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The goal of this paper is predicting the conformational distributions of ligand binding sites using the AlphaFold2 (AF2) protein structure prediction program with stochastic subsampling of the multiple sequence alignment (MSA). We explored the opening of cryptic ligand binding sites in 16 proteins, where the closed and open conformations define the expected extreme points of the conformational variation. Due to the many structures of these proteins in the Protein Data Bank (PDB), we were able to study whether the distribution of X-ray structures affects the distribution of AF2 models. We have found that AF2 generates both a cluster of open and a cluster of closed models for proteins that have comparable numbers of open and closed structures in the PDB and not too many other conformations. This was observed even with default MSA parameters, thus without further subsampling. In contrast, with the exception of a single protein, AF2 did not yield multiple clusters of conformations for proteins that had imbalanced numbers of open and closed structures in the PDB, or had substantial numbers of other structures. Subsampling improved the results only for a single protein, but very shallow MSA led to incorrect structures. The ability of generating both open and closed conformations for six out of the 16 proteins agrees with the success rates of similar studies reported in the literature. However, we showed that this partial success is due to AF2 "remembering" the conformational distributions in the PDB and that the approach fails to predict rarely seen conformations., Competing Interests: Competing interests statement:The authors declare no competing interest.

G protein-coupled receptors (GPCRs) regulate multiple cellular responses and represent highly successful therapeutic targets. The mechanisms by which agonists activate the G protein are unclear for many GPCR families, including the bitter taste receptors (TAS2Rs). We ascertained TAS2R5 properties by live cell-based functional assays, direct binding affinity measurements using optical resonators, and atomistic molecular dynamics simulations. We focus on three agonists that exhibit a wide range of signal transduction in cells despite comparable ligand-receptor binding energies derived from direct experiment and computation. Metadynamics simulations revealed that the critical barrier to activation is ligand-induced opening of the G protein between the α-helical (AH) and Ras-like domains of Gα subunit from a precoupled TAS2R5-G protein state to the fully activated state. A moderate agonist opens the AH-Ras cleft from 22 Å to 31 Å with an energy gain of -4.8 kcal mol -1 , making GDP water-exposed for signaling. A high-potency agonist had an energy gain of -11.1 kcal mol -1 . The low-potency agonist is also exothermic for Gα opening, but with an energy gain of only -1.4 kcal mol -1 . This demonstrates that TAS2R5 agonist-bound functional potencies are derived from energy gains in the transition from a precoupled complex at the level of Gα opening. Our experimental and computational study provides insights into the activation mechanism of signal transduction that provide a basis for rational design of new drugs., Competing Interests: Competing interests statement:The authors declare no competing interest.

NLR family, apoptosis inhibitory proteins (NAIPs) detect bacterial flagellin and structurally related components of bacterial type III secretion systems (T3SS), and recruit NLR family CARD domain containing protein 4 (NLRC4) and caspase-1 into an inflammasome complex that induces pyroptosis. NAIP/NLRC4 inflammasome assembly is initiated by the binding of a single NAIP to its cognate ligand, but a subset of bacterial flagellins or T3SS structural proteins are thought to evade NAIP/NLRC4 inflammasome sensing by not binding to their cognate NAIPs. Unlike other inflammasome components such as NLRP3, AIM2, or some NAIPs, NLRC4 is constitutively present in resting macrophages and not known to be induced by inflammatory signals. Here, we demonstrate that Toll-like receptor (TLR)-dependent p38 mitogen-activated protein kinase signaling up-regulates NLRC4 transcription and protein expression in murine macrophages, which licenses NAIP detection of evasive ligands. In contrast, TLR priming in human macrophages did not up-regulate NLRC4 expression, and human macrophages remained unable to detect NAIP-evasive ligands even following priming. Critically, ectopic expression of either murine or human NLRC4 was sufficient to induce pyroptosis in response to immunoevasive NAIP ligands, indicating that increased levels of NLRC4 enable the NAIP/NLRC4 inflammasome to detect these normally evasive ligands. Altogether, our data reveal that TLR priming tunes the threshold for the murine NAIP/NLRC4 inflammasome to enable inflammasome responses against immunoevasive or suboptimal NAIP ligands. These findings provide insight into species-specific TLR regulation of NAIP/NLRC4 inflammasome activation., Competing Interests: Competing interests statement:The authors declare no competing interest.

Enzymatic mechanisms are typically inferred from structural data. However, understanding enzymes require unravelling the intricate dynamic interplay between dynamics, conformational substates, and multiple protein structures. Here, we use single-molecule nanopore analysis to investigate the catalytic conformational changes of adenylate kinase (AK), an enzyme that catalyzes the interconversion of various adenosine phosphates (ATP, ADP, and AMP). Kinetic analysis validated by hidden Markov models unravels the details of domain motions during catalysis. Our findings reveal that allosteric interactions between ligands and cofactor enable converting binding energies into directional conformational changes of the two catalytic domains of AK. These coordinated motions emerged to control the exact sequence of ligand binding and the affinity for the three different substrates, thereby guiding the reactants along the reaction coordinates. Interestingly, we find that about 10% of enzymes show altered allosteric regulation and ligand affinities, indicating that a subset of enzymes folds in alternative catalytically active forms. Since molecules or proteins might be able to selectively stabilize one of the folds, this observation suggests an evolutionary path for allostery in enzymes. In AK, this complex catalytic framework has likely emerged to prevent futile ATP/ADP hydrolysis and to regulate the enzyme for different energy needs of the cell., Competing Interests: Competing interests: G.M. is founder, director, and shareholder in Portal Biotech Limited, a nanopore company. This work was not supported by Portal Biotech Limited. The remaining authors declare no competing interests’., (© 2024. The Author(s).)

Background: Nanozymes have the advantages of cost effective, simple synthesis, high durability and stability, and have been widely used in various fields. However, only a few nanomaterials with multiple enzyme-like activity have been reported, and most of the currently developed nanozymes are usually used in colorimetric or fluorescence analysis depending on a single colorimetric or fluorescence signal output. In this study, a copper-based dual-ligand biomimetic nanoflower (Cu-MN) was constructed, which demonstrated potential multiple enzyme-like activity, and was applied to the multi-mode detection of 2,4-dichlorophenol (2,4-DP), epinephrine (EP), and H 2 O 2 ., Results: The laccase-like activity of Cu-MN can catalyze the conversion of 2,4-DP and EP, resulting in the formation of red and yellow-brown oxidation products with distinct UV absorption peaks at 510 nm and 485 nm, respectively. Furthermore, the fluorescence emission peak at 426 nm of Cu-MN can be dynamic quenched during substrate oxidation due to the fluorescence internal filtration effect (IFE). Therefore, a dual-mode analysis method was constructed to detect 2,4-DP and EP by fluorescence and ultraviolet colorimetry, which was successfully applied in natural lake water and rabbit plasma analysis, respectively. Furthermore, a colorimetric sensing strategy based on the peroxidase-like activity of Cu-MN was developed and successfully applied to the monitoring of H 2 O 2 in hydrogen peroxide disinfectant. Additionally, the visualization analysis method was also established by RGB reading of the smartphone., Significance and Novelty: In brief, inspired by the fluorescence characteristics of 2-aminoterephthallc acid and the imidazole group of 2-methylimidazole, a novel copper-based dual-ligand biomimetic nanoflower (Cu-MN) was prepared and used to establish multi-mode method for the detection of 2,4-DP, EP, and H 2 O 2 , which opens up new avenues for its applications in bioanalysis and environmental monitoring., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Communication between cells in metazoan organisms is mediated by a remarkably small number of highly conserved signaling pathways. Given this small number of signaling pathways, the existence of multiple related ligands for many of these pathways represents a key evolutionary innovation for encoding complexity into cell-cell signaling. Relatedly, crosstalk between pathways is another critical feature which allows a modest number pathways to ultimately generate an enormously diverse range of outcomes. It would thus be useful to have genetic tools to identify and manipulate not only those cells which express a given signaling ligand, but also those cells that specifically co-express pairs of signaling ligands. We present a collection of split-Gal4 knock-in lines targeting many of the ligands for highly conserved signaling pathways in Drosophila (Notch, Hedgehog, FGF, EGF, TGFβ, JAK/STAT, JNK, and PVR). We demonstrate that these lines faithfully recapitulate the endogenous expression pattern of their targets, and that they can be used to identify cells and tissues that co-express pairs of ligands. As a proof of principle, we demonstrate that the 4th chromosome TGFβ ligands myoglianin and maverick are broadly co-expressed in muscles and other tissues of both larva and adults, and that the JAK/STAT ligands upd2 and upd3 are partially co-expressed from cells of the midgut following gut damage. Together with our previously collection of split-Gal4 lines targeting the seven Wnt ligands, this resource allows Drosophila researchers to identify and genetically manipulate cells that specifically express pairs of conserved ligands from nearly all the major intercellular signaling pathways., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Toxoplasma gondii is capable of being transmitted by nearly all warm-blooded animals, and rodents are a major source of parasite dissemination, yet mechanisms driving its broad host range are poorly understood. Although a phylogenetically close relative of T. gondii , Neospora caninum differs from T. gondii in that it does not infect mice and only infects a small number of ruminant and canine species. We recently showed that T. gondii and N. caninum grow similarly in mice during the first 24 h post-infection, but only N. caninum induces an IFNγ-driven response within hours that controls the infection. The goal of the present study was to understand the cellular basis of this rapid response to N. caninum . To do this, we compared immune cell populations at the site of infection 4 h after T. gondii or N. caninum infection in mice. We found that both parasites induced similar frequencies of peritoneal monocytes, while macrophages and dendritic cell populations were not increased compared to uninfected mice. Through a series of knockout mouse experiments, we show that B, T, and NKT cells are not required for immediate IFNγ production and ultimate control of N. caninum infection, suggesting that natural killer (NK) cells are the primary inducers of immediate IFNγ in response to N. caninum. N. caninum infections exhibited significantly more IFNγ + NK cells in the peritoneum compared with T. gondii -infected and uninfected mice. Finally, we demonstrate that differences in early IFNγ production during N. caninum and T. gondii infections in mice are at least partly due to differences in soluble antigen(s) produced by tachyzoites., Importance: Pathogen differences in host range are poorly understood at the molecular level even though even closely related pathogen species can have dramatically distinct host ranges. Here, we study two related parasite species that have a dramatic difference in their ability to infect mice. Here, we show that soluble proteins from these species determine one driver of this difference: induction of interferon gamma by cells of the innate immune system., Competing Interests: The authors declare no conflict of interest.

Abstract: von Willebrand factor (VWF) is a multimeric protein consisting of covalently linked monomers, which share an identical domain architecture. Although involved in processes such as inflammation, angiogenesis, and cancer metastasis, VWF is mostly known for its role in hemostasis, by acting as a chaperone protein for coagulation factor VIII (FVIII) and by contributing to the recruitment of platelets during thrombus formation. To serve its role in hemostasis, VWF needs to bind a variety of ligands, including FVIII, platelet-receptor glycoprotein Ib-α, VWF-cleaving protease ADAMTS13, subendothelial collagen, and integrin α-IIb/β-3. Importantly, interactions are differently regulated for each of these ligands. How are these binding events accomplished and coordinated? The basic structures of the domains that constitute the VWF protein are found in hundreds of other proteins of prokaryotic and eukaryotic organisms. However, the determination of the 3-dimensional structures of these domains within the VWF context and especially in complex with its ligands reveals that exclusive, VWF-specific structural adaptations have been incorporated in its domains. They provide an explanation of how VWF binds its ligands in a synchronized and timely fashion. In this review, we have focused on the domains that interact with the main ligands of VWF and discuss how elucidating the 3-dimensional structures of these domains has contributed to our understanding of how VWF function is controlled. We further detail how mutations in these domains that are associated with von Willebrand disease modulate the interaction between VWF and its ligands., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

DNA can self-assemble into G-quadruplexes and i-motifs non-canonical secondary structures that are formed by guanine-rich sequences and the cytosine-rich sequences, respectively. G-quadruplexes and i-motifs have been closely linked to cancer development since they can regulate genes expression in various promoter regions. Moreover, these structures have gained attention as viable targets for anticancer treatments because of their physicochemical properties and gene-regulatory functions. As a result, they are attractive molecular targets for innovative cancer therapies. Herein, we review the G-quadruplex and i-motif structures, their dynamic relationship in biological systems, as well as their significance in cancer biology and the potential therapeutic approaches. Furthermore, we also address the simultaneous and mutually exclusive formation of G-quadruplex and i-motif structures in cellular environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Danneskiold-Samsøe and coworkers 1 have developed an in silico screening pipeline based on AlphaFold2 for identifying single-pass transmembrane receptors for secreted peptides that play important roles in cell-cell signaling. Their approach can be used to deorphanize a diverse range of ligands. The overall strategy can be valuable in screening for weak and transient interactions., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Proteins are attractive building blocks for fabricating diverse and precise nanomaterials. However, the facile fabrication of multidimensional artificial assemblies is highly challenging. Here, inspired by the large-scale production technique of inorganic nanomaterials, we demonstrate the application of liquid phase exfoliation (LPE) on native protein ConA by the design of synthetic ligands. These ligands provide distinct in-plane and out-of-plane supramolecular interactions, allowing the generation of multidimensional architectures based on the same protein by dissociating a single interaction in solution, including 3D porous protein crystals, 2D sizable nanosheets, and 1D fibrils. Importantly, the exfoliated 2D sheets were dozens of times larger than the self-assembled nanosheets, resulting in a dramatic enhancement of the intrinsic bioactivity of the building blocks by receptor clustering and less endocytosis. These findings enable the successful application of LPE on biomacromolecules and open up an alternative avenue to generate advanced multidimensional nanomaterials, without the need for complex protein design and careful adjustment of self-assembly conditions.

Secreted proteins play crucial roles in paracrine and endocrine signaling; however, identifying ligand-receptor interactions remains challenging. Here, we benchmarked AlphaFold2 (AF2) as a screening approach to identify extracellular ligands to single-pass transmembrane receptors. Key to the approach is the optimization of AF2 input and output for screening ligands against receptors to predict the most probable ligand-receptor interactions. The predictions were performed on ligand-receptor pairs not used for AF2 training. We demonstrate high discriminatory power and a success rate of close to 90% for known ligand-receptor pairs and 50% for a diverse set of experimentally validated interactions. Further, we show that screen accuracy does not correlate linearly with prediction of ligand-receptor interaction. These results demonstrate a proof of concept of a rapid and accurate screening platform to predict high-confidence cell-surface receptors for a diverse set of ligands by structural binding prediction, with potentially wide applicability for the understanding of cell-cell communication., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Selenium-based nanoparticles exhibit antiviral activity by directly modulating immune function. Despite recent promising developments in utilizing selenium nanoparticles (Se NPs) against viral infections, the impact of surface ligand charge on the conformation and interaction with viral proteins, as well as the effectiveness of Se NPs in anti-Herpes simplex virus 1 (HSV-1) infection remains unexplored. In this study, three types of selenium nanoparticles (CTAB-Se, PVP-Se, SDS-Se) with distinct surface charges were synthesized by modifying the surface ligands. We found that apart from differences in surface charge, the size, morphology, and crystal structure of the three types of Se NPs were similar. Notably, although the lipophilicity and cellular uptake of SDS-Se with a negative charge were lower compared to positively charged CTAB-Se and neutrally charged PVP-Se, SDS-Se exhibited the strongest protein binding force during interaction with HSV-1. Consequently, SDS-Se demonstrated the most potent anti-HSV-1 activity and safeguarded normal cells from damage. The mechanistic investigation further revealed that SDS-Se NPs effectively inhibited the proliferation and assembly of HSV-1 by powerfully suppressing the key genes and proteins of HSV-1 at various stages of viral development. Hence, this study highlights the significant role of surface ligand engineering in the antiviral activity of Se NPs, presenting a viable approach for synthesizing Se NPs with tailored antiviral properties by modulating surface charge. This method holds promise for advancing research on the antiviral capabilities of Se NPs., (© 2024 IOP Publishing Ltd. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

This study explores the intricacies of dopamine receptor-ligand interactions, focusing on the D1R and D5R subtypes. Using molecular modeling techniques, we investigated the binding of the pan-agonist rotigotine, revealing a universal binding mode at the orthosteric binding pocket. Additionally, we analyze the stability of antagonist-receptor complexes with SKF83566 and SCH23390. By examining the impact of specific mutations on ligand-receptor interactions through computational simulations and thermostability assays, we gain insights into binding stability. Our research also delves into the structural and energetic aspects of antagonist binding to D1R and D5R in their inactive states. These findings enhance our understanding of dopamine receptor pharmacology and hold promise for drug development in central nervous system disorders, opening doors to future research and innovation in this field.

Allosteric transcription factors (aTF) regulate gene expression through conformational changes induced by small molecule binding. Although widely used as biosensors, aTFs have proven challenging to design for detecting new molecules because mutation of ligand-binding residues often disrupts allostery. Here, we develop Sensor-seq, a high-throughput platform to design and identify aTF biosensors that bind to non-native ligands. We screen a library of 17,737 variants of the aTF TtgR, a regulator of a multidrug exporter, against six non-native ligands of diverse chemical structures - four derivatives of the cancer therapeutic tamoxifen, the antimalarial drug quinine, and the opiate analog naltrexone - as well as two native flavonoid ligands, naringenin and phloretin. Sensor-seq identifies biosensors for each of these ligands with high dynamic range and diverse specificity profiles. The structure of a naltrexone-bound design shows shape-complementary methionine-aromatic interactions driving ligand specificity. To demonstrate practical utility, we develop cell-free detection systems for naltrexone and quinine. Sensor-seq enables rapid and scalable design of new biosensors, overcoming constraints of natural biosensors., Competing Interests: Competing interests K.K.N., N.N., and S.R. have filed a provisional patent application with the title “Method of identifying allosteric biosensor proteins with new specificities” based on the results generated from this paper through the Wisconsin Alumni Research Foundation (P230343US02). The remaining authors declare no competing interests., (© 2024. The Author(s).)

A stepwise, data-driven approach for Anti-Drug Antibodies (ADA) singlicate assays has been developed to evaluate the feasibility of singlicate ligand binding assay (LBA) method development, qualification and validation to support our clinical programs. With initial precision runs in method validation and subsequent statistical analysis to directly compare duplicate and singlicate formats, our results indicated no meaningful difference in assay precision between duplicate and singlicate. Consequently, the rest of the ADA method validation proceeded with singlicate analysis yielding acceptable validation results. The validated ADA assay in singlicate has been employed to support a Phase I study. The appropriateness of singlicate analyses is further supported by Signal-to-Noise (S/N) data from the three tiers of the confirmatory positive samples, which showed strong correlation in S/N values., Competing Interests: Declaration Conflict of Interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties., (© 2024. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

The concept of targeted protein degradation is at the forefront of modern drug discovery, which aims to eliminate disease-causing proteins using specific molecules. In this paper, we explored the idea to design protein degraders based on the section of ligands that cause protein destabilization, hence that facilitate the cellular breakdown of the target. Our studies present covalent agents targeting Pin1, a cis-trans prolyl isomerase that plays a crucial role in tumorigenesis. Our design strategy entailed iterative optimizations of agents for potency and Pin1 destabilization in vitro. Biophysical and cellular studies suggest that the agents may act like molecular crowbars , displacing protein-stabilizing interactions that open the structure for recognition by the proteasome degradation machinery. This approach resulted in a series of potent and effective Pin1 degraders with potential applications in target validation and in therapeutic development. We propose that our design strategy can identify molecular degraders without engineering bifunctional agents that artificially create interactions between a disease-causing protein and a ubiquitin ligase., Competing Interests: Competing interests statement:M.P. is a co-founder of Armida labs, Inc., Riverside. The Pin1 inhibitors are being patented by UCR.

The dark generation of reactive oxygen species (ROS), particularly hydroxyl radicals (·OH), is crucial in the oxidative transformation of various pollutants. However, the mechanisms behind this process are predominantly linked to direct O 2 activation by reduced substances such as Fe(II) and natural organic matter. In this study, we introduce a previously overlooked dual-ligand mechanism that significantly amplifies ·OH generation on iron oxyhydroxides, facilitated by cysteine and pyrophosphate. Our findings reveal that these ligands collaboratively boost ·OH generation by 99.5-125.7% compared to Fe(II) alone. This enhancement occurs through a two-step electron transfer (ET) process, where cysteine transfers electrons to O 2 through iron oxyhydroxides. The complexation of pyrophosphate with iron oxyhydroxides further reduces the thermodynamic barriers, notably promoting this ET process and significantly improving the electron utilization efficiency for O 2 activation by the electron donor cysteine. Such a process has shown its great potential for effectively driving the oxidative transformation of various pollutants, including As(III), dichlorophenol, and carbamazepine. These findings offer valuable insights for nature-based pollutant mitigation in soil and subsurface environments.

Europium halide perovskites are promising candidates for environmentally benign blue-light emitters with their narrow emission line width. However, the development of high-photoluminescence quantum yield (PLQY) colloidal europium halide perovskite nanocrystals (PNCs) is hindered by limited synthetic methods and elusive reaction mechanisms. Here, we provide an effective synthetic route for achieving high-PLQY deep-blue-emitting colloidal CsEuBr 3 PNCs. Using two Br-organic ligand precursors, oleylammonium bromide (OLAMHBr) and trioctylphosphine dibromide (TOPBr 2 ), we identified distinct phase evolution routes involving Eu 2+ :CsBr, Cs 4 EuBr 6 , and CsEuBr 3 . The OLAMHBr precursor initially promotes the formation of the Eu 2+ :CsBr phase, which reorganizes into the CsEuBr 3 perovskite phase via proton transfer. In contrast, the TOPBr 2 precursor induces the formation of core/shell Cs 4 EuBr 6 /CsBr PNCs, which subsequently transform into CsEuBr 3 through nucleophilic addition. The TOPBr 2 route exhibited enhanced CsEuBr 3 phase homogeneity, resulting in a significantly higher PLQY (40.5%; full width at half-maximum (fwhm) = 24 at 430 nm), compared to the OLAMHBr route (16.5% at 418 nm). Notably, the phase-pure CsEuBr 3 PNCs demonstrated a world-record PLQY among the reported blue-emitting lead-free PNCs that exhibit a narrow emission line width (fwhm <25 nm). This work highlights the significant role of organic ligands in the colloidal synthesis of CsEuBr 3 PNCs and their potential as nontoxic, solution-processable blue-light emitters.

The human carbonic anhydrase II (HCA II) enzyme is a cytosolic protein located in the membrane of red blood cells that reversible hydration of carbon dioxide (CO 2 ). Considering the critical role of the HCA II and the effects of some mutations on the activity and stability of the enzyme in humans, several computational methods are used to study the structure and dynamics of the wild-type and the mutant enzymes with three ligands, CO 2 , 4-nitrophenyl acetate and acetazolamide. Our results of MD simulation of a wild-type enzyme with 4-nitrophenyl acetate show that it created essential effects on the fluctuation of this enzyme and made it more unstable and less compact than the same enzyme without ligand. In the MD of the mutant enzyme with 4-nitrophenyl acetate ligand, no significant difference is observed between with and without ligand. The affinity of the wild-type enzyme to the 4-nitrophenyl acetate is notably higher than the mutant enzyme with the same ligand. Furthermore, results showed that wild-type and mutant enzymes with CO 2 are more favorable in stability and flexibility than the same enzymes without ligand. The MD results of wild-type with acetazolamide indicate instability compare without ligand, but in MD of mutant enzyme with acetazolamide show that it more stable and compact than the same enzyme without ligand. Finally, Comparing protein trajectories to assess the impact of ligands on the stability and activity of HCA II enzymes can have medical applications and can in the engineering and design of new variants of carbonic anhydrase enzyme., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Retinoid X receptors (RXRs) belong to a retinoid-binding subgroup of the nuclear receptor family, and their synthetic agonists have been developed as therapeutics for glucose and lipid metabolism, inflammation, and inflammatory bowel disease, although RXR agonists could cause side effects such as hypothyroidism, hypertriglyceridemia, and hepatomegaly. We previously reported novel full and partial agonists, NEt-3IB and NEt-4IB, which reduce the side effects, but the molecular basis of their different activity was not clear. In this study, we report the crystal structures of the ligand-binding domain of human RXRα complexed with NEt-3IB and NEt-4IB. Detailed comparisons of the two structures showed that the full agonist, NEt-3IB, is more stably accommodated in the ligand-binding pocket due to the interactions of the bulky iso-butoxy group with helices 5 and 7. The stabilization of these helices led to the stabilization of helix 12, which is important for formation of the coactivator-binding site. The structures shed light on the novel mechanism of the regulation of RXR activity through the interaction between the bound agonist and helix 7, an interaction that was not previously considered important., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Bitter taste receptors (TAS2Rs), a subfamily of G-protein coupled receptors (GPCRs) expressed orally and extraorally, elicit signaling in response to a large set of tastants. Among 25 functional TAS2Rs encoded in the human genome, TAS2R14 is the most promiscuous, and responds to hundreds of chemically diverse ligands. Here we present the cryo-electron microscopy (cryo-EM) structure of the human TAS2R14 in complex with its signaling partner gustducin, and bound to flufenamic acid (FFA), a clinically approved nonsteroidal anti-inflammatory drug. The structure reveals an unusual binding mode, where two copies of FFA are bound at distinct pockets: one at the canonical receptor site within the trans-membrane bundle, and the other in the intracellular facet, bridging the receptor with gustducin. Together with a pocket-specific BRET-based ligand binding assay, these results illuminate bitter taste signaling and provide tools for a site-targeted compound design., Competing Interests: Competing interests The authors declare no competing interests, (© 2024. The Author(s).)

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor transcription factor that regulates gene expression programs in response to ligand binding. Endogenous and synthetic ligands, including covalent antagonist inhibitors GW9662 and T0070907, are thought to compete for the orthosteric pocket in the ligand-binding domain (LBD). However, we previously showed that synthetic PPARγ ligands can cooperatively cobind with and reposition a bound endogenous orthosteric ligand to an alternate site, synergistically regulating PPARγ structure and function (Shang et al., 2018). Here, we reveal the structural mechanism of cobinding between a synthetic covalent antagonist inhibitor with other synthetic ligands. Biochemical and NMR data show that covalent inhibitors weaken-but do not prevent-the binding of other ligands via an allosteric mechanism, rather than direct ligand clashing, by shifting the LBD ensemble toward a transcriptionally repressive conformation, which structurally clashes with orthosteric ligand binding. Crystal structures reveal different cobinding mechanisms including alternate site binding to unexpectedly adopting an orthosteric binding mode by altering the covalent inhibitor binding pose. Our findings highlight the significant flexibility of the PPARγ orthosteric pocket, its ability to accommodate multiple ligands, and demonstrate that GW9662 and T0070907 should not be used as chemical tools to inhibit ligand binding to PPARγ., Competing Interests: JS, DK No competing interests declared, (© 2024, Shang and Kojetin.)

The zeolitic imidazolate framework (ZIF) is one of the most explored metal-organic-framework-based systems for nucleic acid delivery to cancer cells. Current nucleic acid delivery tools exhibit several drawbacks, such as high manufacturing costs, endosomal entrapment, toxicity, and immunogenicity. However, the biomimetic mineralization of Zn-based ZIFs offers a low-cost and facile encapsulation of nucleic acids at room temperature in aqueous conditions. The efficiency of nucleic acid delivery and its subsequent impact on inflammation in cells are influenced by the physicochemical properties of the material. The imidazole content determines the formation and crystallinity of ZIF, and an optimal ratio ensures the formation of well-defined and highly crystalline structures. In this study, a series of siRNA-encapsulated ZIFs (siRNA@ZIF) were systematically prepared by varying ligand-to-metal (L/M) molar ratios. Our study demonstrates that variations in ligand concentrations influence the crystalline structures, particle size, and shape of siRNA@ZIF particles. At low L/M, two-dimensional siRNA@ZIF particles form with a size of 1 μm. As the L/M ratio increases gradually, the particle size decreases, resulting in three-dimensional particles ∼200 nm in size. We also observed better stability of siRNA@ZIF in water prepared using high L/M values and time-dependent cellular uptake by the cells. Additionally, no significant impact of the biocomposites on inflammation was found, indicating the lack of an unwanted immune response and nonimmunotoxic nature over longer periods (96 h). These findings highlight the necessity of fine-tuning ligand concentrations and synthesis chemistry in designing efficient and optimal ZIF-based systems as versatile delivery platforms for nucleic acids.

In this work, a dual-ligand functionalized lanthanide-encapsulated selenotungstate [H 2 N(CH 3 ) 2 ] 16 Na 2 H 10 [Ho 6 (H 2 O) 10 (HPACA) 4 W 10 O 28 (Ac) 2 ][SeW 9 O 33 ] 6 · 60H 2 O ( 1 , HPACA = 2-pyrazinecarboxylic acid, HAc = acetic acid) was successfully acquired by simultaneously incorporating rigid HPACA and flexible Ac - ligands to one reaction system. Interestingly, the polyanion [Ho 6 (H 2 O) 10 (HPACA) 4 W 10 O 28 (Ac) 2 ][SeW 9 O 33 ] 6 28- of 1 is composed of six trivacant Keggin-type [B-α-SeW 9 O 33 ] 8- units interconnected through an organic-inorganic hybrid dual-ligand bimetallic [Ho 6 (H 2 O) 10 (HPACA) 4 W 10 O 28 (Ac) 2 ] 20+ cluster. Moreover, the 1 @PNMPy film (PNMPy = poly( N -methylpyrrole)) was successfully prepared through an electrochemical polymerization strategy. The doping of 1 significantly narrows the bandgap in the 1 @PNMPy film, which enables the 1 @PNMPy film to exhibit remarkable conductivity and rapid electron transfer capability. Then, the 1 @PNMPy film-modified glassy carbon electrode was used to construct a 1 @PNMPy-based electrochemical biosensor (ECBS), which achieves sensitive electrochemical detection (a low limit of detection of 0.108 fM and a wide concentration detection range of 10 -8 -10 -15 M) for broad-spectrum tumor marker microRNA-155. Also, the 1 @PNMPy-based ECBS has a good specific recognition performance for microRNA-155 in a variety of interfering media. The research not only contributes to a deeper understanding of the synthetic chemistry of multicomponent polyoxometalate (POM)-based materials but also can further expand innovative applications of multicomponent POM-based materials in electrochemical detection and electrochemical devices.

Biased signaling and ligand bias, often termed functional selectivity or selective nuclear receptor modulation, have been reported for nuclear receptor partial agonists over the past 20 years. Whether signaling differences produced by partial agonists result from less intense modulation, off-target effects, or biased signaling remains unclear. A commonly postulated mechanism for biased signaling is coactivator favoritism, where agonists induce different coactivator recruitment profiles. We find that both GW1929 (full agonist) and MRL24 (partial agonist) favor recruitment of 100 to 300 residue regions from S-motif coactivators compared with a reference full agonist (rosiglitazone), yielding 95% bias value confidence intervals of 0.05-0.17 and 0.29-0.38, respectively. Calculations based on these data indicate that GW1929 and MRL24 would induce 30% to 60% higher S-motif coactivator occupancy at the receptor compared with rosiglitazone. We compare the transcriptional effects of these same three ligands on human adipocytes using RNA sequencing and exploratory Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Only 50% (rosiglitazone) and 77% (GW1929) of all gene expression changes are shared between these full agonists after 3 hours of exposure. After 24 hours of exposure, 13/98 KEGG pathways appear more intensely modulated by rosiglitazone than GW1929 (e.g., 95% confidence interval of bias in the regulation of lipolysis in adipocytes pathway is 0.03-0.09), despite similar signaling for the remaining 85 affected pathways. Similarly, rosiglitazone has an unusually large effect on several lipid metabolism-related pathways compared with the partial agonist MRL24. These data indicate that nuclear receptor full and partial agonists can induce biased signaling, likely through differences in coactivator recruitment. SIGNIFICANCE STATEMENT: Many nuclear receptor partial agonists cause fewer adverse effects and similar efficacy compared with full agonists, potentially by inducing biased agonism. Our data support the idea that partial agonists, and a full agonist, of the nuclear receptor Peroxisome proliferator-activated receptor gamma (PPARγ) are biased agonists, causing different signaling by inducing PPARγ to favor different coactivators. These data indicate that biased agonism can occur in nuclear receptors and should be considered in efforts to develop improved nuclear receptor-targeted drugs., (Copyright © 2024 by The Author(s).)

Riboswitches are metabolite-binding RNA regulators that modulate gene expression at the levels of transcription and translation. One of the hallmarks of riboswitch regulation is that they undergo structural changes upon metabolite binding. While a lot of effort has been put to characterize how the metabolite is recognized by the riboswitch, there is still relatively little information regarding how ligand sensing is performed within a transcriptional context. Here, we study the ligand-dependent cotranscriptional folding of the FMN-sensing ribB riboswitch of Escherichia coli Using RNase H assays to study nascent ribB riboswitch transcripts, DNA probes targeting the P1 and sequestering stems indicate that FMN binding leads to the protection of these regions from RNase H cleavage, consistent with the riboswitch inhibiting translation initiation when bound to FMN. Our results show that ligand sensing is strongly affected by the position of elongating RNA polymerase, which is defining an FMN-binding transcriptional window that is bordered in its 3' extremity by a transcriptional pause site. Also, using successively overlapping DNA probes targeting a subdomain of the riboswitch, our data suggest the presence of a previously unsuspected helical region involving the 3' strand of the P1 stem. Our results show that this helical region is conserved across bacterial species, thus suggesting that this predicted structure, the anti*-P1 stem, is involved in the FMN-free conformation of the ribB riboswitch. Overall, our study further demonstrates that intricate folding strategies may be used by riboswitches to perform metabolite sensing during the transcriptional process., (© 2024 Eschbach et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The creatine transporter-1 (CRT-1/SLC6A8) maintains the uphill transport of creatine into cells against a steep concentration gradient. Cellular creatine accumulation is required to support the ATP-buffering by phosphocreatine. More than 60 compounds have been explored in the past for their ability to inhibit cellular creatine uptake, but the number of active compounds is very limited. Here, we show that all currently known inhibitors are full alternative substrates. We analyzed their structure-activity relationship for inhibition of CRT-1 to guide a rational approach to the synthesis of novel creatine transporter ligands. Measurements of both inhibition of [ 3 H]creatine uptake and transport associated currents allowed for differentiating between full and partial substrates and true inhibitors. This combined approach led to a refined understanding of the structural requirements for binding to CRT-1, which translated into the identification of three novel compounds - i.e., compound 1 (2-( N -benzylcarbamimidamido)acetic acid), MIPA572 (=carbamimidoylphenylalanine), and MIPA573 (=carbamimidoyltryptophane) that blocked CRT-1 transport, albeit with low affinity. In addition, we found two new alternative full substrates, namely MIPA574 (carbamimidoylalanine) and GiDi1257 (1-carbamimidoylazetidine-3-carboxylic acid), which was superior in affinity to all known CTR-1 ligands, and one partial substrate, namely GiDi1254 (1-carbamimidoylpiperidine-4-carboxylic acid). SIGNIFICANCE STATEMENT: The creatine transporter-1 (CRT-1) is required to maintain intracellular creatine levels. Inhibition of CRT-1 has been recently proposed as a therapeutic strategy for cancer, but pharmacological tools are scarce. In fact, all available inhibitors are alternative substrates. We tested existing and newly synthesized guanidinocarboxylic acids for CRT-1 inhibition and identified three blockers, one partial and two full substrates of CRT-1. Our results support a refined structural understanding of ligand binding to CRT-1 and provide a proof-of-principle for blockage of CRT-1., (Copyright © 2024 by The Author(s).)

The field of G protein-coupled receptor (GPCR) research has greatly benefited from the spatiotemporal resolution provided by light controllable, i.e., photoswitchable ligands. Most of the developed tools have targeted the Rhodopsin-like family (Class A), the largest family of GPCRs. However, to date, all such Class A photoswitchable ligands were designed to act at the orthosteric binding site of these receptors. Herein, we report the development of the first photoswitchable allosteric modulators of Class A GPCRs, designed to target the M 1 muscarinic acetylcholine receptor. The presented benzyl quinolone carboxylic acid (BQCA) derivatives, Photo-BQCisA and Photo-BQCtrAns, exhibit complementary photopharmacological behavior and allow reversible control of the receptor using light as an external stimulus. This makes them valuable tools to further investigate M 1 receptor signaling and a proof of concept for photoswitchable allosteric modulators at Class A receptors., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The goal of designing safer, more effective drugs has led to tremendous interest in molecular mechanisms through which ligands can precisely manipulate the signaling of G-protein-coupled receptors (GPCRs), the largest class of drug targets. Decades of research have led to the widely accepted view that all agonists-ligands that trigger GPCR activation-function by causing rearrangement of the GPCR's transmembrane helices, opening an intracellular pocket for binding of transducer proteins. Here we demonstrate that certain agonists instead trigger activation of free fatty acid receptor 1 by directly rearranging an intracellular loop that interacts with transducers. We validate the predictions of our atomic-level simulations by targeted mutagenesis; specific mutations that disrupt interactions with the intracellular loop convert these agonists into inverse agonists. Further analysis suggests that allosteric ligands could regulate the signaling of many other GPCRs via a similar mechanism, offering rich possibilities for precise control of pharmaceutically important targets., Competing Interests: Competing interests A.K., S.S., J.D.S., J.L., S.M.S., J.M.J., and A.M.W. are current or past employees of Merck Research Laboratories. R.O.D. holds equity in Septerna Inc. The remaining authors declare no competing interests., (© 2024. The Author(s).)

In the race to combat ever-evolving diseases, the drug discovery process often faces the hurdles of high-cost and time-consuming procedures. To tackle these challenges and enhance the efficiency of identifying new therapeutic agents, we introduce VirtuDockDL, which is a streamlined Python-based web platform utilizing deep learning for drug discovery. This pipeline employs a Graph Neural Network to analyze and predict the effectiveness of various compounds as potential drug candidates. During the validation phase, VirtuDockDL was instrumental in identifying non-covalent inhibitors against the VP35 protein of the Marburg virus, a critical target given the virus's high fatality rate and limited treatment options. Further, in benchmarking, VirtuDockDL achieved 99% accuracy, an F1 score of 0.992, and an AUC of 0.99 on the HER2 dataset, surpassing DeepChem (89% accuracy) and AutoDock Vina (82% accuracy). Compared to RosettaVS, MzDOCK, and PyRMD, VirtuDockDL outperformed them by combining both ligand- and structure-based screening with deep learning. While RosettaVS excels in accurate docking but lacks high-throughput screening, and PyRMD focuses on ligand-based methods without AI integration, VirtuDockDL offers superior predictive accuracy and full automation for large-scale datasets, making it ideal for comprehensive drug discovery workflows. These results underscore the tool's capability to identify high-affinity inhibitors accurately across various targets, including the HER2 protein for cancer therapy, TEM-1 beta-lactamase for bacterial infections, and the CYP51 enzyme for fungal infections like Candidiasis. To sum up, VirtuDockDL combines user-friendly interface design with powerful computational capabilities to facilitate rapid, cost-effective drug discovery and development. The integration of AI in drug discovery could potentially transform the landscape of pharmaceutical research, providing faster responses to global health challenges. The VirtuDockDL is available at https://github.com/FatimaNoor74/VirtuDockDL ., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Synthetic genome readers/regulators (SynGRs) are bifunctional molecules that are rationally designed to bind specific genomic sequences and engage cellular machinery that regulates the expression of targeted genes. The prototypical SynGR1 targets GAA trinucleotide repeats and recruits the BET family of transcriptional regulatory proteins via a flexibly tethered ligand, JQ1. This pan-BET ligand binds both tandem bromodomains of BET proteins (BD1 and BD2). Second-generation SynGRs, which substituted JQ1 with bromodomain-selective ligands, unexpectedly revealed that BD1-selective ligands failed to functionally engage BET proteins in living cells despite displaying the ability to bind BD1 in vitro. Mechanistically, recruiting a BET protein via BD1- or BD2-selective SynGRs should have resulted in indistinguishable functional outcomes. Here we report the conversion of inactive BD1-targeting SynGRs into functional gene regulators by a structure-guided redesign of the chemical linker that bridges the DNA-binding molecule to the highly selective BD1 ligand GSK778. The results point to an optimal zone for positioning the BD1-selective ligand for functional engagement of BET proteins on chromatin, consistent with the preferred binding of BD1 domains to distal acetyllysine residues on histone tails. The results not only resolve the mechanistic conundrum but also provide insight into domain-selective targeting and nuanced design of chemo probes and therapeutics.

Chronic neuropathic pain is an increasingly prevalent societal issue that responds poorly to existing therapeutic strategies. The α9α10 nicotinic acetylcholine receptor (nAChR) has emerged as a potential target to treat neuropathic pain. However, challenges in expressing functional α9α10 nAChRs in mammalian cell lines have slowed the discovery of α9α10 ligands and studies into the relationship between α9α10 nAChRs and neuropathic pain. Here, we develop a cell line in the HEK293 background that stably expresses functional α9α10 nAChRs. By also developing cell lines expressing only α9 and α10 subunits, we identify distinct receptor pharmacology between homomeric α9 or α10 and heteromeric α9α10 nAChRs. Moreover, we demonstrate that incubation with nAChR ligands differentially regulates the expression of α9- or α10-containing nAChRs, suggesting a possible mechanism by which ligands may modify receptor composition and trafficking in α9- and α10-expressing cells. We then apply our α9α10 cell line in a screen of FDA-approved and investigational drugs to identify α9α10 ligands that provide new tools to probe α9α10 nAChR function. We demonstrate that one compound from this screen, diphenidol, possesses antinociceptive activity in a murine model of neuropathic pain. These results expand our understanding of α9α10 receptor pharmacology and provide new starting points for developing efficacious neuropathic pain treatments.

Nuclear receptors are ligand-induced transcription factors that bind directly to target genes and regulate their expression. Ligand binding initiates conformational changes that propagate to other domains, allosterically regulating their activity. The nature of this interdomain communication in nuclear receptors is poorly understood, largely owing to the difficulty of experimentally characterizing full-length structures. We have applied computational modeling approaches to describe and study the structure of the full-length farnesoid X receptor (FXR), approximated by the DNA binding domain (DBD) and ligand binding domain (LBD) connected by the flexible hinge region. Using extended molecular dynamics simulations (>10 microseconds) and enhanced sampling simulations, we provide evidence that ligands selectively induce domain rearrangement, leading to interdomain contact. We use protein-protein interaction assays to provide experimental evidence of these interactions, identifying a critical role of the hinge in mediating interdomain contact. Our results illuminate previously unknown aspects of interdomain communication in FXR and provide a framework to enable characterization of other full-length nuclear receptors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) has a crucial role in cell death and inflammation. A promising approach to develop novel inhibitors of RIPK1 mediated necroptosis is to mix the different binding modes of the known RIPK1 inhibitors into one molecule. Herein we report the synthesis and biological evaluation of novel mixed type inhibitors. Using Eclitasertib as a starting point, and applying our previous, published knowledge regarding cyclic malonamides, we successfully identified a library of active compounds. The active enantiomer of the most balanced and promising compound was subjected to pharmacokinetics and in vivo hypothermia study in mice., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The 91 kDa oligomeric ring-shaped ligand binding protein TRAP (trp RNA binding attenuation protein) regulates the expression of a series of genes involved in tryptophan (Trp) biosynthesis in bacilli. When cellular Trp levels rise, the free amino acid binds to sites buried in the interfaces between each of the 11 (or 12, depending on the species) protomers in the ring. Crystal structures of Trp-bound TRAP show the Trp ligands are sequestered from solvent by a pair of loops from adjacent protomers that bury the bound ligand via polar contacts to several threonine residues. Binding of the Trp ligands occurs cooperatively, such that successive binding events occur with higher apparent affinity but the structural basis for this cooperativity is poorly understood. We used solution methyl-TROSY NMR relaxation experiments focused on threonine and isoleucine sidechains, as well as magic angle spinning solid-state NMR 13 C- 13 C and 15 N- 13 C chemical shift correlation spectra on uniformly labeled samples recorded at 800 and 1200 MHz, to characterize the structure and dynamics of the protein. Methyl 13 C relaxation dispersion experiments on ligand-free apo TRAP revealed concerted exchange dynamics on the µs-ms time scale, consistent with transient sampling of conformations that could allow ligand binding. Cross-correlated relaxation experiments revealed widespread disorder on fast timescales. Chemical shifts for methyl-bearing side chains in apo- and Trp-bound TRAP revealed subtle changes in the distribution of sampled sidechain rotameric states. These observations reveal a pathway and mechanism for induced conformational changes to generate homotropic Trp-Trp binding cooperativity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The present study examines the possible inhibitory effect of JM-20, a multi-target neuroprotective compound, on the development of morphine-induced hyperalgesia in Male Sprague-Dawley naïve rats. Additionally, the impact of JM-20 on chronic constriction injury (CCI) rats under chronic morphine exposure was investigated, and its efficacy in reducing mechanical hypersensitivity and histopathological changes in the sciatic nerve was assessed. JM-20 (20 mg/kg, per os [p.o.]), administered 60 min before morphine (10 mg/kg, s.c. twice daily at 12 h intervals) for ten days, significantly inhibited the development of morphine-induced hyperalgesia assessed using an electronic pressure-meter paw test, hot-plate, and formalin test, as well as the appearance of spontaneous withdrawal somatic symptoms in rats. Furthermore, JM-20 decreases spinal pro-inflammatory interleukin-1β and restores glutathione to close physiological concentrations, biomarkers directly related to the intensity of mechanical hypernociception. After CCI and sham surgery, co-treatment with JM-20 (10 mg/kg, p.o.) for five days decreased morphine increased-mechanical hypersensitivity, even 12 days after its discontinuation. Continued morphine treatment imposed a neuroinflammatory challenge in CCI animals, further increasing cellularity (>75% immune cell infiltration) with lymphocytes and macrophages. However, JM-20 co-treatment still reduced the presence of cellular infiltrates (51-75%) with a predominance of lymphocytes. Even in the absence of nerve injury, JM-20 attenuated the peripheral neuroinflammatory response observed in morphine-treated sham-operated animals (0% vs. 1-25%). These findings suggest that JM-20 could prevent morphine-induced hyperalgesia by anti-inflammatory and antioxidant mechanisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

ABC transporters are ancient and ubiquitous nutrient transport systems in bacteria and play a central role in defining lifestyles. Periplasmic solute-binding proteins (SBPs) are components that deliver ligands to their translocation machinery. SBPs have diversified to bind a wide range of ligands with high specificity and affinity. However, accurate assignment of cognate ligands remains a challenging problem in SBPs. Urea metabolism plays an important role in the nitrogen cycle; anthropogenic sources account for more than half of global nitrogen fertilizer. We report identification of urea-binding proteins within a large SBP sequence family that encodes diverse functions. By combining genetic linkage between SBPs, ABC transporter components, enzymes or transcription factors, we accurately identified cognate ligands, as we verified experimentally by biophysical characterization of ligand binding and crystallographic determination of the urea complex of a thermostable urea-binding homolog. Using three-dimensional structure information, these functional assignments were extrapolated to other members in the sequence family lacking genetic linkage information, which revealed that only a fraction bind urea. Using the same combined approaches, we also inferred that other family members bind various short-chain amides, aliphatic amino acids (leucine, isoleucine, valine), γ-aminobutyrate, and as yet unknown ligands. Comparative structural analysis revealed structural adaptations that encode diversification in these SBPs. Systematic assignment of ligands to SBP sequence families is key to understanding bacterial lifestyles, and also provides a rich source of biosensors for clinical and environmental analysis, such as the thermostable urea-binding protein identified here., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ‘Homme W. Hellinga and Malin J. Allert are co-inventors on a patent “Urea biosensors and uses thereof” US 11,906,524.’., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Histone lysine-specific demethylase 1 (LSD1) is hyperactive in breast cancer, which is associated with the metastasis of the tumor. Current irreversible LSD1 inhibitors are all synthesized by covalently binding to the flavin adenine dinucleotide cofactor, which often have side effects due to the high affinity for a variety of targets. Here, we identified isoforsythiaside (IFA), a natural phenylpropanoid glycoside isolated from Forsythia suspensa , as a novel covalent inhibitor of LSD1. The target ligand fishing technique and LC-MS/MS analysis identified that IFA could covalently bind to the Ser817 residue of LSD1 by α,β-unsaturated ketone moiety to block the amine oxidase-like domain of LSD1. Moreover, RBMS3/Twist1/MMP2, the downstream signaling pathway of LSD1, was activated after IFA treatment to inhibit the metastasis of MDA-MB-231 cells in vitro and in vivo . This study provided novel molecular templates for development of LSD1 covalence-binding inhibitor and laid a foundation for developing agents against breast carcinoma metastasis for targeting LSD1.

High-affinity monovalent ligands for lectins are challenging to develop due to weak binding interactions. This study investigates the potential of rationally designed covalent ligands targeting the N-terminal domain of BC2L-C lectin from Burkholderia cenocepacia , a pathogen causing severe respiratory infections in immunocompromised patients. Antiadhesion therapy is emerging as a complementary approach against such infections, and bacterial lectins are suitable targets. The fucose-specific BC2L-C-Nt recognizes blood group oligosaccharides on host cells. Using a computational approach, we designed reversible covalent competitive ligands that include a fucoside anchor and a salicylaldehyde warhead targeting Lys108 near the fucose-binding site. Several candidates were synthesized and tested using competition experiments. The most effective ligand improved the IC 50 of methyl-fucoside by 2 orders of magnitude, matching the affinity of the native H-type 1 trisaccharide. Control experiments confirmed the importance of both fucose anchor and salicylaldehyde moiety in the ligand's affinity. Mass analysis confirmed the covalent interaction with Lys108.

The novel photo-crosslinking ligand 6OTD-Bp, bearing an alkylamine benzophenone (Bp) with macrocyclic hexaoxazole (6OTD), was shown to preferentially ligate with hybrid G4s through recognizing both G-quartets and their characteristic wide groove. Higher crosslinking yield was observed for hybrid G4 with wider grooves.

Based on our proposed "pseudosubstrate envelope" concept, 25 benzothiazole-bearing HIV capsid protein (CA) modulators were designed and synthesized under the guidance of free energy perturbation technology. The most potent compound, IC-1k , exhibited an EC 50 of 2.69 nM against HIV-1, being 393 times more potent than the positive control PF74. Notably, IC-1k emerged as the highest ligand efficiency (LE = 0.32) HIV CA modulator, surpassing that of the approved drug lenacapavir (LE = 0.21). Surface plasmon resonance assay and crystallographic analysis confirmed that IC-1k targeted HIV-1 CA within the chemical space of the "pseudosubstrate envelope". Further mechanistic studies revealed a dual-stage inhibition profile: IC-1k disrupted early-stage capsid-host-factor interactions and promoted late-stage capsid misassembly. Preliminary pharmacokinetic evaluations demonstrated significantly improved metabolic stability in human liver microsomes for IC-1k ( T 1/2 = 91.3 min) compared to PF74 ( T 1/2 = 0.7 min), alongside a favorable safety profile. Overall, IC-1k presents a promising lead compound for further optimization.

In this article, we present the synthesis and characterization of three macrocyclic chelators, H 4 PATA, PATAM, and H 4 PATPA, based on a pyridine-azacrown compound. Their complexation with 68 Ga and 177 Lu has been thoroughly investigated using MALDI TOF MS, 1 H NMR spectroscopy, radiolabeling studies, and experiments in vitro with fetal bovine serum and a 1000-fold molar excess of H 4 EDTA. Our studies have shown that the chelators H 4 PATA and H 4 PATPA form complexes at room temperature with both radionuclides (RCY > 80 % and > 90 % for complexes with H 4 PATA and H 4 PATPA after 30 min, respectively). The chelator PATAM requires high temperature (95 °C) for complexation. In vitro stability assays in fetal bovine serum as well as H 4 EDTA-challenge revealed that transchelation occurs for all complexes with 68 Ga. However, complexes of the ligands H 4 PATA and PATAM with 177 Lu were found stable. Thus, taking into account the radiolabeling at room temperature and in vitro stability of the complex [ 177 Lu]Lu·PATA, our investigations revealed the chelator H 4 PATA is a candidate for radiopharmaceutical use with 177 Lu., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Peroxisome proliferator-activated receptor γ (PPAR γ ), a nuclear receptor involved in metabolic processes, inflammation, and energy balance, represents a promising therapeutic target for cardiovascular diseases. Danshensu Bingpian Zhi (DBZ), a chiral compound derived from traditional Chinese medicine, exhibits potential as a PPAR γ agonist. Using an ensemble-based docking approach, molecular dynamics (MD) simulations, and the molecular mechanics generalized born surface area (MM/GBSA) methods, we explored the binding modes and energetics of DBZ stereoisomers with the PPAR γ ligand-binding domain (LBD). The results indicated that the right-handed stereoisomer (DBZR) binds like a full agonist, while the left-handed stereoisomer (DBZS) binds as a partial agonist with stronger binding energies (Δ G bind ), indicating a robust interaction with PPAR γ . Both the stereoisomers stabilize the β-sheet region of PPAR γ -LBD, potentially protecting Ser245 from phosphorylation by Cdk5, a process implicated in atherosclerosis. Principal component analysis (PCA) and dynamic cross-correlation matrices (DCCM) revealed the complex structural dynamics within the Ω loop, β-sheet, and AF-2 region of PPAR γ -LBD upon ligand binding, which may contribute to the unique binding mode and efficacy of DBZS. These findings provide insights into the molecular recognition of PPAR γ -LBD by DBZ stereoisomers and their impact on the conformational dynamics of PPAR γ , highlighting the therapeutic potential of DBZ and the significance of chirality in drug design.

Nucleoside analogues have seen significant advancements in treating viral infections and cancer through ProTide technology, leading to a series of FDA-approved drugs such as sofosbuvir, tenofovir alafenamide, and remdesivir. The stereochemical configuration at the phosphorus center of ProTides significantly influences their pharmacological properties, necessitating efficient stereoselective synthesis. Traditional methods using chiral auxiliaries or nonracemic phosphorylating agents are labor-intensive and inefficient, while recent organocatalytic approaches, despite their promise, still face limitations. Herein, we present a novel approach employing chiral metal complexes for the stereoselective assembly of P-stereogenic ProTides via asymmetric P-O bond formation. This approach leverages a chiral metal catalyst to activate the electrophilic phosphorylating reagent, facilitating a base-promoted nucleophilic replacement pathway. Our protocol, featuring mild reaction conditions and broad applicability, enables the highly stereoselective synthesis of previously inaccessible ( S , R P ) and ( R , S P )-ProTide derivatives. The practical utility of this method is demonstrated through the preparation of pharmaceutically relevant ProTide targets and mechanistic studies were conducted to elucidate the reaction pathway, offering significant advancements for drug development and pharmaceutical research.

CuATSM, a copper(II) complex of a bis(thiosemicarbazone) of diacetyl, prevents oxidative cell death and acts as a neuroprotectant in vivo , prompting its evaluation to treat amyotrophic lateral sclerosis and other neurodegenerative conditions in the clinic. We recently demonstrated that CuATSM functions as a potent radical-trapping antioxidant (RTA), inhibiting lipid peroxidation and associated ferroptotic cell death by a noncanonical mechanism based on radical addition to the ligand backbone. Herein we report our investigations of the generality of this reactivity, which include studies of corresponding complexes of various other metals, including Co, Ru, Ni, Pd, Pt, and Au. Inhibited autoxidations of styrene and dioxane reveal that most of these complexes exhibit RTA activity, consistent with ligand-based reactivity, but the identity of the metal atom nevertheless plays a role. In particular, analyses of the electronic structures of the complexes of metals within the same group (i.e., the group 10 metals Ni, Pd and Pt) highlight how the metal atom can modulate the ligand-based reactivity by enabling spin delocalization to the other thiosemicarbazone moiety. The RTA activity determined in organic solution largely translates to phospholipid bilayers and mammalian cells, where most complexes inhibited lipid peroxidation and associated ferroptotic cell death. A preliminary structure-activity study revealed Pt complexes with potencies eclipsing those of archetype ferroptosis inhibitors ferrostatin-1 and liproxstatin-1, suggesting that Pt (and to a lesser extent Ni) bis(thiosemicarbazone)s may be better suited to optimization for therapeutic development than those based on Cu.

A fundamental understanding of the design of polymer ligands of proximal enzymes is essential for the precise targeting of cancer cells, but it is still in its infancy. In this study, we systematically investigated the contribution of the chain length, ligand density, and ligand ratio of proximal enzyme-targeted polymers to the efficacy, synergy, and selectivity for the inhibition of cancer cell proliferation. The results revealed that employing a moderate chain length as a scaffold allowed for an intrinsically high efficacy and synergy of proximal enzyme-targeted polymers, in contrast to single enzyme-targeted polymers that prefer longer chain length for efficacy. The synergy obtained in proximal enzyme targeting was not provided by the combination of the corresponding small molecules. Moreover, the maturation of the synergistic efficacy of the proximal enzyme-targeted polymers also improved selectivity. This study proposes a rational design for polymer inhibitors and/or ligands for cancer cells with a high efficacy and selectivity.

Receptor kinase (RK) families process information from small molecules, short peptides, or glycan ligands to regulate core cellular pathways in plants. To date, whether individual plant RKs are capable of processing signals from distinct types of ligands remains largely unexplored. Addressing this requires the discovery of structurally unrelated ligands that engage the same receptor. Here, we focus on FLAGELLIN-SENSING 2 (FLS2), an RK that senses a peptide of bacterial flagellin to activate antibacterial immunity in Arabidopsis. We interrogate >20,000 potential interactions between small molecules and the sensory domain of FLS2 using a large-scale reverse chemical screen. We discover two small molecules that interact with FLS2 in atypical ways. The surrogate ligands weakly activate the receptor to drive a functional antibacterial response channeled via unusual gene expression programs. Thus, chemical probes acting as biased ligands can be exploited to discover unexpected levels of output flexibility in RKs signal transduction., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Tracking the dynamic distribution of native proteins in living cells or tissues in real time is essential for understanding the functional mechanisms involved in their physiological or pathological processes. The β3-adrenergic receptor (β3-AR) has important biological functions. It is expected to be a diagnostic indicator for aging, yet current probes for β-ARs are unable to dynamically monitor β3-AR in real time, which impedes the progress of β3-AR research. Here, we developed a ligand-directed anchoring probe, β3-ARP , that precisely covalently anchors with native β3-AR in living cells, for the diagnosis of senescence. The ligand-directed anchoring probe selectively recognizes and traces β3-AR and can achieve dynamic observation and monitoring of β3-AR in living cells, including the interaction between lipid droplets and mitochondria. Moreover, we were able to directly probe the distribution of β3-AR in various organs of aging mice in situ and track the location of native β3-AR in different types of primary neuronal cells using β3-ARP and two-photon imaging, which revealed the aberrant accumulation of lipid droplets and the distribution of β3-AR in neurological diseases. This research has resulted in a new covalently anchoring fluorescent probe, β3-ARP , which could serve as a powerful tool to explore the link between β3-AR and age-related diseases.

Modulating the function of Intrinsically Disordered Proteins (IDPs) with small molecules is of considerable importance given the crucial roles of IDPs in the pathophysiology of numerous diseases. Reported binding affinities for ligands to diverse IDPs vary broadly, and little is known about the detailed molecular mechanisms that underpin ligand efficacy. Molecular simulations of IDP ligand binding mechanisms can help us understand the mode of action of small molecule inhibitors of IDP function, but it is still unclear how binding energies can be modeled rigorously for such a flexible class of proteins. Here, we compare alchemical absolute binding free energy calculations (ABFE) and Markov-State Modeling (MSM) protocols to model the binding of the small molecule 10058-F4 to a disordered peptide extracted from a segment of the oncoprotein c-Myc. The ABFE results produce binding energy estimates that are sensitive to the choice of reference structure. In contrast, the MSM results produce more reproducible binding energy estimates consistent with weak mM binding affinities and transient intermolecular contacts reported in the literature.