Flavonoids: True or promiscuous inhibitors of enzyme? The case of deoxyxylulose phosphate reductoisomerase

Flavonoids, due to their physical and chemical properties (among them hydrophobicity and metal chelation abilities), are potential inhibitors of the 1-deoxyxylulose 5-phosphate reductoisomerase and most of the tested flavonoids effectively inhibited its activity with encouraging IC₅₀ values in the micromolar range. The addition of 0.01% Triton X100 in the assays led however, to a dramatic decrease of the inhibition revealing that a non-specific inhibition probably takes place. Our study highlights the possibility of erroneous conclusions regarding the inhibition of enzymes by flavonoids that are able to produce aggregates in micromolar range. Therefore, the addition of a detergent in the assays prevents possible false positive hits in high throughput screenings.     

“Biosynthesis of psilocybin and its nonnatural derivatives by a promiscuous psilocybin synthesis pathway in Escherichia coli”

Traditional psychedelics are undergoing a transformation from recreational drugs, to promising pharmaceutical drug candidates with the potential to provide an alternative treatment option for individuals struggling with mental illness. Sustainable and economic production methods are thus needed to facilitate enhanced study of these drug candidates to support future clinical efforts. Here, we expand upon current bacterial psilocybin biosynthesis by incorporating the cytochrome P450 monooxygenase, PsiH, to enable the de novo production of psilocybin as well as the biosynthesis of 13 psilocybin derivatives. The substrate promiscuity of the psilocybin biosynthesis pathway was comprehensively probed by using a library of 49 single-substituted indole derivatives, providing biophysical insights to this understudied metabolic pathway and opening the door to the in vivo biological synthesis of a library of previously unstudied pharmaceutical drug candidates.     

Disorderness in Escherichia coli proteome: perception of folding fidelity and protein–protein interactions

Traditionally biased usage of synonymous codons renders selective advantage to proteins expressed at high levels with a few exceptions like in Escherichia coli. Proteome-wide characteristics indicative of trends in highly expressed proteins of E. coli is analyzed in this communication. Implications for the nature of interactions performed by these two groups of highly expressed proteins are discussed here. The group of highly expressed proteins having optimized codon usage through employment of most abundant tRNAs is already shielded from misfolding by their improved error-prone translational machinery. Our data also provide evidence for mechanism by which a significant proportion of highly expressed proteins with high intrinsic disorder evade degradation and successfully carry out their function.     

Structure–activity relationship of boronic acid derivatives of tyropeptin: Proteasome inhibitors

The structure–activity relationship of the boronic acid derivatives of tyropeptin, a proteasome inhibitor, was studied. Based on the structure of a previously reported boronate analog of tyropeptin (2), 41 derivatives, which have varying substructure at the N-terminal acyl moiety and P2 position, were synthesized. Among them, 3-phenoxyphenylacetamide 6 and 3-fluoro picolinamide 22 displayed the most potent inhibitory activity toward chymotryptic activity of proteasome and cytotoxicity, respectively. The replacement of the isopropyl group in the P2 side chain to H or Me had negligible effects on the biological activities examined in this study.     

On the Productivity and Properties of l-Asparaginase from Escherichia coli A–l–3

Oxidation of grayanotoxin (GTX) II with lead (IV) acetate in methanol gave a new derivative, the 1(R)-spiro-3,6(S),14,16-tetra-hydroxy-5-keto derivative. Treatment of GTX-II tetraacetate in acetic acid by using Pb(IV) acetate as an oxidizing agent gave a novel 1,5-seco-GTX derivative, Δ¹⁽¹⁰⁾-1,5-seco-GTX-pentaacetate, together with the 1,5-seco-GTX-1(R) derivative. Oxidation of GTX-II-tetraacetate with Tl(III) acetate in acetic acid or benzene gave the 1,5-seco-GTX-1(S) derivative.     

Mapping of New Escherichia coli K and 15 Restriction Sites on Specific Fragments of Bacteriophage φX174 DNA

We have isolated several new phiX174 mutants which contain sites sensitive to restriction by Escherichia coli. One contains an E. coli 15 restriction site and three are double mutants containing an E. coli K site as well as the E. coli 15 site. The replicative form (RF) DNA of one of the mutants containing a K site has been shown to be restricted in spheroplasts of a K-12 strain. The infectivity of this RF, but not wild-type RF, has also been shown to be inactivated by an E. coli K extract and by purified K restriction enzyme in vitro. The product of the RF treated with purified K restriction enzyme in vitro is a full length linear molecule. The mutant sites have also been localized to specific regions of the phiX174 genome by a fragment mapping technique, making use of specific fragments of phiX174 RF DNA obtained by digestion with a specific endonuclease.     

Increase in synthesis and stability of σ 32 on treatment with inhibitors of DNA gyrase in Escherichia coli

We report here that in Escherichia coli, the anti-bacterial agent nalidixic acid induces transient stabilization and increased synthesis of σ³², accompanied by the induction of heat shock proteins (Dnak and GroEL proteins). The induction of heat shock proteins, increased synthesis of σ³², and stabilization of σ³² observed on treatment of wild-type cells with nalidixic acid were not observed in a nalA26 mutant, a strain that is resistant to nalidixic acid as the result of a mutation in the gyrA gene. Not only oxolinic acid, but also novobiocin, whose targets are the A and B subunits of DNA gyrase, respectively, also induced stabilization and increased synthesis of σ³². Thus, inhibition of the activity of DNA gyrase may cause stabilization and increased synthesis of σ³², resulting in turn in induction of heat shock proteins.     

Recovery of culturability of an HOCl-stressed population of Escherichia coli after incubation in phosphate buffer: resuscitation or regrowth?

An Escherichia coli population harvested in exponential phase at about 10(8) cells/ml was treated in phosphate buffer with HOCl at concentrations ranging from 0.4 to 1 mg/liter (7.7 to 19 microM). The HOCl stress resulted in the appearance of three cell subpopulations: a majority of dead (nonrespiring) cells, a few culturable cells (10(2) to 10(4)), and about 10(7) viable but nonculturable cells. In the absence of any added exogenous nutrient, a culturable population could be recovered after 1 day of incubation in phosphate buffer, and such a population would reach a cell density close to 10% of the initial density of the stressed population, whatever the initial number of survivors. When a small number of untreated cells were mixed with the stressed population, growth of the untreated cells was observed, demonstrating that damaged cells provided nutrients. Similarly, a filtrate and a disrupted-cell filtrate of the stressed population supported growth of untreated cells with the same efficiency. The number of CFU (untreated or stressed) at plateau phase depended on the initial density of the stressed cells. Taken together, these results suggest that recovery in phosphate buffer of an HOCl-stressed population is in large part due to growth of a few culturable cells at the expense of damaged cells. However, comparison of the growth rates of the stressed culturable population and of untreated bacteria growing in filtrate showed significantly faster growth of the stressed cells, a fact not fully compatible with the hypothesis that recovery is only the simple growth of survivors. We suggest, therefore, that in addition to growth of the few culturable stressed cells, there is repair and growth of some mildly injured viable but nonculturable cells.     

Pressure and Temperature Dependence of Growth and Morphology of?Escherichia coli: Experiments and Stochastic Model

We have investigated the growth of Escherichia coli, a mesophilic bacterium, as a function of pressure (P) and temperature (T). Escherichia coli can grow and divide in a wide range of pressure (1–400 atm) and temperature (23–40°C). For T > 30°C, the doubling time of E. coli increases exponentially with pressure and exhibits a departure from exponential behavior at pressures between 250 and 400 atm for all the temperatures studied in our experiments. The sharp change in doubling time is followed by a sharp change in phenotypic transition of E. coli at high pressures where bacterial cells switch to an elongating cell type. We propose a model that this phenotypic change in bacteria at high pressures is an irreversible stochastic process, whereas the switching probability to elongating cell type increases with increasing pressure. The model fits well the experimental data. We discuss our experimental results in the light of structural and thus functional changes in proteins and membranes.     

Discovery of potent and selective rhodanine type IKKβ inhibitors by hit-to-lead strategy

Regulation of NF-κB activation through the inhibition of IKKβ has been identified as a promising target for the treatment of inflammatory and autoimmune disease such as rheumatoid arthritis. In order to develop novel IKKβ inhibitors, we performed high throughput screening toward around 8000 library compounds, and identified a hit compound containing rhodanine moiety. We modified the structure of hit compound to obtain potent and selective IKKβ inhibitors. Throughout hit-to-lead studies, we have discovered optimized compounds which possess blocking effect toward NF-κB activation and TNFα production in cell as well as inhibition activity against IKKβ. Among them, compound 3q showed the potent inhibitory activity against IKKβ, and excellent selectivity over other kinases such as p38α, p38β, JNK1, JNK2, and JNK3 as well as IKKα.     

Synthesis and structure–activity relationship of thiobarbituric acid derivatives as potent inhibitors of urease

A series of thiobarbituric acid derivatives 1–27 were synthesized and evaluated for their urease inhibitory potential. Exciting results were obtained from the screening of these compounds 1–27. Compounds 5, 7, 8, 11, 16, 17, 22, 23 and 24 showed excellent urease inhibition with IC₅₀ values 18.1 ± 0.52, 16.0 ± 0.45, 16.0 ± 0.22, 14.3 ± 0.27, 6.7 ± 0.27, 10.6 ± 0.17, 19.2 ± 0.29, 18.2 ± 0.76 and 1.61 ± 0.18 μM, respectively, much better than the standard urease inhibitor thiourea (IC₅₀ = 21 ± 0.11 μM). Compound 3, 4, 10, and 26 exhibited comparable activities to the standard with IC₅₀ values 21.4 ± 1.04 and 21.5 ± 0.61μM, 22.8 ± 0.32, 25.2 ± 0.63, respectively. However the remaining compounds also showed prominent inhibitory potential The structure–activity relationship was established for these compounds. This study identified a novel class of urease inhibitors. The structures of all compounds were confirmed through spectroscopic techniques such as EI-MS and ¹H NMR.     

Synthesis and structure–activity relationship of pyripyropene A derivatives as potent and selective acyl-CoA:cholesterol acyltransferase 2 (ACAT2) inhibitors: Part 3

In an effort to develop potent and selective inhibitors toward ACAT2, structure–activity relationship studies were carried out using derivatives based on pyripyropene A (PPPA, 1). In particular, we investigated the possibility of introducing appropriate 1,11-O-benzylidene and 7-O-substituted benzoyl moieties into PPPA (1). The new o-substituted benzylidene derivatives showed higher selectivity for ACAT2 than PPPA (1). Among them, 1,11-O-o-methylbenzylidene-7-O-p-cyanobenzoyl PPPA derivative 7q and 1,11-O-o,o-dimethylbenzylidene-7-O-p-cyanobenzoyl PPPA derivative 7z proved to be potent ACAT2 inhibitors with unprecedented high isozyme selectivity.     

Synthesis and structure–activity relationship of pyripyropene A derivatives as potent and selective acyl-CoA:cholesterol acyltransferase 2 (ACAT2) inhibitors: Part 1

In an effort to develop potent and selective inhibitors toward ACAT2, structure–activity relationship studies were carried out using derivatives based on pyripyropene A (PPPA, 1). We have successfully developed novel PPPA derivatives with a 7-O-substituted benzoyl substituent that significantly exhibit more potent ACAT2 inhibitory activity and higher ACAT2 isozyme selectivity than 1.     

Synthesis and structure–activity relationship of pyripyropene A derivatives as potent and selective acyl-CoA:cholesterol acyltransferase 2 (ACAT2) inhibitors: Part 2

Synthesis and structure–activity relationships of 7-O-p-cyanobenzoyl pyripyropene A derivatives with modification at C1 and 11 are described. Regioselective mono-deprotection of di-tert-butylsilylene acetal was critical in their synthesis.     

Gene Cloning, Overproduction and Purification of Escherichia coli tRNA_2~(Arg)

合成的编码大肠杆菌ｔＲＮＡＡｒｇ２的基因,嵌入受ＩＰＴＧ诱导启动子控制的质粒ｐＴｒｃ９９Ｂ中。用上述含目的基因的质粒转化大肠杆菌ＭＴ１０２,得到ｔＲＮＡＡｒｇ２基因序列正确的克隆。诱导表达后,与受体菌相比,转化子中的ｔＲＮＡＡｒｇ的含量高出１０倍,ｔＲＮＡＡｒｇ２的含量高出３０倍,占总ｔＲＮＡ的７０％。ＤＥＡＥＳｅｐｈａｃｅｌ柱层析后,ｔＲＮＡＡｒｇ２的纯度即可达到８８％。再用ｂｅｎｚｙｌＤＥＡＥｃｅｌｕｌｏｓｅ柱层析可得到纯度为９９％、精氨酸接受活力为１６００ｐｍｏｌｅ／Ａ２６０单位的ｔＲＮＡＡｒｇ２。从４升过夜培养液中得到的４０ｍｇ总ｔＲＮＡ。从中可得到１８ｍｇ纯ｔＲＮＡＡｒｇ２,产率为６２％。首次精确地测定了精氨酰ｔＲＮＡ合成酶催化ｔＲＮＡＡｒｇ２时的动力学常数。     

Novel β-dicarbonyl derivatives as inhibitors of aminopeptidase N (APN)

Most zinc metalloproteases are over-expressed in tumor cells and play a critical role in the genesis, development, and metastasis of tumors. Novel zinc binding groups (ZBGs) represent a novel strategy to obtain optimal potency and selectivity for zinc metalloproteases inhibitors. Here we described the design, synthesis, and biological studies of novel β-dicarbonyl derivatives as aminopeptidase N (APN/CD13) inhibitors. The results demonstrated that some compounds exhibited moderate to good inhibitory activities against APN with compound 5c being the most potent, suggesting that 5c could serve as new lead for the future APN inhibitor development. The results further confirm our design rationale of β-dicarbonyl moiety as a new ZBG, which may provide a new direction for the design and discovery of zinc metalloproteases inhibitors as new anti-tumor agents.