Identifying novel selective PPO inhibitors through structure-based virtual screening and bio-evaluation.

Protoporphyrinogen oxidase (PPO) is a key enzyme in chlorophyll and heme biosynthesis, and the development of its inhibitors is of great importance both in the pharmaceutical and pesticide industries. However, the currently developed PPO inhibitors have insignificant bio-selectivity and have a serious impact on non-target organisms. In this study, a docking-based virtual screening approach combined with bio-activity testing was used to obtain novel selective inhibitors of PPO. The results of the bio-activity test showed that thirteen compounds showed 10-fold selectivity over human PPO. And the best selective compound, ZINC70338, has a K i value of 2.21 µM for Nicotiana tabacum PPO and >113-fold selectivity for human PPO. The selectivity mechanism of ZINC70338 in different species of PPO was then analyzed by molecular dynamics simulations to provide a design basis and theoretical guidance for the design of novel selective inhibitors.

Computational study reveals substituted benzimidazole derivatives' binding selectivity to PI3Kδ and PI3Kγ.

Phosphatidylinositol 3-kinase (PI3K) is a key regulatory kinase in the PI3K/AKT/mTOR signaling pathway, which is involved in the regulation of cell proliferation, differentiation, apoptosis, and angiogenesis. Class IA PI3K isoforms γ and δ share a highly homologous ATP binding site and are distinguished by only a few residues around the binding site. Subtype-selective inhibitors have been proven to have great advantages in tumor treatment. Preliminary studies have obtained PI3K inhibitors bearing a benzimidazole structural motif with a certain selectivity for PI3Kδ and PI3Kγ subtypes. On this basis, we investigated the selective inhibitory mechanism of PI3Kδ and PI3Kγ using four developed inhibitors via molecular docking, molecular dynamics, binding free energy calculations, and residue energy decomposition. This study could provide references for the further development of PI3K-isoform-selective inhibitors.

A coumarin-based fluorescent probe for NIR imaging-guided photodynamic therapy against S. aureus-induced infection in mouse models.

A coumarin-based viscosity-responsive fluorescent probe (HZAU800) was designed and synthesized. The probe, containing a strong electron-donating and rigid group on the 7-position of coumarin and a rhodamine derivative containing an oxonium ion on 3-position, could not only shift the emission wavelength to near-infrared region (NIR, λem = 800 nm) but also deliver a good PDT effect due to its high rigid planarity. The NIR fluorescence of HZAU800 can be lighted up in the S. aureus-infected region due to its high viscous environment. Under the laser's irradiation at 690 nm, the PDT effect was effectively triggered up, and the antibacterial evaluation in vitro and in vivo was successfully carried out. This study not only offers a new strategy for constructing coumarin-based phototherapy agents but also facilitates the exploration of the next generation of antibacterial materials based on coumarin architectures.

Design, synthesis and evaluation of tetrahydrocarbazole derivatives as potential hypoglycemic agents.

Two series of tetrahydrocarbazole derivatives have been designed and synthesized based on ZG02, a promising candidate developed in our previous studies. The newly prepared compounds were screened for glucose consumption activity in HepG2 cell lines. Aza-tetrahydrocarbazole compound 12b showed the most potent hypoglycemic activity with a 45% increase in glucose consumption when compared to the solvent control, which had approximately 1.2-fold higher activity than the positive control compounds (metformin and ZG02). An investigation of the potential mechanism indicated that 12b may exhibit hypoglycemic activity via activation of the AMPK pathway. Metabolic stability assays revealed that 12b showed good stability profiles in both artificial gastrointestinal fluids and blood plasma from SD rats. An oral glucose tolerance test (OGTT) was performed and the results further confirmed that 12b was a potent hypoglycemic agent.

Design, synthesis and bioevaluation of novel substituted triazines as potential dual PI3K/mTOR inhibitors.

A series of novel substituted triazines bearing a benzimidazole scaffold were designed and synthesized based on the structures of known anti-cancer agents, namely gedatolisib and alpelisib. All the target compounds were screened for inhibitory activity against PI3Kα and mTOR kinases. Notably, most analogs exhibited IC50 in the nanomolar range. Investigation of the isozyme selectivity indicated that the compounds exhibited remarkable inhibitory activity against PI3Kδ, especially compound 19f showed an IC50 value of 2.3 nM for PI3Kδ and moderate δ-isozyme selectivity over other class I PI3K isoforms and mTOR (with IC50 values of 14.6, 34.0, 849.0 and 15.4 nM for PI3Kα, ß, γ and mTOR, respectively). An in vitro MTT assay was conducted to assess the antiproliferative and cytotoxic effects of the prepared analogs. It was revealed that the compounds displayed significant inhibitory activities against the HCT116 human colon cancer cell line. Compound 19i showed 4.7-fold higher potency than the positive control gedatolisib (0.3 vs. 1.4 µM, IC50 values). Phosphoblot studies demonstrated that 19c and 19i could significantly suppress the PI3K/Akt/mTOR signaling pathway at 10 µM. Moreover, analogs 19b, 19c and 19i displayed better stability in artificial gastric fluids than gedatolisib, while 19i was indicated not very stable in rat liver microsomes, and may occur phase I metabolic transformations.

Ultrafast 2,7-Naphthyridine-Based fluorescent probe for detection of thiophenol with a remarkable Stokes shift and its application In vitro and in vivo.

2,7-Naphthyridine derivatives were developed as fluorophores for the first time to design two fluorescence probes, AND-DNP and ND-DNP, which can be applied for detecting thiophenol in aqueous media. Comparing with ND-DNP, AND-DNP showed more favorable properties such as lower background, larger Stokes shift, and higher fluorescence quantum yield for detecting thiophenol. Moreover, the experimental results were verified by theoretical calculations. Hence, AND-DNP was selected as the superior fluorescence probe to detect thiophenol because of its high sensitivity and selectivity. Based on the experimental results, AND-DNP showed a remarkably larger Stokes shift (225 nm), faster response speed (30 s) and higher fluorescence enhancement (240-fold) than most other fluorescent probes for thiophenol reported in the literature. For an extended application, AND-DNP was applied to detect thiophenol quantitatively in real water samples. Meanwhile, AND-DNP also detected thiophenol via red emission in living A549 cells and zebrafish. All these results proved AND-DNP's potential value as an accurate probe for imaging thiophenol in different environments.

Computational Discovery of Potent and Bioselective Protoporphyrinogen IX Oxidase Inhibitor via Fragment Deconstruction Analysis.

Tuning the binding selectivity through appropriate ways is a primary goal in the design and optimization of a lead toward agrochemical discovery. However, how to achieve rational design of selectivity is still a big challenge. Herein, we developed a novel computational fragment generation and coupling (CFGC) strategy that led to a series of highly potent and bioselective inhibitors targeting protoporphyrinogen IX oxidase. This enzyme plays a vital role in heme and chlorophyll biosynthesis, which has been proven to be associated with many drugs and agrochemicals. However, existing agrochemicals are nonbioselective, resulting in a great threat to nontargeted organisms. To the best of our knowledge, this is the first bioselective inhibitor targeting the tetrapyrrole biosynthesis pathway. In addition, the candidate showed excellent in vivo bioactivity and much better safety toward humans.

Multiple Simulated Annealing-Molecular Dynamics (MSA-MD) for Conformational Space Search of Peptide and Miniprotein.

Protein and peptide structure predictions are of paramount importance for understanding their functions, as well as the interactions with other molecules. However, the use of molecular simulation techniques to directly predict the peptide structure from the primary amino acid sequence is always hindered by the rough topology of the conformational space and the limited simulation time scale. We developed here a new strategy, named Multiple Simulated Annealing-Molecular Dynamics (MSA-MD) to identify the native states of a peptide and miniprotein. A cluster of near native structures could be obtained by using the MSA-MD method, which turned out to be significantly more efficient in reaching the native structure compared to continuous MD and conventional SA-MD simulation.

Synthesis and bioevaluation of pyrazole-benzimidazolone hybrids as novel human 4-Hydroxyphenylpyruvate dioxygenase inhibitors.

4-Hydroxyphenylpyruvate dioxygenase (HPPD), an essential enzyme in tyrosine catabolism, is an important target for treating type I tyrosinemia. Inhibition of HPPD can effectively alleviate the symptoms of type I tyrosinemia. However, only one commercial HPPD inhibitor, 2-(2-nitro-4-trifluoromethylbenzoyl) cyclohexane-1,3-dione (NTBC), has been available for clinical use so far. In the present study, a series of novel pyrazole-benzimidazolone hybrids were designed, synthesized and evaluated as potent human HPPD inhibitors. Most of the new compounds displayed significant inhibitory activity against the recombinant human HPPD. Moreover, compound 9l was identified as the most potent candidate with IC50 value of 0.021 µM against recombinant human HPPD, about 3-fold more potent than NTBC. Thus the pyrazole-benzimidazolone hybrid has great potential to be further developed for the treatment of type I tyrosinemia.

Synthesis and herbicidal evaluation of triketone-containing quinazoline-2,4-diones.

Exploring novel 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) inhibitors is one of the most promising research directions in herbicide discovery. To discover new triketone herbicides with broad-spectrum weed control as well as excellent crop selectivity, a series of (total 52) novel triketone-containing quinazoline-2,4-dione derivatives were synthesized and further bioevaluated. The greenhouse testing indicated that many of the newly synthesized compounds showed better or excellent herbicidal activity against broadleaf and monocotyledonous weeds at the dosages of 37.5-150 g of active ingredient (ai)/ha. The structure and activity relationship in this study indicated that the triketone-containing quinazoline-2,4-dione motif has possessed great impact on herbicide activity and may be used for further optimization. Among the new compounds, III-b and VI-a-VI-d displayed a broader spectrum of weed control than mesotrione. In addition, the compound III-b also demonstrated comparatively superior crop selectivity to mesotrione, thus possessing great potential for weed control in the field.

Syntheses of coumarin-tacrine hybrids as dual-site acetylcholinesterase inhibitors and their activity against butylcholinesterase, Aß aggregation, and ß-secretase.

Exploring small-molecule acetylcholinesterase (AChE) inhibitors to slow the breakdown of acetylcholine (Ach) represents the mainstream direction for Alzheimer's disease (AD) therapy. As the first acetylcholinesterase inhibitor approved for the clinical treatment of AD, tacrine has been widely used as a pharmacophore to design hybrid compounds in order to combine its potent AChE inhibition with other multi-target profiles. In present study, a series of novel tacrine-coumarin hybrids were designed, synthesized and evaluated as potent dual-site AChE inhibitors. Moreover, compound 1g was identified as the most potent candidate with about 2-fold higher potency (Ki=16.7nM) against human AChE and about 2-fold lower potency (Ki=16.1nM) against BChE than tacrine (Ki=35.7nM for AChE, Ki=8.7nM for BChE), respectively. In addition, some of the tacrine-coumarin hybrids showed simultaneous inhibitory effects against both Aß aggregation and ß-secretase. We therefore conclude that tacrine-coumarin hybrid is an interesting multifunctional lead for the AD drug discovery.

Structure-based design of conformationally flexible reverse transcriptase inhibitors to combat resistant HIV.

Reverse transcriptase (RT) is one of the most important targets for HIV drug discovery. However, the emergence of resistant mutants has become one of the biggest challenges in HIV-1 RT drug discovery/development and attracted great research interests worldwide. It is particularly important to develop novel anti-HIV-1 RT agents that have improved potency and efficacy against the wild-type (WT) RT, but also target resistant RT forms. Previous crystal complex structures of HIV-1 RT revealed the interaction mechanism between the enzyme and inhibitors, which promoted the exploitation of inhibitor that had sufficient conformational flexibility to combat resistance. Hence, the potential flexibility of a drug should be part of the strategy considered in the early stages of designing drugs that are intended to be broadly effective against mutated targets associated with drug resistance. This review provides an overview of the state of the art in this field, including design strategies and challenges for medicinal chemists.

Computational and experimental insights into the mechanism of substrate recognition and feedback inhibition of protoporphyrinogen oxidase.

Protoporphyrinogen IX oxidase (PPO; EC 1.3.3.4) is an essential enzyme catalyzing the last common step in the pathway leading to heme and chlorophyll biosynthesis. Great interest in PPO inhibitors arises from both its significance to agriculture and medicine. However, the discovery of PPO inhibitors with ultrahigh potency and selectivity is hampered due to lack of structural and mechanistic understanding about the substrate recognition, which remains a longstanding question central in porphyrin biology. To understand the mechanism, a novel binding model of protogen (protoporphyrinogen IX, the substrate) was developed through extensive computational simulations. Subsequently, amino acid residues that are critical for protogen binding identified by computational simulations were substituted by mutagenesis. Kinetic analyses of these mutants indicated that these residues were critical for protogen binding. In addition, the calculated free energies of protogen binding with these mutants correlated well with the experimental data, indicating the reasonability of the binding model. On the basis of this novel model, the fundamental mechanism of substrate recognition was investigated by performing potential of mean force (PMF) calculations, which provided an atomic level description of conformational changes and pathway intermediates. The free energy profile revealed a feedback inhibition mechanism of proto (protoporphyrin IX, the product), which was also in agreement with experimental evidence. The novel mechanistic insights obtained from this study present a new starting point for future rational design of more efficient PPO inhibitors based on the product-bound PPO structure.

Computational gibberellin-binding channel discovery unraveling the unexpected perception mechanism of hormone signal by gibberellin receptor.

Gibberellins (GAs) are phytohormones essential for many developmental processes in plants. In this work, fundamental mechanism of hormone perception by receptor GID1 has been studied by performing computational simulations, revealing a new GA-binding channel of GID1 and a novel hormone perception mechanism involving only one conformational state of GID1. The novel hormone perception mechanism demonstrated here is remarkably different from the previously proposed/speculated mechanism [Murase et al., Nature 2008, 456, 459] involving two conformational states ("OPEN" and "CLOSED") of GID1. According to the new perception mechanism, GA acts as a "conformational stabilizer," rather than the previously speculated "allosteric inducer," to induce the recognition of protein DELLA by GID1. The novel mechanistic insights obtained in this study provide a new starting point for further studies on the detailed molecular mechanisms of GID1 interacting with DELLA and various hormones and for mechanism-based rational design of novel, potent growth regulators that target crops and ornamental plants.

Design and synthesis of 1-(benzothiazol-5-yl)-1H-1,2,4-triazol-5-ones as protoporphyrinogen oxidase inhibitors.

Protoporphyrinogen oxidase (PPO, E.C. 1.3.3.4) is the action target for several structurally diverse herbicides. A series of novel 4-(difluoromethyl)-1-(6-halo-2-substituted-benzothiazol-5-yl)-3-methyl-1H-1,2,4-triazol-5(4H)-ones 2a-z were designed and synthesized via the ring-closure of two ortho-substituents. The in vitro bioassay results indicated that the 26 newly synthesized compounds exhibited good PPO inhibition effects with K(i) values ranging from 0.06 to 17.79 µM. Compound 2e, ethyl 2-{[5-(4-(difluoromethyl)-3-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-6-fluorobenzo-thiazol-2-yl]thio}acetate, was the most potent inhibitor with K(i) value of 0.06 µM against mtPPO, comparable to (K(i)=0.03 µM) sulfentrazone. Further green house assays showed that compound 2f (K(i)=0.24 µM, mtPPO), ethyl 2-{[5-(4-(difluoromethyl)-3-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-6-fluorobenzothiazol-2-yl]thio}propanoate, showed the most promising post-emergence herbicidal activity with broad spectrum even at concentrations as low as 37.5 gai/ha. Soybean exhibited tolerance to compound 2f at the dosages of 150 gai/ha, whereas they are susceptible to sulfentrazone even at 75 gai/ha. Thus, compound 2f might be a potential candidate as a new herbicide for soybean fields.

Quantitative structure-activity relationships of 1,3,4-thiadiazol-2(3H)-ones and 1,3,4-oxadiazol-2(3H)-ones as human protoporphyrinogen oxidase inhibitors.

Protoporphyrinogen oxidase (Protox, EC 1.3.3.4) has attracted great interest during the last decades due to its unique biochemical characteristics and biomedical significance. As a continuation of our research work on the development of new PPO inhibitors, 23 new 1,3,4-thiadiazol-2(3H)-ones bearing benzothiazole substructure were designed and synthesized. The in vitro assay indicated that the newly synthesized compounds 1a-w displayed good inhibition activity against human PPO (hPPO) with K(i) values ranging from 0.04µM to 245µM. To the knowledge, compound 1a, O-ethyl S-(5-(5-(tert-butyl)-2-oxo-1,3,4-thiadiazol-3(2H)-yl)-6-fluorobenzothiazol-2-yl)carbonothioate, with the K(i) value of 40nM, is so far known as the most potent inhibitor against hPPO. Based on the molecular docking and modified molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) calculations, the quantitative structure-activity relationships of 1,3,4-thiadiazol-2(3H)-ones and 1,3,4-oxadiazol-2(3H)-one derivatives were established with excellent correlation relationships (r(2)=0.81) between the calculated and experimental binding free energies. Some important insights were also concluded for guiding the future rational design of new hPPO inhibitors with improved potency.

Computational simulations of structural role of the active-site W374C mutation of acetyl-coenzyme-A carboxylase: multi-drug resistance mechanism.

Herbicides targeting grass plastidic acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) are selectively effective against graminicides. The intensive worldwide use of this herbicide family has selected for resistance genes in a number of grass weed species. Recently, the active-site W374C mutation was found to confer multi-drug resistance toward haloxyfop (HF), fenoxaprop (FR), Diclofop (DF), and clodinafop (CF) in A. myosuroides. In order to uncover the resistance mechanism due to W374C mutation, the binding of above-mentioned four herbicides to both wild-type and the mutant-type ACCase was investigated in the current work by molecular docking and molecular dynamics (MD) simulations. The binding free energies were calculated by molecular mechanics-Poisson-Boltzmann surface area (MM/PBSA) method. The calculated binding free energy values for four herbicides were qualitatively consistent with the experimental order of IC(50) values. All the computational model and energetic results indicated that the W374C mutation has great effects on the conformational change of the binding pocket and the ligand-protein interactions. The most significant conformational change was found to be associated with the aromatic amino acid residues, such as Phe377, Tyr161' and Trp346. As a result, the π-π interaction between the ligand and the residue of Phe377 and Tyr161', which make important contributions to the binding affinity, was decreased after mutation and the binding affinity for the inhibitors to the mutant-type ACCase was less than that to the wild-type enzyme, which accounts for the molecular basis of herbicidal resistance. The structural role and mechanistic insights obtained from computational simulations will provide a new starting point for the rational design of novel inhibitors to overcome drug resistance associated with W374C mutation.

Protoporphyrinogen oxidase inhibitor: an ideal target for herbicide discovery.

As the last common enzyme in the biosynthetic pathway leading to heme and chlorophyll, protoporphyrinogen oxidase (PPO; EC 1.3.3.4) is an ideal target for herbicide development. Currently, about 30 PPO inhibitors have been developed as agricultural herbicides. PPO inhibitors have displayed environmentally benign, but advantageous characteristics, including low toxicity, low effective concentration, broad herbicidal spectrum (active against both monocotyledon and dicotyledon weeds), quick onset of action, and long lasting effect. Over the last several years, great achievements have been made in revealing the structural biology of PPO. Five PPO crystal structures, four isolated in enzyme-inhibitor complexes and one in the native form, have been determined, including those from Nicotiana tabacum, Myxococcus Xanthus, Bacillus subtilis, and human. Although PPO inhibitors have been developed for over forty years, we continue to uncover exciting future prospects for novel PPO-inhibiting herbicides. In this review, we have summarized the structures of PPOs from plants, human, and bacteria; the interactions between PPOs and inhibitors; the quantitative structure-activity relationships of PPO inhibitors; and the molecular design of new PPO inhibitors.