Structure-Based Design and Development of Phosphine Oxides as a Novel Chemotype for Antibiotics that Dysregulate Bacterial ClpP Proteases.

A series of arylsulfones and heteroarylsulfones have previously been demonstrated to dysregulate the conserved bacterial ClpP protease, causing the unspecific degradation of essential cellular housekeeping proteins and ultimately resulting in cell death. A cocrystal structure of a 2-ß-sulfonylamide analog, ACP1-06, with Escherichia coli ClpP showed that its 2-pyridyl sulfonyl substituent adopts two orientations in the binding site related through a sulfone bond rotation. From this, a new bis-aryl phosphine oxide scaffold, designated as ACP6, was designed based on a "conformation merging" approach of the dual orientation of the ACP1-06 sulfone. One analog, ACP6-12, exhibited over a 10-fold increase in activity over the parent ACP1-06 compound, and a cocrystal X-ray structure with ClpP confirmed its predicted binding conformation. This allowed for a comparative analysis of how different ligand classes bind to the hydrophobic binding site. The study highlights the successful application of structure-based rational design of novel phosphine oxide-based antibiotics.

Computer-Aided Design of α-L-Rhamnosidase to Increase the Synthesis Efficiency of Icariside I.

Icariside I, the glycosylation product of icaritin, is a novel effective anti-cancer agent with immunological anti-tumor activity. However, very limited natural icariside I content hinders its direct extraction from plants. Therefore, we employed a computer-aided protein design strategy to improve the catalytic efficiency and substrate specificity of the α-L-rhamnosidase from Thermotoga petrophila DSM 13995, to provide a highly-efficient preparation method. Several beneficial mutants were obtained by expanding the active cavity. The catalytic efficiencies of all mutants were improved 16-200-fold compared with the wild-type TpeRha. The double-point mutant DH was the best mutant and showed the highest catalytic efficiency (k cat /K M : 193.52 s-1 M-1) against icariin, which was a 209.76-fold increase compared with the wild-type TpeRha. Besides, the single-point mutant H570A showed higher substrate specificity than that of the wild-type TpeRha in hydrolysis of different substrates. This study provides enzyme design strategies and principles for the hydrolysis of rhamnosyl natural products.

The study of citrus-derived flavonoids as effective bitter taste inhibitors.

BACKGROUND: The pericarp of citrus in rutaceae is rich in flavonoids that may possess diverse biological activities. Some citrus flavonoids have been used as natural bitterness inhibitors; however, many citrus flavonoid analogues that possess merit taste amelioration functions have not been reported with respect to utilization in food industry. RESULTS: The effects of 12 citrus flavonoids on the inhibition of the bitter taste of naringin, quinine hydrochloride and stevioside were evaluated both by a sensory panel and electronic tongue analysis. Among the flavonoid compounds evaluated, both neohesperidin dihydrochalcone (NHDC) and neodiosmin were identified to show an excellent bitterness inhibition effect on all three bitterness vehicles tested. The results of the electronic tongue evaluation also showed that the addition of neodiosmin, NHDC or hesperidin dihydrochalcone-7-o-glucoside (HDC-7-G) was able to reduce significantly the bitterness response value of quinine hydrochloride, which is consistent with the sensory panel evaluation. Structure-activity relationship analysis found that the 7-linked neohesperidosyloxy group in the A-ring of the citrus flavonoid skeleton has the best bitterness inhibition effect. In addition, a ternary mixture of NHDC, neodiosmin and naringin, and neodiosmin/ß-cyclodextrin was formulated and it demonstrated, for the first time in the flavor improvement of citrus fruit wine, an enhancement of sweetness and a reduction of bitter taste. CONCLUSION: Twelve citrus flavonoids were found to inhibit the bitter taste of naringin, quinine hydrochloride and stevioside. With respect to the structure-activity relationship analysis, it was found that the 7-linked neohesperidosyloxy group in the A-ring of the citrus flavonoid skeleton possessed the best bitterness inhibition effect. © 2021 Society of Chemical Industry.

ClpP protease activation results from the reorganization of the electrostatic interaction networks at the entrance pores.

Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation and activation of ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in Neisseria meningitidis and Escherichia coli ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering. ACP1 and ADEP induce distinct conformational changes in the ClpP structure. However, reorganization of electrostatic interaction networks at the ClpP entrance pores is necessary and sufficient for activation. Further activation is achieved by formation of ordered N-terminal axial loops and reduction in the structural heterogeneity of the ClpP cylinder. Activating mutations recapitulate the structural effects of small molecule activator binding. Our data, together with previous findings, provide a structural basis for a unified mechanism of compound-based ClpP activation.

The Role of ClpP Protease in Bacterial Pathogenesis and Human Diseases.

In prokaryotic cells and eukaryotic organelles, the ClpP protease plays an important role in proteostasis. The disruption of the ClpP function has been shown to influence the infectivity and virulence of a number of bacterial pathogens. More recently, ClpP has been found to be involved in various forms of carcinomas and in Perrault syndrome, which is an inherited condition characterized by hearing loss in males and females and by ovarian abnormalities in females. Hence, targeting ClpP is a potentially viable, attractive option for the treatment of different ailments. Herein, the biochemical and cellular activities of ClpP are discussed along with the mechanisms by which ClpP affects bacterial pathogenesis and various human diseases. In addition, a comprehensive overview is given of the new classes of compounds in development that target ClpP. Many of these compounds are currently primarily aimed at treating bacterial infections. Some of these compounds inhibit ClpP activity, while others activate the protease and lead to its dysregulation. The ClpP activators are remarkable examples of small molecules that inhibit protein-protein interactions but also result in a gain of function.

Clinical characteristics and neurologic recovery of patients with cervical spinal tuberculosis: should conservative treatment be preferred? A retrospective follow-up study of 115 cases.

OBJECTIVE: To present the clinical characteristics and prognostic factors of neurologic recovery in patients with cervical spinal tuberculosis (CST). METHODS: General description and multivariate analysis were used to detect possible predictors of the outcome of patients with neurologic deficit. Follow-up data were used to generate a Kaplan-Meier curve of neurologic recovery. RESULTS: Protective factors in neurologic recovery included less involved vertebrae, surgery, and higher Japanese Orthopaedic Association score before treatment; not shorter symptom duration was not a protective factor. Normal neurologic function was present in 30% of patients 6 months after treatment, in 56% of patients 12 months after treatment, and in 93% of patients 28 months after treatment. The cumulative complete neurologic recovery rates at 6 months, 12 months, and 28 months were 44%, 68%, and 91.7% in the surgery group and 16.7%, 38.8%, and 94.4% in the nonsurgery group. CONCLUSIONS: Surgery and Japanese Orthopaedic Association score before treatment are important predictors of neurologic recovery in patients with CST. A neurologic recovery curve could predict neurologic recovery in patients with CST and indicate in patients with neurologic deficit how long it will take to achieve complete neurologic recovery. The effect of surgery is quick, and the effect of conservative treatment is slower, but no difference in neurologic recovery was found in the long-term. Conservative treatment should be tried in every patient with CST with no obvious indication for surgery. In contrast to patients with tuberculosis without cervical spine involvement but with more complications, comprehensive conservative therapy should be preferred for patients with neurologic deficit to avoid unnecessary surgery and overtreatment and to conserve medical resources. Indications for surgical intervention for CST should be carefully selected.

[Progress in transgenics on Toxoplasma gondii].

The application of transgenes in Toxoplasma gondii research facilitates its genetic analysis. This article reviews the progress in construction of transgenes vector, transgenics in the research of T. gondii.

9-Substituted berberine derivatives as G-quadruplex stabilizing ligands in telomeric DNA.

The interaction of berberine and its 9-substituted derivatives with human telomeric DNA d[G(3)(T(2)AG(3))(3)](telo21) has been investigated via CD spectroscopy, fluorescence spectroscopy, PCR-stop assay, competitive dialysis, and telomerase repeat amplification protocol (TRAP) assay. The results indicated that these semisynthesized compounds could induce and stabilize the formation of anti-parallel G-quadruplex of telomeric DNA in the presence or absence of metal cations. Compared with berberine, the 9-substituted derivatives exhibit stronger binding affinity with G-quadruplex and higher inhibitory activity for telomerase. Introduction of a side chain with proper length of methylene and terminal amino group to the 9-position of berberine would significantly strengthen the binding affinity with G-quadruplex, resulting in increasing inhibitory effects on the amplification of telo21 DNA and on the telomerase activity.

Synthesis and evaluation of quindoline derivatives as G-quadruplex inducing and stabilizing ligands and potential inhibitors of telomerase.

A new series of quindoline derivatives (4a-j) were designed and synthesized to develop novel and potent telomerase inhibitors. The interaction of the G-quadruplex of human telomere DNA with these newly designed molecules was examined via circular dichroism spectroscopy and electrophoretic mobility shift assay (EMSA). The selectivity between the quindoline derivative (4a) and G-quadruplex or duplex DNA was investigated by competition dialysis. These new compounds as inhibitors of telomerase were also investigated through the utilization of modified telomerase repeat amplification protocol (TRAP) assay. The results revealed that the introduction of electron-donating groups such as substituted amino groups at the C-11 position of quindoline significantly improved the inhibitory effect on telomerase activity ((Tel)IC50 > 138 microM for quindoline, 0.44-12.3 microM for quindoline derivatives 4a-j). The quindoline derivatives not only stabilized the G-quadruplex structure but also induced the G-rich telomeric repeated DNA sequence to fold into quadruplex.

[Molecular cloning of two Rhipicephalus haemaphysaloides haemaphysaloides cathepsin L-like cysteine proteinase gene].

Ticks are obligate ectoparasites and vectors of arboviruses, vickettsiate, spirochetes and parasitil protozoa of humans and domestic animals. Immunological protection of mammalian hosts against tick infestation has been proposed as the most sustainable alternative tick control method to the current use of acaricides. The success of this method is dependent on the identification of key molecules for use as tick vaccine antigens. Proteolytic enzymes are involved in a wide range of cellular processes, thus they can be considered as good target antigens for a tick vaccine. In the present study, we used rapid amplification of cDNA ends protocol and primers that were designed based on the consensus amino acid motifs flanking present in all papain-like cysteine proteinases, to amplify, sequence and characterize two Rhipicephalus haemaphysaloides haemaphysaloides cathepsin L-like cysteine proteinases, named as cysA and cysB. The full length of cysA is 1168bp, encoding a 332 amino acid residue polypeptide with 36.33kD predicted molecular mass; the full length of cysB is 1153bp, encoding a 335 amino acid residue polypeptide with 37.56kD predicted molecular mass. The consensus amino acid motifs flanking presence in both deduced amino acid sequences. And both genes show high sequence homology to other tick cathepsin L-like cysteine proteinase, so they were identified as members of the cysteine proteinase gene family. Expression analysis by RT-PCR revealed that cysA and cysB were expressed differently in different periods of tick development.