Discovery and optimization of dihydropteridone derivatives as novel PLK1 and BRD4 dual inhibitor for the treatment of cancer.

In this study, we have designed, synthesized and tested three series of novel dihydropteridone derivatives possessing isoindolin-1-one or isoindoline moieties as potent inhibitors of PLK1/BRD4. Remarkably, most of the compounds showed preferable inhibitory activity against PLK1 and BRD4. Compound SC10 exhibited excellent inhibitory activity with IC50 values of 0.3 nM and 60.8 nM against PLK1 and BRD4, respectively. Meanwhile, it demonstrated significant anti-proliferative activities against three tumor-derived cell lines (MDA-MB-231 IC50 = 17.3 nM, MDA-MB-361 IC50 = 8.4 nM, and MV4-11 IC50 = 5.4 nM). Moreover, SC10 exhibited moderate rat liver microsomal stability (CLint = 21.3 µL·min-1·mg-1), acceptable pharmacokinetic profile (AUC0-t = 657 ng·h·mL-1, oral bioavailability of 21.4 %) in Sprague-Dawley rats, reduced hERG toxicity, acceptable PPB and CYP450 inhibition. Further research indicated that SC10 could induce MV4-11 cell arrest at the S phase and apoptosis in a dose-dependent manner. This investigation provided us with an initial point for developing novel anticancer agents as dual inhibitors of PLK1 and BRD4.

Development of 2nd generation aminomethyl spectinomycins that overcome native efflux in Mycobacterium abscessus.

Mycobacterium abscessus (Mab), a nontuberculous mycobacterial (NTM) species, is an emerging pathogen with high intrinsic drug resistance. Current standard-of-care therapy results in poor outcomes, demonstrating the urgent need to develop effective antimycobacterial regimens. Through synthetic modification of spectinomycin (SPC), we have identified a distinct structural subclass of N-ethylene linked aminomethyl SPCs (eAmSPCs) that are up to 64-fold more potent against Mab over the parent SPC. Mechanism of action and crystallography studies demonstrate that the eAmSPCs display a mode of ribosomal inhibition consistent with SPC. However, they exert their increased antimicrobial activity through enhanced accumulation, largely by circumventing efflux mechanisms. The N-ethylene linkage within this series plays a critical role in avoiding TetV-mediated efflux, as lead eAmSPC 2593 displays a mere fourfold susceptibility improvement against Mab ΔtetV, in contrast to the 64-fold increase for SPC. Even a minor shortening of the linkage by a single carbon, akin to 1st generation AmSPC 1950, results in a substantial increase in MICs and a 16-fold rise in susceptibility against Mab ΔtetV. These shifts suggest that longer linkages might modify the kinetics of drug expulsion by TetV, ultimately shifting the equilibrium towards heightened intracellular concentrations and enhanced antimicrobial efficacy. Furthermore, lead eAmSPCs were also shown to synergize with various classes of anti-Mab antibiotics and retain activity against clinical isolates and other mycobacterial strains. Encouraging pharmacokinetic profiles coupled with robust efficacy in Mab murine infection models suggest that eAmSPCs hold the potential to be developed into treatments for Mab and other NTM infections.

SNAP25 is a potential target for early stage Alzheimer's disease and Parkinson's disease.

BACKGROUND: Alzheimer's disease (AD) and Parkinson's disease (PD), two common irreversible neurodegenerative diseases, share similar early stage syndromes, such as olfaction dysfunction. Yet, the potential comorbidity mechanism of AD and PD was not fully elucidated. METHODS: The gene expression profiles of GSE5281 and GSE8397 were downloaded from the Gene Expression Omnibus (GEO) database. We utilized a series of bioinformatics analyses to screen the overlapped differentially expressed genes (DEGs). The hub genes were further identified by the plugin CytoHubba of Cytoscape and validated in the hippocampus (HIP) samples of APP/PS-1 transgenic mice and the substantial nigra (SN) samples of A53T transgenic mice by real-time quantitative polymerase chain reaction (RT-qPCR). Meanwhile, the expression of the target genes in the olfactory epithelium/bulb was detected by RT-qPCR. Finally, molecular docking was used to screen potential compounds for the target gene. RESULTS: One hundred seventy-four overlapped DEGs were identified in AD and PD. Five of the top ten enrichment pathways mainly focused on the synapse. Five hub genes were identified and further validated. As a common factor in AD and PD, the changes of synaptosomal-associated protein 25 (SNAP25) mRNA in olfactory epithelium/bulb were significantly decreased and had a strong association with those in the HIP and SN samples. Pazopanib was the optimal compound targeting SNAP25, with a binding energy of - 9.2 kcal/mol. CONCLUSIONS: Our results provided a theoretical basis for understanding the comorbidity mechanism of AD and PD and highlighted that SNAP25 in the olfactory epithelium may serve as a potential target for early detection and intervention in both AD and PD.

A Modified BPaL Regimen for Tuberculosis Treatment replaces Linezolid with Inhaled Spectinamides.

The Nix-TB clinical trial evaluated a new 6-month regimen containing three-oral-drugs; bedaquiline (B), pretomanid (Pa) and linezolid (L) (BPaL regimen) for treatment of tuberculosis (TB). This regimen achieved remarkable results as almost 90% of the multidrug resistant (MDR) or extensively drug resistant (XDR) TB participants were cured but many patients also developed severe adverse effects (AEs). The AEs were associated with the long-term administration of the protein synthesis inhibitor linezolid. Spectinamide 1599 (S) is also a protein synthesis inhibitor of Mycobacterium tuberculosis with an excellent safety profile but which lacks oral bioavailability. Here we hypothesize that inhaled spectinamide 1599, combined with BPa --BPaS regimen--has similar efficacy to that of BPaL regimen while simultaneously avoiding the L-associated AEs. The BPaL and BPaS regimens were compared in the Balb/c and C3HeB/FeJ murine chronic TB efficacy models. After 4-weeks of treatment, both regimens promoted equivalent bactericidal effect in both TB murine models. However, treatment with BPaL resulted in significant weight loss and the complete blood count suggested development of anemia. These effects were not similarly observed in mice treated with BPaS. BPaL treatment also decreased myeloid to erythroid ratio and increased concentration of proinflammatory cytokines in bone marrow compared to mice receiving BPaS regimen. During therapy both regimens improved the lung lesion burden, reduced neutrophil and cytotoxic T cells counts while increased the number of B and helper and regulatory T cells. These combined data suggest that inhaled spectinamide 1599 combined with BPa is an effective TB regimen that avoids L-associated AEs. IMPORTANCE: Tuberculosis (TB) is an airborne infectious disease that spreads via aerosols containing Mycobacterium tuberculosis (Mtb), the causative agent of TB. TB can be cured by administration of 3-4 drugs for 6-9 months but there are limited treatment options for patients infected with multidrug (MDR) and extensively resistant (XDR) strains of Mtb. BPaL is a new all-oral combination of drugs consisting of Bedaquiline (B), Pretomanid (Pa) and Linezolid (L). This regimen was able to cure ∼90% of MDR and XDR TB patients in clinical trials but many patients developed severe adverse effects (AEs) associated to the long-term administration of linezolid. We evaluated a new regimen in which Linezolid in the BPaL regimen was replaced with inhaled spectinamide 1599. In the current study, we demonstrate that 4-weeks of treatment with inhaled spectinamide 1599 in combination with Bedaquiline and Pretomanid has equivalent efficacy to the BPaL drug combination and avoids the L-associated-AEs.

Design and Synthesis of 1,3,5-Triazines or Pyrimidines Containing Dithiocarbamate Moiety as PI3Kα Selective Inhibitors.

Recent studies have shown that phosphoinositide 3-kinase (PI3K) plays a vital role in cell division, and it has become a therapeutic target for many cancers. In this paper, some new 1,3,5-triazine or pyrimidine skeleton derivatives containing dithiocarbamate were designed and synthesized based on the reasonable drug design strategy from the previously effective compound 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK-474), in order to get effective selective PI3Kα inhibitors that have not been reported in the literature. In addition, the inhibitory activities of these compounds on PI3Kα and two tumor cell lines in vitro (HCT-116, U87-MG) were evaluated. The representative compound 13 showed a half-maximal inhibitory concentration (IC50) value of 1.2 nM for PI3Kα and an exciting kinase selectivity. Compound 13 displayed strong efficacy in HCT-116 and U87-MG cell lines with IC50 values of 0.83 and 1.25 µM, respectively. In addition, compound 13 induced obvious tumor regression in the U87-MG cell line xenografts mouse model, with no obvious signs of toxicity after intraperitoneal injection at a dose of 40 mg/kg. Compound 13 can be an effective selective inhibitor of PI3Kα, and it provides patients with an opportunity to avoid the side effects related to the wider inhibition of the class I PI3K family.

Structure-based drug design, synthesis, and biological evaluation of novel 1,3,5-triazine or pyrimidine derivatives containing benzoyl hydrazine moiety as PI3Kα selective inhibitors.

Phosphoinositide 3-kinase (PI3K) was an important cellular signal transducer, while PI3Kα was the most mutated family member in cancer. Selective PI3Kα inhibitors have become the frequent research in recent years because of their excellent curative effect and reduced side effects. Here, we described a series of PI3Kα inhibitors with 1,3,5-triazine or pyrimidine skeleton containing benzoyl hydrazine based on the pan-PI3K inhibitor ZSTK474 relying on the strategies of structure-based drug discovery (SBDD) and computer-aided drug design (CADD). Among them, compound F8 exhibited improved selective PI3Kα inhibition with an IC50 value of 0.14 nM and more significant anti-proliferative activities against three tumor-derived cell lines (PC-3 IC50 = 0.28 µM, HCT-116 IC50 = 0.57 µM, and U87-MG IC50 = 1.37 µM) than ZSTK-474. Compound F-8 induced a great decrease in mitochondrial membrane which caused cell cycle arrest at G1 phase and apoptosis in U87-MG cells in a dose-dependent manner. Furthermore, compound F8 induced significant tumor regressions in a xenograft mouse model of U87-MG cell line with no clear evidence of toxicity following intraperitoneal injection of 40 mg/kg. Compound F8 may serve as a PI3Kα-selective inhibitor and provided the opportunity to spare patients the side effects associated with broader inhibition of the class I PI3K family.

Development of a Minimalistic Physiologically Based Pharmacokinetic (mPBPK) Model for the Preclinical Development of Spectinamide Antibiotics.

Spectinamides 1599 and 1810 are lead spectinamide compounds currently under preclinical development to treat multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis. These compounds have previously been tested at various combinations of dose level, dosing frequency, and route of administration in mouse models of Mycobacterium tuberculosis (Mtb) infection and in healthy animals. Physiologically based pharmacokinetic (PBPK) modeling allows the prediction of the pharmacokinetics of candidate drugs in organs/tissues of interest and extrapolation of their disposition across different species. Here, we have built, qualified, and refined a minimalistic PBPK model that can describe and predict the pharmacokinetics of spectinamides in various tissues, especially those relevant to Mtb infection. The model was expanded and qualified for multiple dose levels, dosing regimens, routes of administration, and various species. The model predictions in mice (healthy and infected) and rats were in reasonable agreement with experimental data, and all predicted AUCs in plasma and tissues met the two-fold acceptance criteria relative to observations. To further explore the distribution of spectinamide 1599 within granuloma substructures as encountered in tuberculosis, we utilized the Simcyp granuloma model combined with model predictions in our PBPK model. Simulation results suggest substantial exposure in all lesion substructures, with particularly high exposure in the rim area and macrophages. The developed model may be leveraged as an effective tool in identifying optimal dose levels and dosing regimens of spectinamides for further preclinical and clinical development.

Characterization of spectinamide 1599 efficacy against different mycobacterial phenotypes.

Spectinamides are a novel series of spectinomycin analogs being developed for the treatment of tuberculosis. The preclinical lead spectinamide 1599 is an antituberculosis drug that possesses robust in vivo efficacy, good pharmacokinetic properties, and excellent safety profiles in rodents. In individuals infected with Mycobacterium tuberculosis or Mycobacterium bovis, causative agents of tuberculosis, the host immune system is capable of restraining these mycobacteria within granulomatous lesions. The harsh microenvironmental conditions of these granuloma lead to phenotypic transformation of mycobacteria. Phenotypically transformed bacteria display suboptimal growth, or complete growth arrest and are frequently associated with drug tolerance. Here we quantified the effect of spectinamide 1599 on log-phase and phenotypically tolerant isoforms of Mycobacterium bovis BCG using various in vitro approaches as a first indicator of spectinamide 1599 activity against various mycobacterial isoforms. We also used the hollow fiber infection model to establish time-kill curves and deployed pharmacokinetic/pharmacodynamic modeling to characterize the activity differences of spectinamide 1599 towards the different phenotypic subpopulations. Our results indicate that spectinamide 1599 is more efficacious against log phase bacteria when compared to its activity against other phenotypically tolerant forms such as acid phase bacteria and hypoxic phase bacteria, a behavior similar to the established antituberculosis drug isoniazid.

Design, synthesis, and biological evaluation of novel dihydropteridone derivatives possessing oxadiazoles moiety as potent inhibitors of PLK1.

Polo like kinase 1 (PLK1) is a serine/threonine kinase that is widely distributed in eukaryotic cells and plays an important role in multiple phases of the cell cycle. Its importance in tumorigenesis has been increasingly recognized in recent years. Herein, we describe the optimization of a series of novel dihydropteridone derivatives (13a-13v and 21g-21l) possessing oxadiazoles moiety as potent inhibitors of PLK1. Compound 21g exhibited improved PLK1 inhibitory capability with an IC50 value of 0.45 nM and significant anti-proliferative activities against four tumor-derived cell lines (MCF-7 IC50 = 8.64 nM, HCT-116 IC50 = 26.0 nM, MDA-MB-231 IC50 = 14.8 nM and MV4-11 IC50 = 47.4 nM) with better pharmacokinetic characteristics than BI2536 in mice (AUC0-t = 11 227 ng h mL-1vs 556 ng h mL-1). Moreover, 21g exhibited moderate liver microsomal stability and excellent pharmacokinetic profile (AUC0-t = 11227 ng h mL-1, oral bioavailability of 77.4%) in Balb/c mice, acceptable PPB, improved PLK1 inhibitory selectivity, and no apparent toxicity was observed in the acute toxicity assay (20 mg/kg). Further investigation showed that 21 g could arrest HCT-116 cells in G2 phase and induce apoptosis in a dose-dependent manner. These results indicate that 21g is a promising PLK1 inhibitor.

Stereoselective Synthesis of Substituted (Z)-N-Allyl Sulfonamides via a Palladium-Catalyzed Three-Component Tandem Reaction.

This paper reports the efficient synthesis of substituted (Z)-N-allyl sulfonamides via a palladium-catalyzed three-component tandem reaction of N-buta-2,3-dienyl sulfonamides with iodides and sulfonyl hydrazide or sulfinic acid sodium salt as nucleophiles. Pd(PPh3)4 (2.5 mol %), K2CO3, and THF were used as the optimal catalyst, base, and solvent, respectively. The substituted (Z)-N-allyl sulfonamides were obtained in a 30-83% overall yield. Mechanistic investigations revealed that the formation of the single (Z)-isomer was controlled by the formation of a six-membered palladacycle intermediate.

Based on 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474), design, synthesis and biological evaluation of novel PI3Kα selective inhibitors.

Based on 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474), three series of novel 1,3,5-triazine or pyrimidine derivatives containing semicarbazones have been designed and synthesized to obtain new potent and selective PI3Kα inhibitors. Their inhibitory activities in vitro were evaluated against PI3Kα and three tumor-derived cell lines (U87-MG, MCF-7, and PC-3). We also tested promising compounds (A4, A6, A10, and B1) for other PI3K class I subtype (PI3Kß, PI3Kδ, and PI3Kγ) activity. The representative compound A10 exhibited an IC50 value of 0.32 nM against PI3Kα, and demonstrated extraordinary subtype selectivity. Furthermore, compound A10 obviously inhibited proliferation of MCF-7 cell lines, induced a great decrease in mitochondrial membrane potential leading to apoptosis of cancer cells, and arrested G2 phase in a dose-dependent manner. Additionally, compound A10 induced significant tumor regressions in a xenograft mouse model of U87-MG cell line without an obvious sign of toxicity upon 20 mg/kg oral administration. Compound A10 may serve as a PI3Kα-selective inhibitor and provide the opportunity to spare patients the side effects associated with broader inhibition of the class I PI3K family.

1,3-Diarylpyrazolyl-acylsulfonamides Target HadAB/BC Complex in Mycobacterium tuberculosis.

Alternative mode-of-inhibition of clinically validated targets is an effective strategy for circumventing existing clinical drug resistance. Herein, we report 1,3-diarylpyrazolyl-acylsulfonamides as potent inhibitors of HadAB/BC, a 3-hydroxyl-ACP dehydratase complex required to iteratively elongate the meromycolate chain of mycolic acids in Mycobacterium tuberculosis (Mtb). Mutations in compound 1-resistant Mtb mutants mapped to HadC (Rv0637; K157R), while chemoproteomics confirmed the compound's binding to HadA (Rv0635), HadB (Rv0636), and HadC. The compounds effectively inhibited the HadAB and HadBC enzyme activities and affected mycolic acid biosynthesis in Mtb, in a concentration-dependent manner. Unlike known 3-hydroxyl-ACP dehydratase complex inhibitors of clinical significance, isoxyl and thioacetazone, 1,3-diarylpyrazolyl-acylsulfonamides did not require activation by EthA and thus are not liable to EthA-mediated resistance. Further, the crystal structure of a key compound in a complex with Mtb HadAB revealed unique binding interactions within the active site of HadAB, providing a useful tool for further structure-based optimization of the series.

Preclinical Evaluation of Inhalational Spectinamide-1599 Therapy against Tuberculosis.

The lengthy treatment time for tuberculosis (TB) is a primary cause for the emergence of multidrug resistant tuberculosis (MDR-TB). One approach to improve TB therapy is to develop an inhalational TB therapy that when administered in combination with oral TB drugs eases and shortens treatment. Spectinamides are new semisynthetic analogues of spectinomycin with excellent activity against Mycobacterium tuberculosis (Mtb), including MDR and XDR Mtb strains. Spectinamide-1599 was chosen as a promising candidate for development of inhalational therapy. Using the murine TB model and intrapulmonary aerosol delivery of spectinamide-1599, we characterized the pharmacokinetics and efficacy of this therapy in BALB/c and C3HeB/FeJ mice infected with the Mtb Erdman strain. As expected, spectinamide-1599 exhibited dose-dependent exposure in plasma, lungs, and ELF, but exposure ratios between lung and plasma were 12-40 times higher for intrapulmonary compared to intravenous or subcutaneous administration. In chronically infected BALB/c mice, low doses (10 mg/kg) of spectinamide-1599 when administered thrice weekly for two months provide efficacy similar to that of higher doses (50-100 mg/kg) after one month of therapy. In the C3HeB/FeJ TB model, intrapulmonary aerosol delivery of spectinamide-1599 (50 mg/kg) or oral pyrazinamide (150 mg/kg) had limited or no efficacy in monotherapy, but when both drugs were given in combination, a synergistic effect with superior bacterial reduction of >1.8 log10 CFU was observed. Throughout the up to eight-week treatment period, intrapulmonary therapy was well-tolerated without any overt toxicity. Overall, these results strongly support the further development of intrapulmonary spectinamide-1599 as a combination partner for anti-TB therapy.

Model-Based Exposure-Response Assessment for Spectinamide 1810 in a Mouse Model of Tuberculosis.

Despite decades of research, tuberculosis remains a leading cause of death from a single infectious agent. Spectinamides are a promising novel class of antituberculosis agents, and the lead spectinamide 1810 has demonstrated excellent efficacy, safety, and drug-like properties in numerous in vitro and in vivo assessments in mouse models of tuberculosis. In the current dose ranging and dose fractionation study, we used 29 different combinations of dose level and dosing frequency to characterize the exposure-response relationship for spectinamide 1810 in a mouse model of Mycobacterium tuberculosis infection and in healthy animals. The obtained data on 1810 plasma concentrations and counts of CFU in lungs were analyzed using a population pharmacokinetic/pharmacodynamic (PK/PD) approach as well as classical anti-infective PK/PD indices. The analysis results indicate that there was no difference in the PK of 1810 in infected compared to healthy, uninfected animals. The PK/PD index analysis showed that bacterial killing of 1810 in mice was best predicted by the ratio of maximum free drug concentration to MIC (fCmax/MIC) and the ratio of the area under the free concentration-time curve to the MIC (fAUC/MIC) rather than the cumulative percentage of time that the free drug concentration is above the MIC (f%TMIC). A novel PK/PD model with consideration of postantibiotic effect could adequately describe the exposure-response relationship for 1810 and supports the notion that the in vitro observed postantibiotic effect of this spectinamide also translates to the in vivo situation in mice. The obtained results and pharmacometric model for the exposure-response relationship of 1810 provide a rational basis for dose selection in future efficacy studies of this compound against M. tuberculosis.

A novel parameter is better than the AHI to assess nocturnal hypoxaemia and excessive daytime sleepiness in obstructive sleep apnoea.

To evaluate whether the percentage of total sleep time spent with apnoea and hypopnoea duration time (AHT%) is better than the apnoea-hypopnoea index (AHI) for the assessment of nocturnal hypoxaemia and excessive daytime sleepiness (EDS) in patients with obstructive sleep apnoea (OSA). Patients with suspected OSA were enrolled. Polysomnography, Epworth Sleepiness Scale, self-administered surveys and anthropometric measures were performed. The efficiency of AHT% and the AHI was evaluated for nocturnal hypoxaemia and EDS. A total of 160 eligible participants were analysed. The median AHT% in normal, mild, moderate and severe OSA patients was significantly different in the four-group patients with OSA. Spearman rank correlations analysis found that the associations were stronger between AHT% with percentage of total sleep time and O2 saturation of < 90% and minimum nocturnal oxygen saturation than these parameters with the AHI. AHT% had a greater area under the curve than the AHI for predicting EDS in patients with OSA. AHT% was significantly higher in the EDS group. We present a novel parameter, AHT%, to evaluate nocturnal hypoxaemia and EDS in OSA patients. AHT% partially compensates for the shortcomings of the AHI. AHT% is better than the AHI for assessing nocturnal hypoxaemia and EDS. AHT% reflects different clinical characteristics associated with OSA from a new perspective.

Synthesis, antibacterial action, and ribosome inhibition of deoxyspectinomycins.

Spectinomycin, an aminocyclitol antibiotic, is subject to inactivation by aminoglycoside modifying enzymes (AMEs) through adenylylation or phosphorylation of the 6-hydroxy group position. In this study, the effects of deoxygenation of the 2- and 6-hydroxy group positions on the spectinomycin actinamine ring are probed to evaluate their relationship to ribosomal binding and the antimicrobial activities of spectinomycin, semisynthetic aminomethyl spectinomycins (amSPCs), and spectinamides. To generate these analogs, an improved synthesis of 6-deoxyspectinomycin was developed using the Barton deoxygenation reaction. 6-Dehydrospectinamide was also synthesized from spectinamide 4 to evaluate the H-bond acceptor character on the C-6 position. All the synthesized analogs were tested for antibacterial activity against a panel of Gram (+) and Gram (-) pathogens, plus Mycobacterium tuberculosis. The molecular contribution of the 2- and 6-hydroxy group and the aryl functionalities of all analogs were examined by measuring inhibition of ribosomal translation and molecular dynamics experiments with MM/GBSA analysis. The results of this work indicate that the 6-hydroxy group, which is the primary target of AMEs, is a required motif for antimicrobial activity in current analogs. Removal of the 6-hydroxy group could be partially rescued by offsetting ribosomal binding contributions made by the aryl side chains found in the spectinamide and amSPCs. This study builds on the knowledge of the structure-activity relationships of spectinomycin analogs and is being used to aid the design of next-generation spectinomycins.

The Discovery and Development of Thienopyrimidines as Inhibitors of Helicobacter pylori That Act through Inhibition of the Respiratory Complex I.

The successful treatment of Helicobacter pylori infections is becoming increasingly difficult due to the rise of resistance against current broad spectrum triple therapy regimens. In the search for narrow-spectrum agents against H. pylori, a high-throughput screen identified two structurally related thienopyrimidine compounds that selectively inhibited H. pylori over commensal members of the gut microbiota. To develop the structure-activity relationship (SAR) of the thienopyrimidines against H. pylori, this study employed four series of modifications in which systematic substitution to the thienopyrimidine core was explored and ultimately side-chain elements optimized from the two original hits were merged into lead compounds. During the development of this series, the mode of action studies identified H. pylori's respiratory complex I subunit NuoD as the target for lead thienopyrimidines. As this enzyme complex is uniquely essential for ATP synthesis in H. pylori, a homology model of the H. pylori NuoB-NuoD binding interface was generated to help rationalize the SAR and guide further development of the series. From these studies, lead compounds emerged with increased potency against H. pylori, improved safety indices, and a good overall pharmacokinetic profile with the exception of high protein binding and poor solubility. Although lead compounds in the series demonstrated efficacy in an ex vivo infection model, the compounds had no efficacy in a mouse model of H. pylori infection. Additional optimization of pharmacological properties of the series to increase solubility and free-drug levels at the sequestered sites of H. pylori infection would potentially result in a gain of in vivo efficacy. The thienopyrimidine series developed in this study demonstrates that NuoB-NuoD of the respiratory complex I can be targeted for development of novel narrow spectrum agents against H. pylori and that thienopyrimines can serve as the basis for future advancement of these studies.

Lack of Specificity of Phenotypic Screens for Inhibitors of the Mycobacterium tuberculosis FAS-II System.

Phenotypic screening of inhibitors of the essential Mycobacterium tuberculosis FAS-II dehydratase HadAB led to the identification of GSK3011724A, a compound previously reported to inhibit the condensation step of FAS-II. Whole-cell-based and cell-free assays confirmed the lack of activity of GSK3011724A against the dehydratase despite evidence of cross-resistance between GSK3011724A and HadAB inhibitors. The nature of the resistance mechanisms is suggestive of alterations in the FAS-II interactome reducing access of GSK3011724A to KasA.

Development of BODIPY FL Thalidomide As a High-Affinity Fluorescent Probe for Cereblon in a Time-Resolved Fluorescence Resonance Energy Transfer Assay.

Ligands for cereblon, a component of a functional E3 ligase complex that targets proteins for proteolysis, are critical for developing molecular glues and proteolysis-targeting chimeras (PROTACs), which have therapeutic implications for various diseases. However, the lack of sensitivity of previously reported assays limits characterization of cereblon ligands. To address this shortcoming, we developed BODIPY FL thalidomide (10) as a high-affinity fluorescent probe for the human cereblon protein, with a Kd value of 3.6 nM. We then used BODIPY FL thalidomide (10) to develop a cereblon time-resolved fluorescence resonance energy transfer (TR-FRET) binding assay. The IC50 values of the cereblon ligand pomalidomide (8) were 6.4 nM in our cereblon TR-FRET binding assay, 264.8 nM in a previously reported Cy5-conjugated thalidomide (7)-mediated fluorescence polarization (FP) assay, and 1.2 µM in a previously reported Cy5-conjugated cereblon modulator (compound 7) (9)-mediated TR-FRET assay, indicating that our cereblon TR-FRET binding assay is 41- and 187-fold more sensitive than these two previously published assays. With our cereblon TR-FRET binding assay, we detected binding of cereblon ligands but not binding of bromodomain-containing protein 4 or von Hippel-Lindau ligands, thereby demonstrating its selectivity. Our cereblon TR-FRET binding assay was very stable and detected changes in phthalimide activity due to thalidomide isomerization. Therefore, the BODIPY FL thalidomide (10)-mediated cereblon TR-FRET binding assay we designed is highly sensitive, selective, and stable and will aid the development and characterization of novel cereblon ligands.

Synthesis and antiproliferative activity of sulfonamide-based peptidomimetic calpain inhibitors.

The calpains are a conserved family of cysteine proteases that includes several isoforms of which µ-calpain and m-calpain are the most widely distributed in mammalian cells. Calpains have been implicated in normal physiological processes as well as cellular abnormalities such as neurodegenerative disorders, cataract, and cancer. Therefore, calpain inhibitors are of interest as potential therapeutic agents. We have synthesized four new sulfonamide-based peptidomimetic compounds 2-5 as inhibitors of µ-calpain that incorporate (E)-1-(phenyl)-2-phenyldiazene and (E)-1-(phenyl)-2-phenylethene functionalities as the N-terminal capping groups of the inhibitors. Compound 5 with Ki value of 9 nM versus µ-calpain was the most potent member of the group. The compounds were predicted to be more lipophilic compared to MDL28170 based on CLogP estimation. They displayed moderate to good antiproliferative activity versus melanoma cell lines (A-375 and B-16F1) and PC-3 prostate cancer cells in vitro. Additionally, one member of the group (compound 3) inhibited DU-145 cell invasion by 80% at 2 µM concentration in the Matrigel cell invasion assay.