Discovery and evaluation of novel nitrodihydroimidazooxazoles as promising anti-tuberculosis agents.

New analogues of antitubercular drug Delamanid were prepared, seeking drug candidates with enhanced aqueous solubility and high efficacy. The strategy involved replacement of phenoxy linker proximal to the 2-nitroimidazooxazole of Delamanid by piperidine fused 5 or 6-membered ring heterocycles (ring A). The new compounds were all more hydrophilic than Delamanid, and several class of analogues showed remarkable activities against M. bovis. And among these series, the tetrahydro-naphthyridine-linked nitroimidazoles displayed excellent antimycobacterial activity against both replicating (MABA) and nonreplicating (LORA) M. tb H37Rv and low cytotoxicity. Compared to Delamanid, these new compounds (6, 7, 45) demonstrated dramatically improved physicochemical properties and are suitable for further in vitro and in vivo evaluation.

Mechanistic insights into the allosteric regulation of Pseudomonas aeruginosa aspartate kinase.

In plants and microorganisms, aspartate kinase (AK) catalyzes an initial commitment step of the aspartate family amino acid biosynthesis. Owing to various structural organizations, AKs from different species show tremendous diversity and complex allosteric controls. We report the crystal structure of AK from Pseudomonas aeruginosa (PaAK), a typical α2ß2 hetero-tetrameric enzyme, in complex with inhibitory effectors. Distinctive features of PaAK are revealed by structural and biochemical analyses. Essentially, the open conformation of Lys-/Thr-bound PaAK structure clarifies the inhibitory mechanism of α2ß2-type AK. Moreover, the various inhibitory effectors of PaAK have been identified and a general amino acid effector motif of AK family is described.

Metabolism of SKLB-TB1001, a Potent Antituberculosis Agent, in Animals.

Tuberculosis is a major global health problem, and the emergence of multidrug-resistant and extensively drug-resistant strains has increased the difficulty of treating this disease. Among the novel antituberculosis drugs in the pipeline, decaprenylphosphoryl-beta-d-ribose-2-epimerase (DprE1) inhibitors such as BTZ043 and pBTZ169 exhibited extraordinary antituberculosis potency. Here, the metabolites of the new DprE1 inhibitor SKLB-TB1001 in vivo and its inhibition of cytochrome P450 isoforms and plasma protein binding (PPB) in vitro were studied. The results showed that rapid transformation and high PPB resulted in inadequate exposure in vivo and thus led to the moderate potency of SKLB-TB1001 in vivo This study provided explanations for the discrepant potency of this scaffold in vivo and in vitro Meanwhile, it also provides a rationale for lead optimization of this very promising scaffold of antituberculosis agents to prevent them from being metabolized, thus improving their exposure in vivo.

Synthesis and antitubercular evaluation of 4-carbonyl piperazine substituted 1,3-benzothiazin-4-one derivatives.

Tuberculosis (TB) remains a major human health problem. New therapeutic antitubercular agents are urgent needed to control the global tuberculosis pandemic. We synthesized a new series of 4-carbonyl piperazine substituted 1,3-benzothiazin-4-one derivatives and evaluated their anti-mycobacterial activities against Mycobacterium tuberculosis H37Ra as well as their druggabilities. The results showed that most of these derivatives, especially the compounds with simple alkyl side chains, exhibited good antitubercular activities and favorable aqueous solubilities with no obvious cytotoxicity. It suggested that the 4-carbonyl piperazine substituents in benzothiazinone scaffold were well tolerated, in which the compound 8h, with an antitubercular activity of MIC 0.008 µM, exhibited an excellent aqueous solubility of 104 µg/mL, which was 100-fold better than the potent DprE1 inhibitor Comp.1 (BTZ038), also more soluble than PBTZ169.

Reductions in myeloid-derived suppressor cells and lung metastases using AZD4547 treatment of a metastatic murine breast tumor model.

BACKGROUND: AZD4547, a small-molecule inhibitor targeting the tyrosine kinase of Fibroblast Growth Factor Receptors (FGFRs), is currently under phase II clinical study for human subjects having breast cancer, while the underlying mechanism remains elusive. The aim of this study is to explore the potential mechanism by which AZD4547 inhibits breast tumor lung metastases at the level of the tumor microenvironment. METHODS: First, through in vitro experiments, we investigated the efficacy of the FGFRs inhibitor AZD4547 on 4T1 tumor cells for their proliferation, apoptosis, migration, and invasion. Second, by in vivo animal experiments, we evaluated the effects of AZD4547 on tumor growth and lung metastases in 4T1 tumor-bearing mice. Finally, we examined the impact of AZD4547 on the infiltration of myeloid-derived suppressor cells (MDSCs) in lung, spleens, peripheral blood and tumor. RESULTS: Through this study we found that AZD4547 could efficiently suppress tumor 4T1 cells through restraining their proliferation, blocking migration and invasion, and inducing apoptosis in vitro. In animal model we also demonstrated that AZD4547 was able to inhibit tumor growth and lung metastases, consistent with the decreased MDSCs accumulation in the tumor and lung tissues, respectively. Moreover, the reduced number of MDSCs in peripheral blood and spleens were also observed in the AZD4547-treated mice. Importantly, through the AZD4547 treatment, the CD4(+) and CD8(+) T-cells were significantly increased in tumor and spleens. CONCLUSION: Our studies showed that AZD4547 can inhibit breast cancer cell proliferation, induce its apoptosis and block migration and invasion in vitro and suppress tumor growth and lung metastases by modulating the tumor immunologic microenvironment in vivo.

Discovery and structure-activity relationships study of novel thieno[2,3-b]pyridine analogues as hepatitis C virus inhibitors.

Current treatment for hepatitis C is barely satisfactory, there is an urgent need to develop novel agents for combating hepatitis C virus infection. This study discovered a new class of thieno[2,3-b]pyridine derivatives as HCV inhibitors. First, a hit compound characterized by a thienopyridine core was identified in a cell-based screening of our privileged small molecule library. And then, structure activity relationship study of the hit compound led to the discovery of several potent compounds without obvious cytotoxicity in vitro (12c, EC50=3.3µM, SI >30.3, 12b, EC50=3.5µM, SI >28.6, 10l, EC50=3.9µM, SI >25.6, 12o, EC50=4.5µM, SI >22.2, respectively). Although the mechanism of them had not been clearly elucidated, our preliminary optimization of this class of compounds had provided us a start point to develop new anti-HCV agents.

Synthesis and structure-activity relationships evaluation of benzothiazinone derivatives as potential anti-tubercular agents.

N-Alkyl and heterocycle substituted 1,3-benzothiazin-4-one (BTZ) derivatives were synthesized. The anti-mycobacterial activities of these compounds were evaluated by determination of minimal inhibitory concentration (MIC) for Mycobacterium tuberculosis H37Ra and M. tuberculosis H37Rv. It was found that an extended or branched alkyl chain analog could enhance the potency, and activities of N-alkyl substituted BTZs were not affected by either nitro or trifluoromethyl at 6-position. Trifluoromethyl plays an important role in maintaining anti-tubercular activity in the piperazine or piperidine analogs. Compound 8o, which contains an azaspirodithiolane group, showed a MIC of 0.0001 µM against M. tuberculosis H37Rv, 20-fold more potent than BTZ043 racemate. These results suggested that the volume and lipophilicity of the substituents were important in maintaining activity. In addition, compound 8o was nontoxic to Vero cells and orally bioavailable in a preliminary pharmacokinetics study.

Structural characterization of a Δ3, Δ2-enoyl-CoA isomerase from Pseudomonas aeruginosa: implications for its involvement in unsaturated fatty acid metabolism.

Gene PA4980 from Pseudomonas aeruginosa encodes a putative enoyl-coenzyme A hydratase/isomerase that is associated with the function of the biofilm dispersion-inducing signal molecule cis-2-decenoic acid. To elucidate the role of PA4980 in cis-2-decenoic acid biosynthesis, we reported the crystal structure of its protein product at 2.39 Å. The structural analysis and substrate binding prediction suggest that it acts as a monofunctional enoyl-coenzyme A isomerase, implicating an alternative pathway of the cis-2-decenoic acid synthesis.

Identification of a new series of benzothiazinone derivatives with excellent antitubercular activity and improved pharmacokinetic profiles.

Nitrobenzothiazinone (BTZ) is a promising scaffold with potent activity against M. tuberculosis by inhibiting decaprenylphosphoryl-beta-d-ribose 2'-oxidase (DprE1). But unfavorable durability poses a challenge to further development of this class of agents. Herein, a series of BTZs bearing a variety of different substituents at the C-2 position were designed and synthesized. Compounds were screened for their antimycobacterial activity against Mycobacterium tuberculosis H37Ra and were profiled for metabolic stability, plasma protein-binding capacity and pharmacokinetics in vivo. In general, these new BTZs containing N-piperazine, N-piperidine or N-piperidone moiety have excellent antitubercular activity and low cytotoxicity. Several of the compounds showed improved microsomal stability and lower plasma protein-binding, opening a new direction for further lead optimization. And we obtained compound 3o, which maintained good anti-tuberculosis activity (MIC = 8 nM) and presented better in vitro ADME/T and in vivo pharmacokinetic profiles than reported BTZ compound PBTZ169, which may serve as a candidate for the treatment of tuberculosis.

Structural and functional studies on Pseudomonas aeruginosa DspI: implications for its role in DSF biosynthesis.

DspI, a putative enoyl-coenzyme A (CoA) hydratase/isomerase, was proposed to be involved in the synthesis of cis-2-decenoic acid (CDA), a quorum sensing (QS) signal molecule in the pathogen Pseudomonas aeruginosa (P. aeruginosa). The present study provided a structural basis for the dehydration reaction mechanism of DspI during CDA synthesis. Structural analysis reveals that Glu126, Glu146, Cys127, Cys131 and Cys154 are important for its enzymatic function. Moreover, we show that the deletion of dspI results in a remarkable decreased in the pyoverdine production, flagella-dependent swarming motility, and biofilm dispersion as well as attenuated virulence in P. aeruginosa PA14. This study thus unravels the mechanism of DspI in diffusible signal factor (DSF) CDA biosynthesis, providing vital information for developing inhibitors that interfere with DSF associated pathogenicity in P. aeruginosa.

Author Correction: Structural and functional studies on Pseudomonas aeruginosa DspI: implications for its role in DSF biosynthesis.

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Structure-Function Relationship of Aminopeptidase P from Pseudomonas aeruginosa.

PepP is a virulence-associated gene in Pseudomonas aeruginosa, making it an attractive target for anti-P. aeruginosa drug development. The encoded protein, aminopeptidases P (Pa-PepP), is a type of X-prolyl peptidase that possesses diverse biological functions. The crystal structure verified its canonical pita-bread fold and functional tetrameric assembly, and the functional studies measured the influences of different metal ions on the activity. A trimetal manganese cluster was observed at the active site, elucidating the mechanism of inhibition by metal ions. Additionally, a loop extending from the active site appeared to be important for specific large-substrate binding. Based on the structural comparison and bacterial invasion assays, we showed that this non-conserved surface loop was critical for P. aeruginosa virulence. Taken together, these findings can extend our understanding of the catalytic mechanism and virulence-related functions of Pa-PepP and provide a solid foundation for the design of specific inhibitors against pathogenic-bacterial infections.

A novel benzothiazinethione analogue SKLB-TB1001 displays potent antimycobacterial activities in a series of murine models.

New chemotherapeutic compounds and regimens are needed to combat multidrug-resistant Mycobacterium tuberculosis. Here, we used a series of murine models to assess an antitubercular lead compound SKLB-TB1001. In the Mycobacterium bovis bacillus Calmette-Guérin and the acute M. tuberculosis H37Rv infection mouse models, SKLB-TB1001 significantly attenuated the mycobacterial load in lungs and spleens. The colony forming unit counts and histological examination of lungs from H37Rv infected mice revealed that the benzothiazinethione analogue SKLB-TB1001 as a higher dose level was as effective as isoniazid. Moreover, in a multidrug-resistant (MDR)-TB mouse model, SKLB-TB1001 showed significant activity in a dose-dependent manner and was more effective than streptomycin. These results suggested that SKLB-TB1001 could be an antitubercular drug candidate worth further investigation.

Discovery of imidazo[2,1-b]thiazole HCV NS4B inhibitors exhibiting synergistic effect with other direct-acting antiviral agents.

The design, synthesis, and SAR studies of novel inhibitors of HCV NS4B based on the imidazo[2,1-b]thiazole scaffold were described. Optimization of potency with respect to genotype 1b resulted in the discovery of two potent leads 26f (EC50 = 16 nM) and 28g (EC50 = 31 nM). The resistance profile studies revealed that 26f and 28g targeted HCV NS4B, more precisely the second amphipathic α helix of NS4B (4BAH2). Cross-resistance between our 4BAH2 inhibitors and other direct-acting antiviral agents targeting NS3/4A, NS5A, and NS5B was not observed. For the first time, the synergism of a series of combinations based on 4BAH2 inhibitors was evaluated. The results demonstrated that our 4BAH2 inhibitor 26f was synergistic with NS3/4A inhibitor simeprevir, NS5A inhibitor daclatasvir, and NS5B inhibitor sofosbuvir, and it could also reduce the dose of these drugs at almost all effect levels. Our study suggested that favorable effects could be achieved by combining 4BAH2 inhibitors such as 26f with these approved drugs and that new all-oral antiviral combinations based on 4BAH2 inhibitors were worth developing to supplement or even replace current treatment regimens for curing HCV infection.