ACS Bio & Med Chem Au最新文献

Labeled β-nicotinamide adenine dinucleotide (NAD) analogues have been critical for uncovering new biochemical connections and quantitating enzymatic activity. They function as tracers for enzymology, flux analyses, and in situ measurements. Nevertheless, there is limited availability of specific types of analogues, especially radiolabeled NAD isotopologues. Here, we describe an improved enzymatic synthesis reaction for ³²P- NAD⁺ with a yield of 98% ± 1%, using lowered concentrations of reactants and standard equipment. This represents the highest reported yield for the enzymatic synthesis of NAD⁺ to date. With the high yield we were able to directly use the reaction product to generate derivatives, such as ³²P-NADP. The high-yield enzymatic synthesis is versatile for a broad variety of labels and NAD derivatives. Its advantages include lowered concentrations of reactants, providing sufficient amounts of product for downstream applications, and minimizing intermediate purification steps.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are of increasing interest in natural products as well as drug discovery. This empowers not only the unique chemical structures and topologies in natural products but also the excellent bioactivities such as antibacteria, antifungi, antiviruses, and so on. Advances in genomics, bioinformatics, and chemical analytics have promoted the exponential increase of RiPPs as well as the evaluation of biological activities thereof. Furthermore, benefiting from their relatively simple and conserved biosynthetic logic, RiPPs are prone to be engineered to obtain diverse analogues that exhibit distinct physiological activities and are difficult to synthesize. This Review aims to systematically address the variety of biological activities and/or the mode of mechanisms of novel RiPPs discovered in the past decade, albeit the characteristics of selective structures and biosynthetic mechanisms are briefly covered as well. Almost one-half of the cases are involved in anti-Gram-positive bacteria. Meanwhile, an increasing number of RiPPs related to anti-Gram-negative bacteria, antitumor, antivirus, etc., are also discussed in detail. Last but not least, we sum up some disciplines of the RiPPs’ biological activities to guide genome mining as well as drug discovery and optimization in the future.

Targeted protein degradation (TPD) is emerging as one of the most innovative strategies to tackle infectious diseases. Particularly, proteolysis-targeting chimera (PROTAC)-mediated protein degradation may offer several benefits over classical anti-infective small-molecule drugs. Because of their peculiar and catalytic mechanism of action, anti-infective PROTACs might be advantageous in terms of efficacy, toxicity, and selectivity. Importantly, PROTACs may also overcome the emergence of antimicrobial resistance. Furthermore, anti-infective PROTACs might have the potential to (i) modulate “undruggable” targets, (ii) “recycle” inhibitors from classical drug discovery approaches, and (iii) open new scenarios for combination therapies. Here, we try to address these points by discussing selected case studies of antiviral PROTACs and the first-in-class antibacterial PROTACs. Finally, we discuss how the field of PROTAC-mediated TPD might be exploited in parasitic diseases. Since no antiparasitic PROTAC has been reported yet, we also describe the parasite proteasome system. While in its infancy and with many challenges ahead, we hope that PROTAC-mediated protein degradation for infectious diseases may lead to the development of next-generation anti-infective drugs.

Asthma is a chronic respiratory disease initiated by a variety of factors, including allergens. During an asthma attack, the secretion of C-X-C-motif chemokine 10 (CXCL10) and chemokine ligand 5 (CCL5) causes the migration of immune cells, including platelets, into the lungs and airway. Platelets, which contain three classes of chemical messenger-filled granules, can secrete vasodilators (adenosine diphosphate and adenosine triphosphate), serotonin (a vasoconstrictor and a vasodilator, depending on the biological system), platelet-activating factor, N-formylmethionyl-leucyl-phenylalanine ((fMLP), a bacterial tripeptide that stimulates chemotaxis), and chemokines (CCL5, platelet factor 4 (PF4), and C-X-C-motif chemokine 12 (CXCL12)), amplifying the asthma response. The goal of this work was threefold: (1) to understand if and how the antibody immunoglobulin E (IgE), responsible for allergic reactions, affects platelet response to the common platelet activator thrombin; (2) to understand how allergen stimulation compares to thrombin stimulation; and (3) to monitor platelet response to fMLP and the chemokines CXCL10 and CCL5. Herein, high-pressure liquid chromatography with electrochemical detection and/or carbon-fiber microelectrode amperometry measured granular secretion events from platelets with and without IgE in the presence of the allergen 2,4,6-trinitrophenyl-conjugated ovalbumin (TNP-Ova), thrombin, CXCL10, or CCL5. Platelet adhesion and chemotaxis were measured using a microfluidic platform in the presence of CXCL10, CCL5, or TNP-OVA. Results indicate that IgE binding promotes δ-granule secretion in response to platelet stimulation by thrombin in bulk. Single-cell results on platelets with exogenous IgE exposure showed significant changes in the post-membrane–granule fusion behavior during chemical messenger delivery events after thrombin stimulation. In addition, TNP-Ova allergen stimulation of IgE-exposed platelets secreted serotonin to the same extent as thrombin platelet stimulation. Enhanced adhesion to endothelial cells was demonstrated by TNP-Ova stimulation. Finally, only after incubation with IgE did platelets secrete chemical messengers in response to stimulation with fMLP, CXCL10, and CCL5.

Nonmutational mechanisms were recently discovered leading to reversible drug tolerance. Despite the rapid elimination of a majority of tumor cells, a small subpopulation of “‘drug-tolerant”’ cells remain viable with lethal drug exposure, which may further lead to resistance or tumor relapse. Several signaling pathways are involved in the local or systemic inflammatory responses contributing to drug-induced phenotypic switch. Here, we report that Toll-like receptor 4 (TLR4)-interacting lipid docosahexaenoic acid (DHA) restores the cytotoxic effect of doxorubicin (DOX) in the lipopolysaccharide-treated breast tumor cell line 4T1, preventing the phenotypic switch to drug-tolerant cells, which significantly reduces primary tumor growth and lung metastasis in both 4T1 orthotopic and experimental metastasis models. Importantly, DHA in combination with DOX delays and inhibits tumor recurrence following surgical removal of the primary tumor. Furthermore, the coencapsulation of DHA and DOX in a nanoemulsion significantly prolongs the survival of mice in the postsurgical 4T1 tumor relapse model with significantly reduced systemic toxicity. The synergistic antitumor, antimetastasis, and antirecurrence effects of DHA + DOX combination are likely mediated by attenuating TLR4 activation, thus sensitizing tumor cells to standard chemotherapy.

Fungal infections are a major public health problem resulting from the lack of public policies addressing these diseases, toxic and/or expensive therapeutic tools, scarce diagnostic tests, and unavailable vaccines. In this Perspective, we discuss the need for novel antifungal alternatives, highlighting new initiatives based on drug repurposing and the development of novel antifungals.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus that causes COVID-19, continues to evolve resistance to vaccines and existing antiviral therapies at an alarming rate, increasing the need for new direct-acting antiviral drugs. Despite significant advances in our fundamental understanding of the kinetics and mechanism of viral RNA replication, there are still open questions regarding how the proofreading exonuclease (NSP10/NSP14 complex) contributes to replication fidelity and resistance to nucleoside analogs. Through single turnover kinetic analysis, we show that the preferred substrate for the exonuclease is double-stranded RNA without any mismatches. Double-stranded RNA containing a 3′-terminal remdesivir was hydrolyzed at a rate similar to a correctly base-paired cognate nucleotide. Surprisingly, single-stranded RNA or duplex RNA containing a 3′-terminal mismatch was hydrolyzed at rates 125- and 45-fold slower, respectively, compared to the correctly base-paired double-stranded RNA. These results define the substrate specificity and rate of removal of remdesivir for the exonuclease and outline rigorous kinetic assays that could help in finding next-generation exonuclease inhibitors or nucleoside analogs that are able to evade excision. These results also raise important questions about the role of the polymerase/exonuclease complex in proofreading during viral replication. Addressing these questions through rigorous kinetic analysis will facilitate the search for desperately needed antiviral drugs to combat COVID-19.

Chemically induced proximity between certain endogenous enzymes and a protein of interest (POI) inside cells may cause post-translational modifications to the POI with biological consequences and potential therapeutic effects. Heterobifunctional (HBF) molecules that bind with one functional part to a target POI and with the other to an E3 ligase induce the formation of a target-HBF-E3 ternary complex, which can lead to ubiquitination and proteasomal degradation of the POI. Targeted protein degradation (TPD) by HBFs offers a promising approach to modulate disease-associated proteins, especially those that are intractable using other therapeutic approaches, such as enzymatic inhibition. The three-way interactions among the HBF, the target POI, and the ligase─including the protein–protein interaction between the POI and the ligase─contribute to the stability of the ternary complex, manifested as positive or negative binding cooperativity in its formation. How such cooperativity affects HBF-mediated degradation is an open question. In this work, we develop a pharmacodynamic model that describes the kinetics of the key reactions in the TPD process, and we use this model to investigate the role of cooperativity in the ternary complex formation and in the target POI degradation. Our model establishes the quantitative connection between the ternary complex stability and the degradation efficiency through the former’s effect on the rate of catalytic turnover. We also develop a statistical inference model for determining cooperativity in intracellular ternary complex formation from cellular assay data and demonstrate it by quantifying the change in cooperativity due to site-directed mutagenesis at the POI-ligase interface of the SMARCA2-ACBI1-VHL ternary complex. Our pharmacodynamic model provides a quantitative framework to dissect the complex HBF-mediated TPD process and may inform the rational design of effective HBF degraders.

Tricarbocyanine dyes have become popular tools in life sciences and medicine. Their near-infrared (NIR) fluorescence makes them ideal agents for imaging of thick specimens or in vivo imaging, e.g., in fluorescence-guided surgery. Among other types of cyanine dyes, meso-Cl tricarbocyanine dyes have received a surge of interest, as it emerged that their high reactivity makes them inherently tumor-targeting. As such, significant research efforts have focused on conjugating these to functional moieties. However, the syntheses generally suffer from low yields. Hereby, we report on the reaction of meso-Cl dyes with a small selection of coupling reagents to give the corresponding keto-polymethines, potentially explaining low yields and the prevalence of monofunctionalized cyanine conjugates in the current state of the art of functional near-infrared dyes. We present the synthesis and isolation of the first keto-polymethine-based conjugate and present preliminary investigation in the prostate cancer cell lines PC3 and DU145 by confocal microscopy and discuss changes to optical properties in biological media.