomplexe molekulare Strukturen im Rahmen vielschichtiger Biosynthesewegen konstruiert, ist durch chemische Synthesen nur schwer zu übertreffen. Da die Biosynthese jedoch auf Kaskaden nativer enzymatischer Umwandlungen angewiesen ist, um bestimmte Zielstrukturer zu erzeugen, ist ihr Spektrum auch erheblich eingeschränkt. In dieser Arbeit werden nicht‐natürliche biokatalytische Module, konkret eine Peroxidase‐vermittelte Achmatowicz‐Umlagerung und eine Dehydrogenase‐katalysierte “Borrowing Hydrogen”‐Isomerisierung, erfolgreich in einen künstlichen Stoffwechsel implementiert, der die Vorteile der traditionellen Retrosynthese mit der Eleganz und Effizienz biosynthetischer Systeme verbindet. Die Totalsynthese des tricyclischen Angiopterlactons B gelingt so in nur zwei Schritten ausgehend von Acetylfuran, wobei das Verfahren vollständig in wässriger Umgebung abläuft und hauptsächlich auf Enzyme als wichtige Reaktionsvermittler angewiesen ist.

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.

Gene knockdown bysiRNA offers an unrestricted choice of targetsand specificity based on the principle of complementary Watson-Crickbase pairing with mRNA. However, the negative charge, large molecularsize, and susceptibility to enzymatic degradation of siRNA impedeits successful transfection, hence limiting its potential for therapeuticuse. The development of efficient and safe siRNA transfection agentsis, therefore, critical for siRNA-based therapy. Herein, we developeda protein-based biodynamic polymer (biodynamer) that showed potentialas a siRNA transfection vector, owing to its excellent biocompatibility,easy tunability, and dynamic polymerization under acidic environments.The positively charged biodynamers formed stable dynamic nanocomplexes(XL-DPs, hydrodynamic diameter of approximately 104 nm) with siRNAvia electrostatic interactions and chemical cross-linking. As a proofof concept, the optimized XL-DPs were stable in physiological conditionswith serum proteins and demonstrated significant pH-dependent sizechange and degradability, as well as siRNA release capability. Theminimal cytotoxicity and excellent cellular uptake of XL-DPs effectivelysupported the intracellular delivery of siRNA. Our study demonstratedthat the XL-DPs in survivin siRNA delivery enabled potent knockdownof survivin mRNA and induced notable apoptosis of carcinoma cells(2.2 times higher than a lipid-based transfection agent, Lipofectamine2000). These findings suggested that our XL-DPs hold immense potentialas a promising platform for siRNA delivery and can be considered strongcandidates in the advancement of next-generation transfection agents.

Icumazoles are potent antifungal polyketides with intriguing struc-tural features. Here, we present the polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) hybrid biosynthetic gene clus-ter of icumazoles. Surprisingly, an unusual nonterminal thioesterase domain divides the PKS/NRPS assembly line. The succeeding PKS modules potentially form a rare precursor 4-methyl-2-hexenoyl-ACP, thus deviating from the previously proposed polyoxypeptin pathway. The 4-methyl-2-hexenoyl-ACP is further reductively carboxylated to 2-methylbutylmalonyl-ACP, essential for icumazole biosynthesis by IcuL. We characterize IcuL and its homologs TgaD and Leu10 in vitro, suggesting a stricter substrate specificity of this new family of crotonyl carboxylases/reductases (CCRs) than found in canonical ones. Intriguingly, we also find that TgaD unpre-cedently utilizes both nicotinamide adenine dinucleotide phosphate (NADPH) and NADH as cofactors with similar efficiency, diverging from the NADPH-specific characteristic of canonical CCRs. Further-more, a sequence similarity network-based and phylogenetic bio-informatic survey reveals that the IcuL-like CCRs are evolutionarily separated from canonical CCRs.

Nature's way to construct highly complex molecular entities as part of biosynthetic pathways is unmatched by any chemical synthesis. Yet, relying on a cascade of native enzymatic transformations to achieve a certain target structure, biosynthesis is also significantly limited in its scope. In this study, non-natural biocatalytic modules, a peroxidase-mediated Achmatowicz rearrangement and a dehydrogenase-catalyzed borrowing-hydrogen-type isomerization were successfully incorporated into an artificial metabolism, combining the benefits of traditional retrosynthesis with the elegance and efficacy of biosynthetic networks. In a highly streamlined process, the total synthesis of tricyclic angiopterlactone B was achieved in two steps operating entirely in an aqueous environment while relying mainly on enzymes as key reaction mediators.

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.

We report structure elucidation and total synthesis of five unprecedented terpenoid-alkaloids, the sandacrabins, alongside with the first description of their producing organism Sandaracinus defensii MSr10575, which expands the Sandaracineae family by only its second member. The genome sequence of S. defensii as presented in this study was utilized to identify enzymes responsible for sandacrabin formation, whereby dimethylbenzimidazol, deriving from cobalamin biosynthesis, was identified as key intermediate. Biological activity profiling revealed that all sandacrabins except congener A exhibit potent antiviral activity against the human pathogenic coronavirus HCoV229E in the three digit nanomolar range. Investigation of the underlying mode of action discloses that the sandacrabins inhibit the SARS-CoV-2 RNA-dependent RNA polymerase complex, highlighting them as structurally distinct non-nucleoside RNA synthesis inhibitors. The observed segregation between cell toxicity at higher concentrations and viral inhibition represents a good starting point for their medicinal chemistry optimization towards selective inhibitors.

Here, we report on a potent class of substituted ureidothiophenes targeting energy-coupling factor (ECF) transporters, an unexplored target that is not addressed by any antibiotic in the market. Since the ECF module is crucial for the vitamin transport mechanism, the prevention of substrate uptake should ultimately lead to cell death. By utilizing a combination of virtual and functional whole-cell screening of our in-house library, the membrane-bound protein mediated uptake of folate could be effectively inhibited. Structure-based optimization of our hit yielded low-micromolar inhibitors, whereby the most active compounds showed in addition potent antimicrobial activities against a panel of clinically relevant Gram-positive pathogens without significant cytotoxic effects.

Small macrocyclic peptides are promising candidates for new anti-infective drugs. To date, such peptides have been poorly studied in the context of anti-virulence targets. Using phage display and a self-designed peptide library, we identified a cyclic heptapeptide that can bind the carbon storage regulator A (CsrA) from Yersinia pseudotuberculosis and displace bound RNA. This disulfide-bridged peptide, showed an IC50 value in the low micromolar range. Upon further characterization, cyclisation was found to be essential for its activity. To increase metabolic stability, a series of disulfide mimetics were designed and a redox-stable 1,4-disubstituted 1,2,3-triazole analogue displayed activity in the double-digit micromolar range. Further experiments revealed that this triazole peptidomimetic is also active against CsrA from Escherichia coli and RsmA from Pseudomonas aeruginosa. This study provides an ideal starting point for medicinal chemistry optimization of this macrocyclic peptide and might pave the way towards broad-acting virulence modulators.

Nature's way to construct highly complex molecular entities with virtue as part of biosynthetic pathways is unmatched by any chemical synthesis. Yet, relying on a cascade of native enzymatic transformations to achieve a certain target structure, biosynthesis is also significantly limited in its scope. In this work, non-natural biocatalytic modules are successfully implemented into an artificial metabolism, combining the benefits of traditional retrosynthesis with the elegance and efficacy of biosynthetic networks. In a highly streamlined process, a fully enzymatic total synthesis of the tricyclic angiopterlactone B is achieved.