Two natural compounds as potential inhibitors against the Helicobacter pylori and Acinetobacter baumannii IspD enzymes.

In a vast majority of bacteria, protozoa and plants, the methylerythritol phosphate (MEP) pathway is utilized for the synthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), which are precursors for isoprenoids. Isoprenoids, such as cholesterol and coenzyme Q, play a variety of crucial roles in physiological activities, including cell-membrane formation, protein degradation, cell apoptosis, and transcription regulation. In contrast, humans employ the mevalonate (MVA) pathway for the production of IDP and DMADP, rendering proteins in the MEP pathway appealing targets for antimicrobial agents. This pathway consists of seven consecutive enzymatic reactions, of which 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD) and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) catalyze the third and fifth steps, respectively. In this study, we characterized the enzymatic activities and protein structures of Helicobacter pylori IspDF and Acinetobacter baumannii IspD. Then, using the direct interaction-based thermal shift assay, we conducted a compound screening of an approved drug library and identified 27 hit compounds potentially binding to AbIspD. Among them, two natural products, rosmarinic acid and tanshinone IIA sodium sulfonate, exhibited inhibitory activities against HpIspDF and AbIspD, by competing with one of the substrates, MEP. Moreover, tanshinone IIA sodium sulfonate also demonstrated certain antibacterial effects against H. pylori. In summary, we identified two IspD inhibitors from approved ingredients, broadening the scope for antibiotic discovery targeting the MEP pathway.

Cyclic peptides discriminate BCL-2 and its clinical mutants from BCL-XL by engaging a single-residue discrepancy.

Overexpressed pro-survival B-cell lymphoma-2 (BCL-2) family proteins BCL-2 and BCL-XL can render tumor cells malignant. Leukemia drug venetoclax is currently the only approved selective BCL-2 inhibitor. However, its application has led to an emergence of resistant mutations, calling for drugs with an innovative mechanism of action. Herein we present cyclic peptides (CPs) with nanomolar-level binding affinities to BCL-2 or BCL-XL, and further reveal the structural and functional mechanisms of how these CPs target two proteins in a fashion that is remarkably different from traditional small-molecule inhibitors. In addition, these CPs can bind to the venetoclax-resistant clinical BCL-2 mutants with similar affinities as to the wild-type protein. Furthermore, we identify a single-residue discrepancy between BCL-2 D111 and BCL-XL A104 as a molecular "switch" that can differently engage CPs. Our study suggests that CPs may inhibit BCL-2 or BCL-XL by delicately modulating protein-protein interactions, potentially benefiting the development of next-generation therapeutics.

SAR study of 1,2-benzisothiazole dioxide compounds that agonize HIF-2 stabilization and EPO production.

Benzisothiazole dioxide compound was reported to agonize HIF-2 stabilization and improve EPO production, thus conceiving a potential strategy to treat disease with chronic hypoxia exemplified by renal anemia. Herein, on the bases of multiple molecular and cellular assays, a series of benzisothiazole derivatives have been synthesized and their structure-activity relationship was evaluated. The SAR and molecular docking studies have revealed the structural insights on the rational design of HIF-2 agonist and discovered a more potential 5-bromine substituted analogue, which showed 2-4 times improvement of HIF-2 downstream gene transcriptions, including EPO production. The present results suggest the therapeutic potential of the compounds for diseases related to EPO insufficiency.

Structures of NPAS4-ARNT and NPAS4-ARNT2 heterodimers reveal new dimerization modalities in the bHLH-PAS transcription factor family.

Neuronal PER-ARNT-SIM (PAS) domain protein 4 (NPAS4) is a protective transcriptional regulator whose dysfunction has been linked to a variety of neuropsychiatric and metabolic diseases. As a member of the basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) transcription factor family, NPAS4 is distinguished by an ability to form functional heterodimers with aryl hydrocarbon receptor nuclear translocator (ARNT) and ARNT2, both of which are also bHLH-PAS family members. Here, we describe the quaternary architectures of NPAS4-ARNT and NPAS4-ARNT2 heterodimers in complexes involving DNA response elements. Our crystallographic studies reveal a uniquely interconnected domain conformation for the NPAS4 protein itself, as well as its differentially configured heterodimeric arrangements with both ARNT and ARNT2. Notably, the PAS-A domains of ARNT and ARNT2 exhibit variable conformations within these two heterodimers. The ARNT PAS-A domain also forms a set of interfaces with the PAS-A and PAS-B domains of NPAS4, different from those previously noted in ARNT heterodimers formed with other class I bHLH-PAS family proteins. Our structural observations together with biochemical and cell-based interrogations of these NPAS4 heterodimers provide molecular glimpses of the NPAS4 protein architecture and extend the known repertoire of heterodimerization patterns within the bHLH-PAS family. The PAS-B domains of NPAS4, ARNT, and ARNT2 all contain ligand-accessible pockets with appropriate volumes required for small-molecule binding. Given NPAS4's linkage to human diseases, the direct visualization of these PAS domains and the further understanding of their relative positioning and interconnections within the NPAS4-ARNT and NPAS4-ARNT2 heterodimers may provide a road map for therapeutic discovery targeting these complexes.

Structural basis for the allosteric inhibition of hypoxia-inducible factor (HIF)-2 by belzutifan.

Hypoxia-inducible factor (HIF)-2α and its obligate heterodimerization partner aryl hydrocarbon receptor nuclear translocator (ARNT), are both members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factor family. Previous studies have identified HIF-2α as a key oncogenic driver in clear cell renal cell carcinoma (ccRCC), rendering it a promising drug target for this type of kidney cancer. Belzutifan is the first HIF-2α inhibitor approved for treating ccRCC and other cancers associated with the von Hippel-Lindau (VHL) disease. However, the detailed inhibitory mechanism of belzutifan at molecular level is still unclear. Here we obtained the crystal structure of HIF-2α-ARNT heterodimer in complex with belzutifan at 2.75 Å resolution. The complex structure shows that belzutifan binds into the PAS-B pocket of HIF-2α, and it destabilizes the dimerization of HIF-2α and ARNT through allosteric effects mainly mediated by the key residue M252 of HIF-2α near the dimer interface. We further explored the inhibitory effects of belzutifan using biochemical and functional assays. The time-resolved fluorescence energy transfer (TR-FRET)-based binding assay showed that belzutifan disrupts the dimerization of HIF-2α and ARNT with a Ki value of 20 nM. The luciferase reporter assay indicated that belzutifan can efficiently inhibit the transcriptional activity of HIF-2α with an IC50 value of 17 nM. Besides, the real-time PCR assay illustrated that belzutifan can reduce the expression of HIF-2α downstream genes in 786-O kidney cancer cells in a dose-dependent manner. Our work reveals the molecular mechanism by which belzutifan allosterically inhibits HIF-2α and provides valuable information for the subsequent drug development targeting HIF-2α. Significance Statement The bHLH-PAS family of transcription factors are an emerging group of small-molecule drug targets. Belzutifan, originally developed by Peloton Therapeutics, is the first FDA-approved drug directly binding to a bHLH-PAS protein, the hypoxia-inducible factor (HIF)-2α. Based on the protein-drug complex structure, biochemical binding assays, and functional profiling of downstream gene expression, this study reveals the regulatory mechanism of how belzutifan allosterically destabilizes HIF-2α's heterodimerization with its obligate partner protein, thus reducing their transcriptional activity that links to tumor progression.

Identification of oleoylethanolamide as an endogenous ligand for HIF-3α.

Hypoxia-inducible factors (HIFs) are α/ß heterodimeric transcription factors modulating cellular responses to the low oxygen condition. Among three HIF-α isoforms, HIF-3α is the least studied to date. Here we show that oleoylethanolamide (OEA), a physiological lipid known to regulate food intake and metabolism, binds selectively to HIF-3α. Through crystallographic analysis of HIF-3 α/ß heterodimer in both apo and OEA-bound forms, hydrogen-deuterium exchange mass spectrometry (HDX-MS), molecular dynamics (MD) simulations, and biochemical and cell-based assays, we unveil the molecular mechanism of OEA entry and binding to the PAS-B pocket of HIF-3α, and show that it leads to enhanced heterodimer stability and functional modulation of HIF-3. The identification of HIF-3α as a selective lipid sensor is consistent with recent human genetic findings linking HIF-3α with obesity, and demonstrates that endogenous metabolites can directly interact with HIF-α proteins to modulate their activities, potentially as a regulatory mechanism supplementary to the well-known oxygen-dependent HIF-α hydroxylation.

Docking-guided rational engineering of a macrolide glycosyltransferase glycodiversifies epothilone B.

Glycosyltransferases typically display acceptor substrate flexibility but more stringent donor specificity. BsGT-1 is a highly effective glycosyltransferase to glycosylate macrolides, including epothilones, promising antitumor compounds. Here, we show that BsGT-1 has three major regions significantly influencing the glycodiversification of epothilone B based on structural molecular docking, "hot spots" alanine scanning, and site saturation mutagenesis. Mutations in the PSPG-like motif region and the C2 loop region are more likely to expand donor preference; mutations of the flexible N3 loop region located at the mouth of the substrate-binding cavity produce novel epothilone oligosaccharides. These "hot spots" also functioned in homologues of BsGT-1. The glycosides showed significantly enhanced water solubility and decreased cytotoxicity, although the glycosyl appendages of epothilone B also reduced drug permeability and attenuated antitumor efficacy. This study laid a foundation for the rational engineering of other GTs to synthesize valuable small molecules.

Engineering and elucidation of the lipoinitiation process in nonribosomal peptide biosynthesis.

Nonribosomal peptide synthetases containing starter condensation domains direct the biosynthesis of nonribosomal lipopeptides, which generally exhibit wide bioactivities. The acyl chain has strong impacts on bioactivity and toxicity, but the lack of an in-depth understanding of starter condensation domain-mediated lipoinitiation limits the bioengineering of NRPSs to obtain novel derivatives with desired acyl chains. Here, we show that the acyl chains of the lipopeptides rhizomide, holrhizin, and glidobactin were modified by engineering the starter condensation domain, suggesting a workable approach to change the acyl chain. Based on the structure of the mutated starter condensation domain of rhizomide biosynthetic enzyme RzmA in complex with octanoyl-CoA and related point mutation experiments, we identify a set of residues responsible for the selectivity of substrate acyl chains and extend the acyl chains from acetyl to palmitoyl. Furthermore, we illustrate three possible conformational states of starter condensation domains during the reaction cycle of the lipoinitiation process. Our studies provide further insights into the mechanism of lipoinitiation and the engineering of nonribosomal peptide synthetases.

Structural insights into a novel Ca2+-independent PL-6 alginate lyase from Vibrio OU02 identify the possible subsites responsible for product distribution.

Alginate lyases have a wide range of industrial applications, such as oligosaccharide preparation, medical treatment, and bioconversion. Therefore, the discovery and characterization of novel alginate lyases are extremely important. PL-6 alginate lyases are classified into two groups: those with a single domain or two domains. However, only one structure of a two-domain alginate lyase has been determined to date. In this study, we characterized a novel single-domain PL-6 alginate lyase (named AlyF). According to the biochemical analysis, AlyF possesses unique features compared with other PL-6 enzymes, including (1) a Ca2+-independent catalytic mechanism and (2) a PolyG-specific cleavage specificity that predominantly produces trisaccharides. The structures of AlyF and its complexes described here reveal the structural basis for these unique features and substrate binding mechanisms, which were further confirmed using mutagenesis. More importantly, we determined the possible subsites specifying the predominantly trisaccharide products of AlyF, which may facilitate the rational design of AlyF for potential applications in preparing a single alginate oligomer.

Single-Stranded DNA-Binding Protein and Exogenous RecBCD Inhibitors Enhance Phage-Derived Homologous Recombination in Pseudomonas.

The limited efficiency of the available tools for genetic manipulation of Pseudomonas limits fundamental research and utilization of this genus. We explored the properties of a lambda Red-like operon (BAS) from Pseudomonas aeruginosa phage Ab31 and a Rac bacteriophage RecET-like operon (RecTEPsy) from Pseudomonas syringae pv. syringae B728a. Compared with RecTEPsy, the BAS operon was functional at a higher temperature indicating potential to be a generic system for Pseudomonas. Owing to the lack of RecBCD inhibitor in the BAS operon, we added Redγ or Pluγ and found increased recombineering efficiencies in P. aeruginosa and Pseudomonas fluorescens but not in Pseudomonas putida and P. syringae. Overexpression of single-stranded DNA-binding protein enhanced recombineering in several contexts including RecET recombination in E. coli. The utility of these systems was demonstrated by engineering P. aeruginosa genomes to create an attenuated rhamnolipid producer. Our work enhances the potential for functional genomics in Pseudomonas.

Bidirectional modulation of HIF-2 activity through chemical ligands.

Hypoxia-inducible factor-2 (HIF-2) is a heterodimeric transcription factor formed through dimerization between an oxygen-sensitive HIF-2α subunit and its obligate partner subunit ARNT. Enhanced HIF-2 activity drives some cancers, whereas reduced activity causes anemia in chronic kidney disease. Therefore, modulation of HIF-2 activity via direct-binding ligands could provide many new therapeutic benefits. Here, we explored HIF-2α chemical ligands using combined crystallographic, biophysical, and cell-based functional studies. We found chemically unrelated antagonists to employ the same mechanism of action. Their binding displaced residue M252 from inside the HIF-2α PAS-B pocket toward the ARNT subunit to weaken heterodimerization. We also identified first-in-class HIF-2α agonists and found that they significantly displaced pocket residue Y281. Its dramatic side chain movement increases heterodimerization stability and transcriptional activity. Our findings show that despite binding to the same HIF-2α PAS-B pocket, ligands can manifest as inhibitors versus activators by mobilizing different pocket residues to allosterically alter HIF-2α-ARNT heterodimerization.