A New Golden Age of Natural Products Drug Discovery.

The 2015 Nobel Prize in Physiology or Medicine has been awarded to William C. Campbell, Satoshi Omura, and Youyou Tu for the discovery of avermectins and artemisinin, respectively, therapies that revolutionized the treatment of devastating parasite diseases. With the recent technological advances, a New Golden Age of natural products drug discovery is dawning.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Trastuzumab does not bind rat or mouse ErbB2/neu: implications for selection of non-clinical safety models for trastuzumab-based therapeutics.

PURPOSE: Assessment of non-clinical safety signals relies on understanding species selectivity of antibodies. This is particularly important with antibody-drug conjugates, where it is key to determine target-dependent versus target-independent toxicity. Although it appears to be widely accepted that trastuzumab does not bind mouse or rat HER2/ErbB2/neu, numerous investigators continue to use mouse models to investigate safety signals of trastuzumab and trastuzumab emtansine (T-DM1). We, therefore, conducted a broad array of both binding and biologic studies to demonstrate selectivity of trastuzumab for human HER2 versus mouse/rat neu. METHODS: Binding of anti-neu and anti-HER2 antibodies was assessed by ELISA, FACS, IHC, Scatchard, and immunoblot methods in human, rat, and mouse cell lines. In human hepatocytes, T-DM1 uptake and catabolism were measured by LC-MS/MS; cell viability changes were determined using CellTiter-Glo. RESULTS: Our data demonstrate, using different binding methods, lack of trastuzumab binding to rat or mouse neu. Structural studies show important amino acid differences in the trastuzumab-HER2 binding interface between mouse/rat and human HER2 ECD. Substitution of these rodent amino acid residues into human HER2 abolish binding of trastuzumab. Cell viability changes, uptake, and catabolism of T-DM1 versus a DM1 non-targeted control ADC were comparable, indicating target-independent effects of the DM1-containing ADCs. Moreover, trastuzumab binding to human or mouse hepatocytes was not detected. CONCLUSIONS: These data, in total, demonstrate that trastuzumab, and by extension T-DM1, do not bind rat or mouse neu, underscoring the importance of species selection for safety studies investigating trastuzumab or trastuzumab-based therapeutics.

Personalized Cancer Vaccines: Clinical Landscape, Challenges, and Opportunities.

Tremendous innovation is underway among a rapidly expanding repertoire of promising personalized immune-based treatments. Therapeutic cancer vaccines (TCVs) are attractive systemic immunotherapies that activate and expand antigen-specific CD8+ and CD4+ T cells to enhance anti-tumor immunity. Our review highlights key issues impacting TCVs in clinical practice and reports on progress in development. We review the mechanism of action, immune-monitoring, dosing strategies, combinations, obstacles, and regulation of cancer vaccines. Most trials of personalized TCVs are ongoing and represent diverse platforms with predominantly early investigations of mRNA, DNA, or peptide-based targeting strategies against neoantigens in solid tumors, with many in combination immunotherapies. Multiple delivery systems, routes of administration, and dosing strategies are used. Intravenous or intramuscular administration is common, including delivery by lipid nanoparticles. Absorption and biodistribution impact antigen uptake, expression, and presentation, affecting the strength, speed, and duration of immune response. The emerging trials illustrate the complexity of developing this class of innovative immunotherapies. Methodical testing of the multiple potential factors influencing immune responses, as well as refined quantitative methodologies to facilitate optimal dosing strategies, could help resolve uncertainty of therapeutic approaches. To increase the likelihood of success in bringing these medicines to patients, several unique development challenges must be overcome.

Microtubule and tubulin binding and regulation of microtubule dynamics by the antibody drug conjugate (ADC) payload, monomethyl auristatin E (MMAE): Mechanistic insights into MMAE ADC peripheral neuropathy.

Monomethyl auristatin E (MMAE) is a potent anti-cancer microtubule-targeting agent (MTA) used as a payload in three approved MMAE-containing antibody drug conjugates (ADCs) and multiple ADCs in clinical development to treat different types of cancers. Unfortunately, MMAE-ADCs can induce peripheral neuropathy, a frequent adverse event leading to treatment dose reduction or discontinuation and subsequent clinical termination of many MMAE-ADCs. MMAE-ADC-induced peripheral neuropathy is attributed to non-specific uptake of the ADC in peripheral nerves and release of MMAE, disrupting microtubules (MTs) and causing neurodegeneration. However, molecular mechanisms underlying MMAE and MMAE-ADC effects on MTs remain unclear. Here, we characterized MMAE-tubulin/MT interactions in reconstituted in vitro soluble tubulin or MT systems and evaluated MMAE and vcMMAE-ADCs in cultured human MCF7 cells. MMAE bound to soluble tubulin heterodimers with a maximum stoichiometry of ~1:1, bound abundantly along the length of pre-assembled MTs and with high affinity at MT ends, introduced structural defects, suppressed MT dynamics, and reduced the kinetics and extent of MT assembly while promoting tubulin ring formation. In cells, MMAE and MMAE-ADC (via nonspecific uptake) suppressed proliferation, mitosis and MT dynamics, and disrupted the MT network. Comparing MMAE action to other MTAs supports the hypothesis that peripheral neuropathy severity is determined by the precise mechanism(s) of each individual drug-MT interaction (location of binding, affinity, effects on morphology and dynamics). This work demonstrates that MMAE binds extensively to tubulin and MTs and causes severe MT dysregulation, providing convincing evidence that MMAE-mediated inhibition of MT-dependent axonal transport leads to severe peripheral neuropathy.

Submerged fermentation of Streptomyces uncialis providing a biotechnology platform for uncialamycin biosynthesis, engineering, and production.

Uncialamycin (UCM) belongs to the anthraquinone-fused subfamily of 10-membered enediyne natural products that exhibits an extraordinary cytotoxicity against a wide spectrum of human cancer cell lines. Antibody-drug conjugates, utilizing synthetic analogues of UCM as payloads, are in preclinical development. UCM is exclusively produced by Streptomyces uncialis DCA2648 on solid agar medium with low titers (∼0.019 mg/l), limiting its supply by microbial fermentation and hampering its biosynthetic and engineering studies by in vivo pathway manipulation. Here, we report cultivation conditions that enable genetic manipulation of UCM biosynthesis in vivo and allow UCM production, with improved titers, by submerged fermentation of the engineered S. uncialis strains. Specifically, the titer of UCM was improved nearly 58-fold to ∼1.1 mg/l through the combination of deletion of biosynthetic gene clusters encoding unrelated metabolites from the S. uncialis wild-type, chemical mutagenesis and manipulation of pathway-specific regulators to generate the engineered S. uncialis strains, and finally medium optimization of the latter for UCM production. Genetic manipulation of UCM biosynthesis was demonstrated by inactivating selected genes in the engineered S. uncialis strains, one of which afforded a mutant strain accumulating tiancimycin B, a common biosynthetic intermediate known for the anthraquinone-fused subfamily of enediyne natural products. These findings highlight a biotechnology platform for UCM biosynthesis, engineering, and production that should facilitate both its fundamental studies and translational applications.

Discovery of ammosesters by mining the Streptomyces uncialis DCA2648 genome revealing new insight into ammosamide biosynthesis.

The ammosamides (AMMs) are a family of pyrroloquinoline alkaloids that exhibits a wide variety of bioactivities. A biosynthetic gene cluster (BGC) that is highly homologous in both gene content and genetic organization to the amm BGC was identified by mining the Streptomyces uncialis DCA2648 genome, leading to the discovery of a sub-family of new AMM congeners, named ammosesters (AMEs). The AMEs feature a C-4a methyl ester, differing from the C-4a amide functional group characteristic to AMMs, and exhibit modest cytotoxicity against a broad spectrum of human cancer cell lines, expanding the structure-activity relationship for the pyrroloquinoline family of natural products. Comparative analysis of the ame and amm BGCs supports the use of a scaffold peptide as an emerging paradigm for the biosynthesis of the pyrroloquinoline family of natural products. AME and AMM biosynthesis diverges from a common intermediate by evolving the pathway-specific Ame24 O-methyltransferase and Amm20 amide synthetase, respectively. These findings will surely inspire future efforts to mimic Nature's combinatorial biosynthetic strategies for natural product structural diversity.

Cryptic Sulfur Incorporation in Thioangucycline Biosynthesis.

Sulfur incorporation into natural products is a critical area of biosynthetic studies. Recently, a subset of sulfur-containing angucyclines has been discovered, and yet, the sulfur incorporation step is poorly understood. In this work, a series of thioether-bridged angucyclines were discovered, and a cryptic epoxide Michael acceptor intermediate was revealed en route to thioangucyclines (TACs) A and B. However, systematic gene deletion of the biosynthetic gene cluster (BGC) by CRISPR/Cas9 could not identify any gene responsible for the conversion of the epoxide intermediate to TACs. Instead, a series of in vitro and in vivo experiments conclusively showed that the conversion is the result of two non-enzymatic steps, possibly mediated by endogenous hydrogen sulfide. Therefore, the TACs are proposed to derive from a detoxification process. These results are expected to contribute to the study of both angucyclines and the utilization of inorganic sulfur in natural product biosynthesis.

The LnmK Bifunctional Acyltransferase/Decarboxylase Specifying (2R)-Methylmalonyl-CoA and Employing Substrate-Assisted Catalysis for Polyketide Biosynthesis.

We previously showed that the bifunctional LnmK acyltransferase/decarboxylase (AT/DC) catalyzed the formation of a propionyl-S-acyl carrier protein (ACP) from methylmalonyl-CoA, but its substrate specificity to (2S)-, (2R)-, or (2RS)-methylmalonyl CoA was not known. We subsequently revealed that LnmK AT and DC activities share the same active site, employing a Tyr as the catalytic residue for AT, but failed to identify a general base within the vicinity of the active site for LnmK catalysis. We now show that (i) LnmK specifies (2R)-methylmalonyl-CoA and (ii) the AT and DC activities are coupled, featuring substrate-assisted catalysis via the enolate to account for the missing general base within the LnmK active site. LnmK and its homologues are the only bifunctional AT/DC enzymes known to date and are widespread. These findings, therefore, enrich PKS chemistry and enzymology. Since only the (2S)-methylmalonyl-CoA enantiomer has been established previously as a substrate for polyketide biosynthesis by PKSs, we now establish a role for both (2R)- and (2S)-methylmalonyl-CoA in polyketide biosynthesis, and (2R)-methylmalonyl-CoA should be considered as a substrate in future efforts for engineered production of polyketides by combinatorial biosynthesis or synthetic biology strategies in model hosts.

Leveraging a large microbial strain collection for natural product discovery.

Throughout history, natural products have significantly contributed to the discovery of novel chemistry, drug leads, and tool molecules to probe and address complex challenges in biology and medicine. Recent microbial genome sequencing efforts have uncovered many microbial biosynthetic gene clusters without an associated natural product. This means that the natural products isolated to date do not fully reflect the biosynthetic potential of microbial strains. This observation has rejuvenated the natural product community and inspired a return to microbial strain collections. Mining large microbial strain collections with the most current technologies in genome sequencing, bioinformatics, and high-throughput screening techniques presents new opportunities in natural product discovery. In this review, we report on the newly expanded microbial strain collection at The Scripps Research Institute, which represents one of the largest and most diverse strain collections in the world. Two complementary approaches, i.e. structure-centric and function-centric, are presented here to showcase how to leverage a large microbial strain collection for natural product discovery and to address challenges and harness opportunities for future efforts. Highlighted examples include the discovery of alternative producers of known natural products with superior growth characteristics and high titers, novel analogs of privileged scaffolds, novel natural products, and new activities of known and new natural products. We anticipate that this large microbial strain collection will facilitate the discovery of new natural products for many applications.

Platensimycin-Encapsulated Poly(lactic-co-glycolic acid) and Poly(amidoamine) Dendrimers Nanoparticles with Enhanced Anti-Staphylococcal Activity in Vivo.

Serious bacterial infections by multi-drug-resistant pathogens lead to human losses and endanger public health. The discovery of antibiotics with new modes of action, in combination with nanotechnology, might offer a promising route to combat multi-drug-resistant pathogens. Platensimycin (PTM), a potent inhibitor of FabB/FabF for bacterial fatty acid biosynthesis, is a promising drug lead against many drug-resistant bacteria. However, the clinical development of PTM is hampered by its poor pharmacokinetics. Herein, we report a nanostrategy that encapsulated PTM in two types of nanoparticles (NPs) poly(lactic-co-glycolic acid) (PLGA) and poly(amidoamine) (PAMAM) dendrimer to enhance its antibacterial activity in vitro and in vivo. The PTM-encapsulated NPs were effective to inhibit Staphylococcus aureus biofilm formation, and killed more S. aureus in a macrophage cell infection model over free PTM. The pharmacokinetic studies showed that PTM-loaded PLGA and PAMAM NPs exhibited increased AUC0-t (area under the curve) (∼4- and 2-fold) over free PTM. In a mouse peritonitis model, treatment of methicillin-resistant S. aureus infected mice using both PTM-loaded NPs (10 mg/kg) by intraperitoneal injection led to their full survival, while all infected mice died when treated by free PTM (10 mg/kg). These results not only suggest that PTM-loaded NPs may hold great potential to improve the poor pharmacokinetic properties of PTM, but support the rationale to develop bacterial fatty acid synthase inhibitors as promising antibiotics against drug-resistant pathogens.