Click Chemistry and Multicomponent Reaction for Linker Diversification of Zinc Dipicolylamine-Based Drug Conjugates.

An efficient Ugi multicomponent reaction with strain promoted azide-alkyne cycloaddition protocol has been utilized in concert or independently to prepare a small family of bioactive zinc(II) dipicolylamine (ZnDPA)-based SN-38 conjugates. With sequential click chemistry coupling between the cytotoxic payload and phosphatidylserine-targeting ZnDPA ligand derived from structurally diverse carboxylic acids, aldehyde or ketones, and isocyanides, we demonstrated that this convergent synthetic strategy could furnish conjugates harnessing diversified linkers that exhibited different pharmacokinetic profiles in systemic circulation in vivo. Among the eight new conjugates, comparative studies on in vitro cytotoxicities, plasma stabilities, in vivo pharmacokinetic properties, and maximum tolerated doses were then carried out to identify a potent ZnDPA-based SN-38 conjugate that resulted in pancreatic cancer growth regression with an 80% reduction of cytotoxic payload used when compared to that of the marketed irinotecan. Our work provided the roadmap to construct a variety of theranostic agents in a similar manner for cancer treatment.

Effect of a Neuropilin-1-Derived Virus Receptor Trap on Enterovirus A71 Infection In Vitro.

We discovered that neuropilin 1 (NRP1) is a new receptor candidate to mediate enterovirus A71 (EVA71) into cells. In the engineered form as a decoy receptor, NRP1 was able to recognize and neutralize EVA71 but not enterovirus D68 or coxsackievirus B3 (CVB3). NRP1 recognizes EVA71 through a novel domain on the VP3 capsid protein. The principle in the design, engineering, and refinement of the NRP1-based decoy receptor described in this study represents a general and well-suited antiviral strategy.

Discovery of M Protease Inhibitors Encoded by SARS-CoV-2.

The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (Mpro, also called 3C-like protease [3CLpro]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting Mpro in the picornavirus-like supercluster, is a potent inhibitor for the Mpro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC50) of 26.4 ± 1.1 nM. In this study, we also show that GC376 inhibits SARS-CoV-2 replication with a half-maximum effective concentration (EC50) of 0.91 ± 0.03 µM. Only a small portion of SARS-CoV-2 Mpro was covalently modified in the excess of GC376 as evaluated by mass spectrometry analysis, indicating that improved inhibitors are needed. Subsequently, molecular docking analysis revealed that the recognition and binding groups of GC376 within the active site of SARS-CoV-2 Mpro provide important new information for the optimization of GC376. Given that sufficient safety and efficacy data are available for GC376 as an investigational veterinary drug, expedited development of GC376, or its optimized analogues, for treatment of SARS-CoV-2 infection in human is recommended.

Workflow and Strategies for Recruitment and Retention in Longitudinal 3D Craniofacial Imaging Study.

Longitudinal epidemiological studies are considered the gold standard for understanding craniofacial morphologic development, but participant recruitment and retention can be challenging. This study describes strategies used to recruit and maintain a high level of participation in a longitudinal study involving annual three-dimensional (3D) craniofacial soft-tissue imaging from healthy Taiwanese Chinese elementary school students aged 6 to 12 years. The key aspects for project delineation, implementation, and the initial three-year practical experiment are portrayed in an integrated multistep workflow: ethics- and grant-related issues; contact, approval, and engagement from partners of the project (school stakeholders and parents); a didactic approach to recruit the students; research staff composition with task design; three station-based data collection days with two educative activities (oral hygiene and psychosocial interaction stations) and one 3D craniofacial imaging activity; and reinforcement tactics to sustain the longitudinal annual participation after the first enrollment. Randomly selected students and teachers answered an experience satisfaction questionnaire (five-point Likert scale ranging from one to five) designed to assist in understanding what they think about the data collection day. Measures of frequency (percentage) and central tendency (mean) were adopted for descriptive analysis. Six of seven contacted schools accepted participation in the project. All parents who attended the explanatory meetings agreed to join the project. A cohort of 676 students (336 girls) participated at baseline enrollment, with a follow-up rate of 96% in the second data collection. The average questionnaire-related scores were 4.2 ± 0.7 and 4.4 ± 0.6 for teachers and students, respectively. These 3D craniofacial norms will benefit multidisciplinary teams managing cleft-craniofacial deformities in the globally distributed ethnic Chinese population, particularly useful for phenotypic variation characterization, conducting quantitative morphologic comparisons, and therapeutic planning and outcome assessment. The described pathway model will assist other groups to establish their own age-, sex-, and ethnic-specific normative databases.

Rationally designed divalent caffeic amides inhibit amyloid-ß fibrillization, induce fibril dissociation, and ameliorate cytotoxicity.

One of the pathologic hallmarks in Alzheimer's disease (AD) is extracellular senile plaques composed of amyloid-ß (Aß) fibrils. Blocking Aß self-assembly or disassembling Aß aggregates by small molecules would be potential therapeutic strategies to treat AD. In this study, we synthesized a series of rationally designed divalent compounds and examined their effects on Aß fibrillization. A divalent amide (2) derived from two molecules of caffeic acid with a propylenediamine linker of ∼5.0 Šin length, which is close to the distance of adjacent ß sheets in Aß fibrils, showed good potency to inhibit Aß(1-42) fibrillization. Furthermore, compound 2 effectively dissociated the Aß(1-42) preformed fibrils. The cytotoxicity induced by Aß(1-42) aggregates in human neuroblastoma was reduced in the presence of 2, and feeding 2 to Aß transgenic C. elegans rescued the paralysis phenotype. In addition, the binding and stoichiometry of 2 to Aß(1-40) were demonstrated by using electrospray ionization-traveling wave ion mobility-mass spectrometry, while molecular dynamic simulation was conducted to gain structural insights into the Aß(1-40)-2 complex.

Inhibition of formyl peptide-stimulated phospholipase D activation by Fal-002-2 via blockade of the Arf6, RhoA and protein kinase C signaling pathways in rat neutrophils.

Three recently developed selective phospholipase D (PLD) inhibitors N-(2-(4-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)ethyl)-2-naphthamide (VU0155056), (S)-N-(1-(4-(5-chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)propan-2-yl)-2-naphthamide (VU0155069), and N-(2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]decan-8-yl)ethyl)quinoline-3-carboxamide (VU0285655-1) inhibited O2 (•-) generation in formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils. A novel 2-phenyl-4-quinolone compound 6-chloro-2-(2-chlorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (Fal-002-2), which inhibited O2 (•-) generation, also reduced the fMLP- but not phorbol ester-stimulated PLD activity (IC50 16.0 ± 5.0 µM). Fal-002-2 attenuated the interaction of PLD1 with ADP-ribosylation factor (Arf) 6, Ras homology (Rho) A and protein kinase C (PKC) isoforms (α, ßI, and ßII), and also inhibited the membrane recruitment of Arf6 and RhoA in fMLP-stimulated neutrophils, but not in GTPγS-stimulated cell-free system. The cellular levels of GTP-bound Arf6 and GTP-bound RhoA were reduced by Fal-002-2. Fal-002-2 also attenuated the membrane recruitment of Rho-associated protein kinase 1, phosphorylation of myosin light chain 2 at Thr18/Ser19 and PLD1 at Thr147, and the interaction of Arf6 with both arfaptin 1 and phosphatidylinositol 4-phosphate 5-kinase 1A. The association between RhoA and Vav, the interaction of Vav with both Lyn and Lck, the membrane recruitment of Vav, and the phosphorylation of Vav at Tyr174, but not Src family at Tyr416, were all attenuated by Fal-002-2 in fMLP-stimulated neutrophils. These results indicate that Fal-002-2 is not a direct PLD inhibitor, but the inhibition of fMLP-stimulated PLD activity by Fal-002-2, which partly accounts for its suppression of O2 (•-) generation, is attributable to the blockade of both Arf6 and RhoA activation and attenuation of the interaction of Arf6, RhoA and PKC isoforms with PLD1 in rat neutrophils.

Inhibition of formyl peptide-stimulated superoxide anion generation by Fal-002-2 occurs mainly through the blockade of the p21-activated kinase and protein kinase C signaling pathways in ratneutrophils.

In formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils, a synthetic compound, 6-chloro-2-(2-chlorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (Fal-002-2), inhibited superoxide anion (O2(•-)) generation with an IC50 value of about 11µM, which was not mediated by scavenging the generated O2(•-) or by a cytotoxic effect on neutrophils. Fal-002-2 effectively attenuated the phosphorylation of Ser residues in p47(phox) and the association between p47(phox) and p22(phox) in fMLP-stimulated neutrophils. The interaction of p47(phox) with protein kinase C (PKC) isoforms (α, ßI, ßII, δ and ζ) was attenuated by Fal-002-2 with a similar IC50 value to that required for inhibition of O2(•-) generation, whereas Fal-002-2 had no prominent effect on PKC isoform membrane translocation and did not affect the kinase activity. Moreover, Fal-002-2 had no effect on the phosphorylation of Akt and downstream glycogen synthase kinase-3ß, only slightly affected the intracellular free Ca(2+) concentration, phosphorylation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase (MAPK), but effectively attenuated the downstream MAPK-activated protein kinase-2 phosphorylation. The interaction of p21-activated kinase (PAK) 1with p47(phox), phosphorylation of PAK1 (Thr423/Ser144) and the membrane recruitment of PAK1 were effectively inhibited by Fal-002-2. Fal-002-2 also blocked the activation of Rac1 and Cdc42 in a concentration range that effectively inhibited PAK activation. Taken together, these results suggest that Fal-002-2 inhibits fMLP-stimulated O2(•-) generation in neutrophils mainly through the blockade of PKC and PAK signaling pathways and partly through p38 MAPK signaling.

p38 Mitogen-activated protein kinase and extracellular signal-regulated kinase signaling pathways are not essential regulators of formyl peptide-stimulated p47(phox) activation in neutrophils.

Three structurally unrelated p38 mitogen-activated protein kinase (MAPK) inhibitors, (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole (SB203580), 1-5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl] urea (BIRB 796) and 5-(2,6-dichlorophenyl)-2-[2,4-difluorophenyl]thio]-6H-pyrimido[1,6-b]pyridazin-6-one (VX 745) showed approximately 40% inhibition of formyl-Met-Leu-Phe (fMLP)-stimulated neutrophil superoxide anion (O2(•-)) generation at concentrations that greatly diminished p38 MAPK activity. However, a significant inhibition of p47(phox) activation occurred at concentrations much higher than the corresponding IC50 values of these inhibitors in blocking p38 MAPK activity. 4-Ethyl-2(p-methoxyphenyl)-5-(4'-pyridyl)-IH-imidazole (SB202474), an inactive analogue of SB203580, at a concentration (30µM) which significantly attenuated p38 MAPK activity, had no effect on p47(phox) activation, whereas it inhibited O2(•-) generation with an IC50 value of approximately 16µM. Moreover, both SB203580 and BIRB 796 had no effect on protein kinase B (PKB)/Akt Ser473 phosphorylation and S100A9 protein membrane translocation at concentrations that effectively blocked p38 MAPK activity. Pretreatment of cells with two structurally unrelated MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors, 2-(2-amino-3-methoxy-phenyl)-chromen-4-one (PD 98059) and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), at concentrations that effectively blocked MEK activity, attenuated p47(phox) phosphorylation but did not affect the recruitment of p47(phox) to p22(phox) or O2(•-) generation. Both p47(phox) activation and O2(•-) generation were attenuated by a protein kinase C (PKC) inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X) in the concentration range that effectively blocked PKC activity. Taken together, these results suggest that the ERK-mediated Ser phosphorylation of p47(phox) is not implicated in the assembly of NADPH oxidase or O2(•-) generation, and that O2(•-) generation is partly attributable to p38 MAPK signaling through mechanisms other than p47(phox) activation, Akt activation and S100A9 membrane recruitment in fMLP-stimulated neutrophils.

The signaling mechanisms mediating the inhibitory effect of TCH-1116 on formyl peptide-stimulated superoxide anion generation in neutrophils.

In fMLP (formyl-Met-Leu-Phe)-stimulated rat neutrophils, a mixture of regioisomers benzo[a]furo[2,3-c]phenazine-10-carboxylic acid and benzo[a]furo[2,3-c]phenazine-11-carboxylic acid (TCH-1116) inhibited O(2)(-) (superoxide anion) generation, which was not mediated by scavenging the generated O(2)(-) or by a cytotoxic effect on neutrophils. TCH-1116 had no effect on the arachidonic acid-induced NADPH oxidase activation in a cell-free system, whereas it effectively attenuated the phosphorylation of Ser residues in p47(phox) and the association between p47(phox) and p22(phox) in fMLP-stimulated neutrophils. The interaction of p47(phox) with PKC (protein kinase C) isoforms (α, ßI, ßII, δ and ζ) was attenuated by TCH-1116, whereas TCH-1116 did not affect the PKC isoforms membrane translocation, phosphorylation (Ser660) and kinase activity. TCH-1116 effectively attenuated the association between PKB/Akt (protein kinase B) and p47(phox), Akt phosphorylation (Thr308/Ser473) and kinase activities of Akt and human recombinant PDK (3-phosphoinositide-dependent kinase) 1, whereas it had no effect on recruitment of Akt, phospho-PDK1 (Ser241) and p110γ to membrane. Moreover, the interaction of p21-activated kinase (PAK) 1 with p47(phox) and the phosphorylation of PAK1 (Thr423 but not Ser144) were inhibited by TCH-1116, but without affecting the membrane recruitment of PAK1. The cellular cyclic AMP level was not changed by TCH-1116. Taken together, these results suggest that TCH-1116 inhibits fMLP-stimulated O(2)(-) generation in rat neutrophils through the blockade of PKC, Akt and PAK signaling pathways.

Signaling mechanisms of inhibition of phospholipase D activation by CHS-111 in formyl peptide-stimulated neutrophils.

A selective phospholipase D (PLD) inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) inhibited the O(2)(-) generation and cell migration but not degranulation in formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils. A novel benzyl indazole compound 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111), which inhibited O(2)(-) generation and cell migration, also reduced the fMLP- but not phorbol ester-stimulated PLD activity (IC(50) 3.9±1.2µM). CHS-111 inhibited the interaction of PLD1 with ADP-ribosylation factor (Arf) 6 and Ras homology (Rho) A, and reduced the membrane recruitment of RhoA in fMLP-stimulated cells but not in GTPγS-stimulated cell-free system. CHS-111 reduced the cellular levels of GTP-bound RhoA, membrane recruitment of Rho-associated protein kinase 1 and the downstream myosin light chain 2 phosphorylation, and attenuated the interaction between phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and Arf6, whereas it only slightly inhibited the guanine nucleotide exchange activity of human Dbs (DH/PH) protein and did not affect the arfaptin binding to Arf6. CHS-111 inhibited the interaction of RhoA with Vav, the membrane association and the phosphorylation of Vav. CHS-111 had no effect on the phosphorylation of Src family kinases (SFK) but attenuated the interaction of Vav with Lck, Hck, Fgr and Lyn. CHS-111 also inhibited the interaction of PLD1 with protein kinase C (PKC) α, ßI and ßII isoenzymes, and the phosphorylation of PLD1. These results indicate that inhibition of fMLP-stimulated PLD activity by CHS-111 is attributable to the blockade of RhoA activation via the interference with SFK-mediated Vav activation, attenuation of the interaction of Arf6 with PLD1 and PIP5K, and the activation of Ca(2+)-dependent PKC in rat neutrophils.