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Traditional Chinese Medicines (TCMs) have been historically used to treat bacterial infections. However, the molecules responsible for these anti-infective properties and their potential mechanisms of action have remained elusive. Using a high-throughput assay for type III protein secretion in Salmonella enterica serovar Typhimurium, we discovered that several TCMs can attenuate this key virulence pathway without affecting bacterial growth. Among the active TCMs, we discovered that baicalein, a specific flavonoid from Scutellaria baicalensis, targets S. Typhimurium pathogenicity island-1 (SPI-l) type III secretion system (T3SS) effectors and translocases to inhibit bacterial invasion of epithelial cells. Structurally related flavonoids present in other TCMs, such as quercetin, also inactivated the SPI-1 T3SS and attenuated S. Typhimurium invasion. Our results demonstrate that specific plant metabolites from TCMs can directly interfere with key bacterial virulence pathways and reveal a previously unappreciated mechanism of action for anti-infective medicinal plants. [ABSTRACT FROM AUTHOR]

Cation-π interactions have been proposed to be important contributors to protein structure and function. In particular, these interactions have been suggested to provide significant stability at the solventexposed surface of a protein. We have investigated the magnitude of cation-π interactions between phenylalanine (Phe) and lysine (Lys), ornithine (Orn), and diaminobutanoic acid (Dab) in the context of an α-helix and have found that only the Phe···Orn interaction provides significant stability to the helix, stabilizing it by -0.4 kcal/mol. This interaction energy is in the same range as a salt bridge in an α-helix, and equivalent to the recently reported Trp···Arg interaction in an α-helix, despite the fact that Trp···guanidinium interactions have been proposed to be stronger than Phe···ammonium interactions. These results indicate that even the simplest cation-π interaction can provide significant stability to protein structure and demonstrate the subtle factors that can influence the observed interaction energies in designed systems. [ABSTRACT FROM AUTHOR]

The article discusses the use of a carboxypeptidase G2 (CPG2)-based reporter system to detect the type III protein secretion from Gram-negative bacterial pathogens. Researchers synthesize a glutamate (Glu)-modified derivative of 2-dicyanomethylene-3-cyano-2,5-dihydrofuran (CyFur) to be used as a fluorogenic substrate for CPG2 and explore the sensitivity of the CPG:Gu-CyFur reporter system. It is noted that this system can monitor protein expression and secretion with sensitivity.

We report the design, synthesis and evaluation of a class of phosphatidylserine-targeting zinc (II) dipicolylamine drug conjugates and show that conjugate 16b elicits immune cell infiltration and remodels the "cold" hepatic tumor microenvironment to the inflamed "hot" tumor. Structure-property relationship study via linker modifications and subsequent pharmacokinetics profiling were carried out to improve the solubility and stability of the conjugates in vivo. In a spontaneous hepatocellular carcinoma mouse model, we showed that conjugate 16b exhibited better antitumor efficacy than sorafenib. In particular, significant increase of CD8+ T cell infiltration and granzyme B level was observed, providing insights in sensitizing tumors from intrinsic immune suppressive microenvironment. Evaluation of tumor inflammation-related mRNA expression profile revealed that conjugate 16b, through inductions of key gene expressions including STAT1, CXCL9, CCL5, and PD-L1, rejuvenated tumor microenvironment with enhancement in T cell-, macrophage-, NK cell-, chemokines and cytokines'- functions. Our study establishes that an apoptosis-targeting theranostic enables enrichment of multifaceted immune cells into the tumor mass, which provides potential therapeutic strategies in the combination with immune checkpoint blockade treatment. [Display omitted] • Designing phosphatidylserine-targeting zinc (II) dipicolylamine drug conjugates. • Reducing hepatic tumor burden and increasing immune cells infiltration. • Remodeling of hepatic tumor microenvironment to inflamed "hot" tumor. [ABSTRACT FROM AUTHOR]

Head and neck cancer is a major cancer type, with high motility rates that reduce the quality of life of patients. Herein, we investigated the effectiveness and mechanism of a combination therapy involving TLR9 activator (CpG-2722) and phosphatidylserine (PS)-targeting prodrug of SN38 (BPRDP056) in a syngeneic orthotopic head and neck cancer animal model. The results showed a cooperative antitumor effect of CpG-2722 and BPRDP056 owing to their distinct and complementary antitumor functions. CpG-2722 induced antitumor immune responses, including dendritic cell maturation, cytokine production, and immune cell accumulation in tumors, whereas BPRDP056 directly exerted cytotoxicity toward cancer cells. We also discovered a novel function and mechanism of TLR9 activation, which increased PS exposure on cancer cells, thereby attracting more BPRDP056 to the tumor site for cancer cell killing. Killed cells expose more PS in tumor for BPRDP056 targeting. Tumor antigens released from the dead cells were taken up by antigen-presenting cells, which enhanced the CpG-272-promoted T cell-mediated tumor-killing effect. These form a positive feed-forward antitumor effect between the actions of CpG-2722 and BPRDP056. Thus, the study findings suggest a novel strategy of utilizing the PS-inducing function of TLR9 agonists to develop combinational cancer treatments using PS-targeting drugs., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Nitro-fatty acids are a class of endogenous electrophilic lipid mediators with anti-inflammatory and cytoprotective effects in a wide range of inflammatory and fibrotic disease models. While these beneficial biological effects of nitro-fatty acids are mainly attributed to their ability to form covalent adducts with proteins, only a small number of proteins are known to be nitro-alkylated and the scope of protein nitro-alkylation remains undetermined. Here we describe the synthesis and application of a clickable nitro-fatty acid probe for the detection and first global identification of mammalian proteins that are susceptible to nitro-alkylation. 184 high confidence nitro-alkylated proteins were identified in THP1 macrophages, majority of which are novel targets of nitro-fatty acids, including extended synaptotagmin 2 (ESYT2), signal transducer and activator of transcription 3 (STAT3), toll-like receptor 2 (TLR2), retinoid X receptor alpha (RXRα) and glucocorticoid receptor (NR3C1). In particular, we showed that 9-nitro-oleate covalently modified and inhibited dexamethasone binding to NR3C1. Bioinformatic analyses revealed that nitro-alkylated proteins are highly enriched in endoplasmic reticulum and transmembrane proteins, and are overrepresented in lipid metabolism and transport pathways. This study significantly expands the scope of protein substrates targeted by nitro-fatty acids in living cells and provides a useful resource towards understanding the pleiotropic biological roles of nitro-fatty acids as signaling molecules or as multi-target therapeutic agents., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Zinc(II)-dipicolylamine (Zn-DPA) has been shown to specifically identify and bind to phosphatidylserine (PS), which exists in bulk in the tumor microenvironment. BPRDP056, a Zn-DPA-SN38 conjugate was designed to provide PS-targeted drug delivery of a cytotoxic SN38 to the tumor microenvironment, thereby allowing a lower dosage of SN38 that induces apoptosis in cancer cells. Micro-Western assay showed that BPRDP056 exhibited apoptotic signal levels similar to those of CPT-11 in the treated tumors growing in mice. Pharmacokinetic study showed that BPRDP056 has excellent systemic stability in circulation in mice and rats. BPRDP056 is accumulated in tumors and thus increases the cytotoxic effects of SN38. The in vivo antitumor activities of BPRDP056 have been shown to be significant in subcutaneous pancreas, prostate, colon, liver, breast, and glioblastoma tumors, included an orthotopic pancreatic tumor, in mice. BPRDP056 shrunk tumors at a lower (~20% only) dosing intensity of SN38 compared to that of SN38 conjugated in CPT-11 in all tumor models tested. A wide spectrum of antitumor activities is expected to treat all cancer types of PS-rich tumor microenvironments. BPRDP056 is a first-in-class small molecule drug conjugate for cancer therapy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A patent (international application number: PCT/US2015/031386) has been filed in relation to this work. The authors, Y.S.C., L.K.T., and C.T.C, are also co-inventors in the patent. The authors declare no additional competing financial interests., (Copyright © 2020. Published by Elsevier Inc.)

The function of the CXCR4/CXCL12 axis accounts for many disease indications, including tissue/nerve regeneration, cancer metastasis, and inflammation. Blocking CXCR4 signaling with its antagonists may lead to moving out CXCR4 + cell types from bone marrow to peripheral circulation. We have discovered a novel series of pyrimidine-based CXCR4 antagonists, a representative (i.e., 16) of which was tolerated at a higher dose and showed better HSC-mobilizing ability at the maximal response dose relative to the approved drug 1 (AMD3100), and thus considered a potential drug candidate for PBSCT indication. Docking compound 16 into the X-ray crystal structure of CXCR4 receptor revealed that it adopted a spider-like conformation striding over both major and minor subpockets. This putative binding mode provides a new insight into CXCR4 receptor-ligand interactions for further structural modifications.

A series of zinc(II) dipicolylamine (ZnDPA)-based drug conjugates have been synthesized to probe the potential of phosphatidylserine (PS) as a new antigen for small molecule drug conjugate (SMDC) development. Using in vitro cytotoxicity and plasma stability studies, PS-binding assay, in vivo pharmacokinetic studies, and maximum tolerated dose profiles, we provided a roadmap and the key parameters required for the development of the ZnDPA based drug conjugate. In particular, conjugate 24 induced tumor regression in the COLO 205 xenograft model and exhibited a more potent antitumor effect with a 70% reduction of cytotoxic payload compared to that of the marketed irinotecan when dosed at the same regimen. In addition to the validation of PS as an effective pharmacodelivery target for SMDC, our work also provided the foundation that, if applicable, a variety of therapeutic agents could be conjugated in the same manner to treat other PS-associated diseases.

Series of N -substituted carbazole analogues bearing an indole ring were synthesized as anti-methicillin-resistant Staphylococcus aureus (MRSA) agents from a molecular hybridization approach. The representative compound 19 showed an MIC = 1 μg/mL against a panel of MRSA clinical isolates as it possessed comparable in vitro activities to that of vancomycin. Moreover, compound 19 also exhibited MIC = 1 μg/mL activities against a recent identified Z172 MRSA strain (vancomycin-intermediate and daptomycin-nonsusceptible phenotype) and the vancomycin-resistant Enterococcus faecalis (VRE) strain. In a mouse model with lethal infection of MRSA (4N216), a 75% survival rate was observed after a single dose of compound 19 was intravenously administered at 20 mg/kg. In light of their equipotent activities against different MRSA isolates and VRE strain, the data underscore the importance of designed hybrid series for the development of new N -substituted carbazoles as potential anti-MRSA agents.

We have discovered a novel series of quinazoline-based CXCR4 antagonists. Of these, compound 19 mobilized CXCR4(+) cell types, including hematopoietic stem cells and endothelial progenitor cells, more efficiently than the marketed 1 (AMD3100) with subcutaneous administration at the same dose (6 mg/kg) in mice. This series of compounds thus provides a set of valuable tools to study diseases mediated by the CXCR4/SDF-1 axis, including myocardial infarction, ischemic stroke, and cancer metastasis. More importantly, treatment with compound 19 significantly lowered levels of blood urea nitrogen and serum creatinine in rats with renal ischemia-reperfusion injury, providing evidence for its therapeutic potential in preventing ischemic acute kidney injury. CXCR4 antagonists such as 19 might also be useful to increase circulating levels of adult stem cells, thereby exerting beneficial effects on damaged and/or inflamed tissues in diseases that currently are not treated by standard approaches.

Motivated by the pivotal role of CXCR4 as an HIV entry co-receptor, we herein report a de novo hit-to-lead effort on the identification of subnanomolar purine-based CXCR4 antagonists against HIV-1 infection. Compound 24, with an EC50 of 0.5 nM against HIV-1 entry into host cells and an IC50 of 16.4 nM for inhibition of radioligand stromal-derived factor-1α (SDF-1α) binding to CXCR4, was also found to be highly selective against closely related chemokine receptors. We rationalized that compound 24 complementarily interacted with the critical CXCR4 residues that are essential for binding to HIV-1 gp120 V3 loop and subsequent viral entry. Compound 24 showed a 130-fold increase in anti-HIV activity compared to that of the marketed CXCR4 antagonist, AMD3100 (Plerixafor), whereas both compounds exhibited similar potency in mobilization of CXCR4(+)/CD34(+) stem cells at a high dose. Our study offers insight into the design of anti-HIV therapeutics devoid of major interference with SDF-1α function.

Protein S-prenylation is a lipid modification that regulates membrane-protein and protein-protein interactions in cell signaling. Though sites of protein S-prenylation can be predicted based upon conserved C-terminal CaaX or CC/CXC motifs, biochemical detection of protein S-prenylation in cells is still challenging. Herein, we report an alkynyl-isoprenol chemical reporter (alk-FOH) as an efficient substrate for prenyltransferases in mammalian cells that enables sensitive detection of S-farnesylated and S-geranylgeranylated proteins using bioorthogonal ligation methods. Fluorescent detection alleviates the need to deplete cellular isoprenoids for biochemical analysis of S-prenylated proteins and enables robust characterization of S-prenylated proteins, such as effectors that are injected into host cells by bacterial pathogens. This alkynyl-prenylation reporter provides a sensitive tool for biochemical analysis and rapid profiling of prenylated proteins in cells.

Recent years have seen the rapid advancement of new therapeutic agents against hepatitis C virus (HCV) in response to the need for treatment that is unmet by interferon (IFN)-based therapies. Most antiviral drugs discovered to date are small molecules that modulate viral enzyme activities. In the search for highly selective protein-binding molecules capable of disrupting the viral life cycle, we have identified a class of anionic tetraphenylporphyrins as potent and specific inhibitors of the HCV replicons. Based on the structure-activity relationship studies reported herein, meso-tetrakis-(3,5-dicarboxy-4,4'-biphenyl) porphyrin was found to be the most potent inhibitor of HCV genotype 1b (Con1) replicon systems but was less effective against the genotype 2a (JFH-1) replicon. This compound induced a reduction of viral RNA and protein levels when acting in the low nanomolar range. Moreover, the compound could suppress replicon rebound in drug-treated cells and exhibited additive to synergistic effects when combined with protease inhibitor BILN 2061 or with IFN-alpha-2a. Our results demonstrate the potential use of tetracarboxyphenylporphyrins as potent anti-HCV agents.

Proteins in nature fold into native conformations in which combinations of peripherally projected aliphatic, aromatic and ionic functionalities direct a wide range of properties. Alpha-helices, one of the most common protein secondary structures, serve as important recognition regions on protein surfaces for numerous protein-protein, protein-DNA and protein-RNA interactions. These interactions are characterized by conserved structural features within the alpha-helical domain. Rational design of structural mimetics of these domains with synthetic small molecules has proven an effective means to modulate such protein functions. In this tutorial review we discuss strategies that utilize synthetic small-molecule antagonists to selectively target essential protein-protein interactions involved in certain diseases. We also evaluate some of the protein-protein interactions that have been or are potential targets for alpha-helix mimetics.