ACS Chemical Biology最新文献

The Bcl-2 family of proteins governs mitochondrial outer membrane (MOM) permeabilization, a critical step in apoptosis that is dysfunctional in many cancers. Although cellular studies have long implicated direct interactions between the pore-forming apoptotic Bax protein and its opponent, the antiapoptotic Bcl-2 protein in apoptosis regulation, the underlying basic principles behind this control remained unresolved. To provide in-depth insight, we carried out a systematic biophysical study in which we utilized neutron reflectometry (NR) and ATR-FTIR to elucidate the molecular communication between those proteins in and around the mitochondrial membrane environment. The spatial and temporal changes across model MOM surfaces were resolved during the interaction of Bax with Bcl-2. The NR-derived membrane surface Bax distributions suggested that Bcl-2 mediated Bax sequestration through both Bcl-2/Bax heterodimerization and Bax/Bax oligomerization. Kinetic analysis revealed a two-step process: rapid formation of Bcl-2/Bax heterodimers, followed by slower Bax oligomerization on these complexes. Importantly, this sequestration mechanism was also observed in the presence of cardiolipin, a lipid known to promote the formation of an apoptotic pore by Bax in the absence of Bcl-2. These findings suggest a fundamental mechanism by which cancer cells may evade apoptosis by exploiting Bcl-2's ability to neutralize Bax through structural entrapment, even if excess Bax is present, either in response to treatment or natural death signals.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disorder with no approved therapeutics targeting the disease mechanism. DM1 is caused by the expression of expanded CUG repeat RNA (CUG_exp), which sequester the muscleblind-like (MBNL) family of RNA binding proteins leading to dysregulated alternative splicing and a host of downstream impacts. While previous studies showed that diamidines rescued DM1 dysregulated alternative splicing events, their potential was limited by toxicity and off-target effects. A new class of modified polycyclic compounds (MPCs), based on diamidines, were created and screened in DM1 patient-derived cell lines. This approach identified MPC03 and MPC04 as being capable of rescuing DM1 dysregulated splicing events at low nanomolar concentrations with no obvious toxicity and limited off-target effects. In a DM1 mouse model, treatment with MPC03 and MPC04 reduced CUG_exp RNA levels and partially rescued DM1 mis-splicing. Binding data and modeling showed that lead MPCs bind to CUG_exp RNA, and in cells lacking CUG repeats, MPC activity was absent, suggesting that these compounds displace sequestered MBNL proteins from CUG_exp RNA. Taken together, MPCs show therapeutic promise across multiple DM1 models.

Bioluminescent reporters are widely used to monitor and image biological processes. Among these, NanoLuc luciferase and its complementation variants (LgBiT/SmBiT and LgBiT/HiBiT) are commonly used due to their brightness, sensitivity, and compatibility with prolonged kinetic measurements. However, the single-channel emission of these NanoLuc-based systems (460 nm peak) limits their use in multiplexed assays. Prior efforts to shift NanoLuc's emission employed bioluminescence resonance energy transfer (BRET) to a proximal fluorescent protein or organic fluorophore. Building on this concept, we engineered high-efficiency BRET reporters, termed NanoPrism luciferases, by inserting circularly permuted NanoLuc or LgBiT into a surface loop of the self-labeling HaloTag protein. These NanoPrisms achieve a ∼90% BRET efficiency by optimally positioning NanoLuc variants near a fluorophore covalently bound to HaloTag. The binary design further supports high- and low-affinity complementation, allowing applications in HiBiT knock-in cells and tracking protein-protein interactions, respectively. Pairing red-shifted NanoPrisms with unmodified NanoLuc or its complementation variants, we created a two-color bioluminescent reporter platform featuring bright signals of similar intensity and >100 nm spectral separation, allowing quantitative, simultaneous measurement of two molecular readouts within the same sample. Here, we demonstrate the platform's utility for monitoring a degradation target alongside a control protein and for tracking two distinct events within a biological pathway, using plate-based detection and bioluminescence imaging. By enabling concurrent measurements within the same sample, the system provides insights into cellular dynamics while reducing variability and complexity associated with parallel single-channel assays.

OSW-1, a steroidal disaccharide isolated from the bulbs of Ornithogalum saundersiae, has been extensively studied for its extremely potent cytotoxicity against the National Cancer Institute's 60 cancer cell lines with an average IC₅₀ of 0.78 nM, while exhibiting selectivity toward normal cells. Although OSBP and ORP4L have been identified as its binding targets, their known functions appear insufficient to account for the compound's exceptional potency, suggesting the involvement of additional mechanisms and targets. Therefore, elucidating novel target proteins associated with its activity is essential for the further development of this molecule. Here, we disclose that OSW-1 can block the glycolytic pathway and trigger compensatory mitochondrial oxidative phosphorylation. This previously uncharacterized mechanism is relevant to the key rate-limiting enzyme, enolase 1 (ENO1), which shows subnanomolar affinity with OSW-1. Our study repurposes OSW-1 to be a small-molecule probe to investigate the function of ENO1 and a promising candidate for metabolism-targeted anticancer therapy.

Glycoconjugates facilitate a myriad of biological processes, including cell-cell recognition and immune response, and they are generated by enzymes that transfer glycans. The orthologs MraY and DPAGT1 are dimeric phosphoglycosyltransferases involved in oligosaccharide biosynthesis for either bacterial peptidoglycan or eukaryotic N-linked glycans, respectively. Both enzymes play central regulatory roles, making them attractive targets for antibacterial and anticancer therapies. In our prior studies, a muraymycin A1-derived inhibitor termed APPB (aminouridyl phenoxypiperidinbenzyl butanamide) was developed. It exhibits sub-100 nM IC₅₀ values against both MraY and DPAGT1 and has demonstrated efficacy against DPAGT1-dependent cancers, making it an excellent starting point for next-generation inhibitors. To guide their development, we determined cryo-EM structures of APPB bound to MraY or DPAGT1 at a resolution of 2.9 Å using single-particle analysis. The structures reveal that APPB, composed of a nucleoside, a central amide, and a lipid-mimetic, adopts two conformations in each protein, which correlate with the local hydrogen-bonding contacts of the central amide carbonyl. Examination of the amide carbonyl environments guides conformer selection for future DPAGT1-targeting anticancer agents. Further, comparisons of APPB-bound geometries and nucleoside interactions inform opportunities for antibacterial agents targeting MraY. Overall, our study provides design principles for MraY- or DPAGT1-specific drugs and motivates the utility of simultaneously characterizing inhibitor-bound orthologs for selective therapeutics.

RNA molecules play essential roles in all aspects of cellular function, but their complex secondary and tertiary structures pose significant challenges for selective targeting. Traditional antisense strategies often avoid these structured regions, focused instead on unstructured sequences. In this study, we present an enhanced Self-Avoiding Molecular Recognition System designed to selectively recognize and bind structured RNA elements, offering an alternative approach for targeting biologically relevant RNA conformations with improved specificity and selectivity. This is achieved by incorporating established self-avoidance nucleobases (t and c) along with a deazapurine series (a and g)─designed to provide greater flexibility in fine-tuning binding affinity─into a conformationally preorganized gamma peptide nucleic acid backbone. Despite possessing self-complementary arms (b and b'), the system resists self-hybridization and selectively binds to the intended stem-loop RNA target (bcb'). Thermal stability measurements, electrophoretic mobility assays, and mismatch specificity analyses confirm the effectiveness of this approach, offering a general strategy for targeting structured RNA with precision.

Here, we report a reversible chemical strategy for regulating RNA function through a Staudinger reaction-mediated postsynthetic modification. We designed a bifunctional azide reagent, 1,3-diazidopropan-2-yl 1H-imidazol-1-carboxylate (DAPIC), which specifically modifies the 2'-hydroxyl of RNA, thereby disrupting RNA structure and function. Treatment with 2-diphenylphosphinoethylamine (DPPEA) reactivates the modified RNA through an efficient Staudinger reduction. This approach enables reversible modulation of RNA folding, hybridization, and protein-binding interactions, and can be applied to guide RNAs in the CRISPR-Cas9 system. DAPIC modification completely abrogates Cas9-mediated DNA cleavage, which is restored in a DPPEA concentration-dependent manner both in vitro and in living cells. Compared with monoazide derivatives, DAPIC exhibits enhanced reactivity and reduced reagent requirements. This Staudinger-based RNA regulation platform establishes a robust and generalizable chemical tool for conditional gene editing and studies of RNA function in complex biological environments.

Graspetides represent a class of ribosomally synthesized and post-translationally modified peptide (RiPP) that can contain macrolactone/macrolactam linkages from amino acid side chains. Many predicted graspetide biosynthetic gene clusters (BGCs) contain untapped tailoring enzymes, including some with the potential to modify macrocyclic peptide scaffolds. In this work, we investigated several of these BGCs and discovered the first examples of partner protein-dependent graspetide biosynthesis and the installation of an unprecedented cyclized 5-hydroxyisopeptide moiety. We first updated the bioinformatic tool RODEO to robustly identify diverse graspetides with additional tailoring enzymes. Using this algorithm to survey available genomic data, a data set of >20,000 predicted graspetides was generated and a large-scale bioinformatic analysis was performed on proteins in or near graspetide BGCs. From this analysis, two groups of graspetides with strictly conserved co-occurring proteins were prioritized for characterization. These graspetides contained novel ring connectivities and their biosynthesis was dependent on co-occurring partner proteins, a feature unprecedented among characterized graspetides. The first graspetide, rosaritide, features three interlocking macrolactone linkages. The activity and stability of the rosaritide graspetide synthetase was dependent on a partner protein which copurifies to form a catalytically active complex. The second graspetide, corallotide, is unusually large and contains five repeated motifs in which a Lys is first macrocyclized into an isopeptide bond and then hydroxylated at the δ-carbon by a divergent 2OG-Fe(II)-dependent oxygenase. The biosynthesis and biosynthetic enzymes from these BGCs were then characterized in vitro. Overall, this study expands our understanding of graspetide biosynthesis and the ability to predict graspetide BGCs.

Enzymatic catalysis promises the achievement of functional group reactivity selectivity with the conjunctional expansive acquirement of reactivity diversity through the conformal mutagenesis tuning of catalytic cavity. However, essentially all of the functional group reactivity diversification systems demonstrated thus far are centered on the intersubstrate-shift regime, with the enzymatic catalysis adapted to alternative substrate and associated target functional group in totality. Herein, we report an enzymatic stringency-relaxation strategy for effecting reactivity diversification into the intrasubstrate orthogonal functional group reactivity selectivity regime. The stringency-relaxation strategy operates in either the amino acid relaxation or functional group relaxation format by a working sequence of initial stringency amino acid catalytic access to the catalytically most demanding functional group and subsequent relaxation amino acid catalytic access to the catalytically less demanding functional group. In particular, herein, through this strategy, α,β-unsaturated carbonyls have been reduced with ketoreductase, in a selective manner, at either the carbonyl group site or the alkenyl group site. A broad substrate scope has been established for the alkenyl reduction of α-cyano-α,β-unsaturated esters, showcasing enzymatic stringency-relaxation as a prospective platform for programming reactivity diversification and reaction development.

Poly(ADP-ribosyl)ation (PARylation) is a highly dynamic post-translational modification mediated by poly(ADP-ribose) polymerases (PARPs), influencing DNA damage response, transcription, and cell death. Previously, we showed that noncovalent interactions between PAR and the intrinsically disordered regions (IDPRs) of the p53 C-terminal domain (CTD) mediate the PARP1-dependent covalent modification of its target proteins. In this study, we test whether this mechanism also applies to noncovalent interactions involving the highly basic RGG IDPR of the FUS protein, as well as to domains lacking IDPRs, such as WWE, PBZ, and the macrodomain. We employed a chemical biology approach using a fluorescently labeled NAD⁺ analogue together with fusion constructs that contain defined ADP-ribose-binding domains and validated PARylation acceptors. We find that the p53-CTD, RGG, WWE, and PBZ domains support efficient covalent PARylation, whereas binding through the macrodomain protein Af1521 results in only very weak PARylation. These findings suggest that the mode of PAR binding influences how effectively proteins are directed toward covalent PARylation. This work broadens our molecular understanding of the interplay of noncovalent and covalent PARylation mechanisms and highlights the importance of distinct PAR-binding modes, which may inform future therapeutic strategies aimed at modulating PAR signaling in disease.