RSC Chemical Biology最新文献

The chemical logic associated with assembly of many bacterial terpenoids remains poorly understood. We focused our efforts on the early-stage biosynthesis of the phenalinolactone diterpenoids, demonstrating that the anti/anti/syn-perhydrophenanthrene core is constructed by sequential prenylation, epoxidation, and cyclization. The functions and timing of PlaT1–PlaT3 were assigned by comprehensive heterologous reconstitution. We illustrated that the UbiA prenyltransferase PlaT3 acts on geranylgeranyl diphosphate (GGPP) in the first step of phenalinolactone biosynthesis, prior to epoxidation by the flavin-dependent monooxygenase PlaT1 and cyclization by the type II terpene cyclase PlaT2. Finally, we isolated eight new-to-nature terpenoids, expanding the scope of the bacterial terpenome. The biosynthetic strategy employed in the assembly of the phenalinolactone core, with cyclization occurring after prenylation, is rare in bacteria and resembles fungal meroterpenoid biosynthesis. The findings presented here set the stage for future discovery, engineering, and enzymology efforts in bacterial meroterpenoids.

Two human mitochondrial membrane CYP11B enzymes play a pivotal role in steroidogenesis. CYP11B1 generates the major glucocorticoid cortisol, while CYP11B2 catalysis yields the primary mineralocorticoid aldosterone. Catalysis by both requires electron delivery by a soluble iron–sulfur adrenodoxin redox partner. However recent studies have shown that adrenodoxin/CYP11B interaction alone allosterically increases substrate and inhibitor affinity as exhibited by decreased dissociation constant (K_d) values. The current study moves beyond such equilibrium studies, by defining adrenodoxin effects on the rates of P450 ligand binding and release separately. Stopped-flow data clearly demonstrate that adrenodoxin interaction with the P450 proximal surfaces increases ligand binding in both P450 CYP11B active sites by increasing the on rate constant and decreasing the off rate constant. As substrate entry and exit from the sequestered P450 active site requires conformational changes on the distal side of the P450 enzyme, a likely explanation is that adrenodoxin binding allosterically modulates CYP11B conformational changes. The 93% identical CYP11B enzymes can bind and hydroxylate each other's native substrates differing only by a hydroxyl. However, CYP11B1 exhibits monophasic substrate binding and CYP11B2 biphasic substrate binding, even when the substrates are swapped. This indicates that small differences in amino acid sequence between human CYP11B1 and CYP11B2 enzymes are more functionally important in ligand binding and could suggest avenues for more selective inhibition of these drug targets. Both protein/protein interactions and protein/substrate interactions are most likely to act by modulating CYP11B conformational dynamics.

Triplex DNA formation is a useful genomic targeting tool that is expected to have a wide range of applications, including the antigene method; however, there are fundamental limitations in its forming sequence. We recently extended the triplex DNA-forming sequence to methylated DNA sequences containing ^5mCG base pairs by developing guanidino-dN, which is capable of recognizing a ^5mCG base pair with high affinity. We herein investigated the effect of triplex DNA formation using TFOs with guanidino-dN on methylated DNA sequences at the promoter of the RASSF1A gene, whose expression is epigenetically suppressed by DNA methylation in MCF-7 cells, on gene expression. Interestingly, triplex DNA formation increased the expression of the RASSF1A gene at the transcript and protein levels. Furthermore, RASSF1A-activated MCF-7 cells exhibited cell growth suppressing activity. Changes in the expression of various genes associated with the promotion of apoptosis and breast cancer survival accompanied the activation of RASSF1A in cells exhibited antiproliferative activity. These results suggest the potential of increases in gene expression through triplex DNA formation as a new genomic targeting tool.

Gene editing by CRISPR/Cas9 offers great therapeutic opportunities but requires delivering large plasmid DNA (pDNA) into cells, a task for which transfection reagents are better suited than viral vectors. Here we performed a structure–activity relationship study of Z22, a D-enantiomeric, arginine containing, lipidated peptide dendrimer developed for pDNA transfection of a CRISPR/Cas9 plasmid co-expressing GFP. While all dendrimer analogs tested bound pDNA strongly and internalized their cargo into cells, D-chirality proved essential for transfection by avoiding proteolysis of the dendrimer structure required for endosome escape and possibly crossing of the nuclear envelope. Furthermore, a cysteine residue at the core of Z22 proved non-essential and was removed to yield the more active analog Z34. This dendrimer shows >83% GFP transfection efficiency in HEK cells with no detrimental effect on cell viability and promotes functional CRISPR/Cas9 mediated gene editing. It is accessible by solid-phase peptide synthesis and therefore attractive for further development.

The mycobacterial membrane protein large 3 (MmpL3) transports key precursor lipids to the outer membrane of Mycobacterium species. Multiple structures of MmpL3 from both M. tuberculosis and M. smegmatis in various conformational states indicate that the protein is both structurally and functionally monomeric. However, most other resistance, nodulation and cell division (RND) transporters structurally characterised to date are either dimeric or trimeric. Here we present an in depth biophysical and computational analysis revealing that MmpL3 from M. smegmatis exists as a dimer in a variety of membrane mimetic systems (SMALPs, detergent-based solution and nanodiscs). Sucrose gradient separation of MmpL3 populations from M. smegmatis, reconstituted into nanodiscs, identified monomeric and dimeric populations of the protein using laser induced liquid bead ion desorption (LILBID), a native mass spectrometry technique. Preliminary cryo-EM analysis confirmed that MmpL3 forms physiological dimers. Untargeted lipidomics experiments on membrane protein co-purified lipids revealed PE and PG lipid classes were predominant. Molecular dynamics (MD) simulations, in the presence of physiologically-relevant lipid compositions revealed the likely dimer interface.

Tryptophan 2-monooxygenase (TMO) is an FAD-bound flavoenzyme which catalyzes the oxidative decarboxylation of L-tryptophan to produce indole-3-acetamide (IAM) and carbon dioxide. The reaction of TMO is the first step of indole-3-acetic acid (IAA) biosynthesis. Although TMO is of interest for mechanistic studies and synthetic biology applications, the enzyme has low thermostability and soluble expression yield. Herein, we employed a combined approach of rational design using computational tools with site-saturation mutagenesis to screen for TMO variants with significantly improved thermostability properties and soluble protein expression. The engineered TMO variants, TMO-PWS and TMO-PWSNR, possess melting temperatures (T_m) of 65 °C, 17 °C higher than that of the wild-type enzyme (TMO-WT). At 50 °C, the stabilities (t_1/2) of TMO-PWS and TMO-PWSNR were 85-fold and 92.4-fold higher, while their soluble expression yields were 1.4-fold and 2.1-fold greater than TMO-WT, respectively. Remarkably, the kinetic parameters of these variants were similar to those of the wild-type enzymes, illustrating that they are promising candidates for future studies. Molecular dynamic simulations of the wild-type and thermostable TMO variants identified key interactions for enhancing these improvements in the biophysical properties of the TMO variants. The introduced mutations contributed to hydrogen bond formation and an increase in the regional hydrophobicity, thereby, strengthening the TMO structure.

N ⁶-methyladenosine (m⁶A) is an abundant modification in mammalian mRNAs and plays important regulatory roles in gene expression, primarily mediated through specific recognition by “reader” proteins. YTH family proteins are one major family of known m⁶A readers, which specifically recognize m⁶A-modified transcripts via the YTH domains. Despite the significant relevance of YTH-m⁶A recognition in biology and diseases, few small molecule inhibitors are available for specifically perturbing this interaction. Here we report the discovery of a new inhibitor (“N-7”) for YTH-m⁶A RNA recognition, from the screening of a nucleoside analogue library against the YTH domain of the YTHDF1 protein. N-7 is characterized to be a pan-inhibitor in vitro against five YTH domains from human YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2 proteins, with IC₅₀ values in the range of 30–48 μM measured using a fluorescence polarization competition assay. We demonstrated that N-7 directly interacts with the YTH domain proteins via a thermal shift assay. N-7 expands the chemical structure landscape of the m⁶A YTH domain-containing reader inhibitors and potentiates future inhibitor development for reader functional studies and therapeutic efforts in targeting the epitranscriptome.

We previously showed that the proteostasis regulator compound AA147 (N-(2-hydroxy-5-methylphenyl)benzenepropanamide) potently protects against neurotoxic insults, such as glutamate-induced oxytosis. Though AA147 is a selective activator of the ATF6 arm of the unfolded protein response in non-neuronal cells, AA147-dependent protection against glutamate toxicity in cells of neuronal origin is primarily mediated through activation of the NRF2 oxidative stress response. AA147 activates NRF2 through a mechanism involving metabolic activation of AA147 by endoplasmic reticulum (ER) oxidases, affording an AA147-based quinone methide that covalently targets the NRF2 repressor protein KEAP1. Previous results show that the 2-amino-p-cresol A-ring of AA147 is required for NRF2 activation, while the phenyl B-ring of AA147 is amenable to modification. Here we explore whether the protease-sensitive amide linker between the A- and B-rings of this molecule can be modified to retain NRF2 activation. We show that replacement of the amide linker of AA147 with a carbamate linker retains NRF2 activation in neuronal cells and improves protection against neurotoxic insults, including glutamate-induced oxytosis and erastin-induced ferroptosis. Moreover, we demonstrate that inclusion of this carbamate linker facilitates identification of next-generation AA147 analogs with improved cellular tolerance and activity in disease-relevant assays.

Rlig1 is the first RNA ligase identified in humans utilising a classical 5′–3′ ligation mechanism. It is a conserved enzyme in all vertebrates and is mutated in various cancers. During our initial research on Rlig1, we observed that Rlig1-knockout (KO) HEK293 cells are more sensitive to the stress induced by menadione than their WT counterpart, representing a type of chemical synthetic lethality. To gain further insight into the biological pathways in which Rlig1 may be involved, we aimed at identifying new synthetically lethal small molecules. To this end, we conducted a high-throughput screening with a compound library comprising over 13 000 bioactive small molecules. This approach led to the identification of compounds that exhibited synthetic lethality in combination with Rlig1-KO. In addition to the aforementioned novel compounds that diverge structurally from menadione, we also tested multiple small molecules containing a naphthoquinone scaffold.

The gut microbiome plays critical roles in human homeostasis, disease progression, and pharmacological efficacy through diverse metabolic pathways. Gut bacterial β-glucuronidase (GUS) enzymes reverse host phase 2 metabolism, in turn releasing active hormones and drugs that can be reabsorbed into systemic circulation to affect homeostasis and promote toxic side effects. The FMN-binding and loop 1 gut microbial GUS proteins have been shown to drive drug and toxin reactivation. Here we report the structure–activity relationships of two selective piperazine-containing bacterial GUS inhibitors. We explore the potency and mechanism of action of novel compounds using purified GUS enzymes and co-crystal structures. Our results establish the importance of the piperazine nitrogen placement and nucleophilicity as well as the presence of a cyclohexyl moiety appended to the aromatic core. Using these insights, we synthesized an improved microbial GUS inhibitor, UNC10206581, that potently inhibits both the FMN-binding and loop 1 GUS enzymes in the human gut microbiome, does not inhibit bovine GUS, and is non-toxic within a relevant dosing range. Kinetic analyses demonstrate that UNC10206581 undergoes a slow-binding and substrate-dependent mechanism of inhibition similar to that of the parent scaffolds. Finally, we show that UNC10206581 displays potent activity within the physiologically relevant systems of microbial cultures and human fecal protein lysates examined by metagenomic and metaproteomic methods. Together, these results highlight the discovery of more effective bacterial GUS inhibitors for the alleviation of microbe-mediated homeostatic dysregulation and drug toxicities and potential therapeutic development.