Discovery of isoquinolinone and naphthyridinone-based inhibitors of poly(ADP-ribose) polymerase-1 (PARP1) as anticancer agents: Structure activity relationship and preclinical characterization

The exploitation of GLU988 and LYS903 residues in PARP1 as targets to design isoquinolinone (I & II) and naphthyridinone (III) analogues is described. Compounds of structure I have good biochemical and cellular potency but suffered from inferior PK. Constraining the linear propylene linker of structure I into a cyclopentene ring (II) offered improved PK parameters, while maintaining potency for PARP1. Finally, to avoid potential issues that may arise from the presence of an anilinic moiety, the nitrogen substituent on the isoquinolinone ring was incorporated as part of the bicyclic ring. This afforded a naphthyridinone scaffold, as shown in structure III. Further optimization of naphthyridinone series led to identification of a novel and highly potent PARP1 inhibitor 34, which was further characterized as preclinical candidate molecule. Compound 34 is orally bioavailable and displayed favorable pharmacokinetic (PK) properties. Compound 34 demonstrated remarkable antitumor efficacy both as a single-agent as well as in combination with chemotherapeutic agents in the BRCA1 mutant MDA-MB-436 breast cancer xenograft model. Additionally, compound 34 also potentiated the effect of agents such as temozolomide in breast cancer, pancreatic cancer and Ewing’s sarcoma models.     

A novel potent metal-binding NDM-1 inhibitor was identified by fragment virtual,SPR and NMR screening

NDM-1 can hydrolyze nearly all available β-lactam antibiotics, including carbapenems. NDM-1 producing bacterial strains are worldwide threats. It is still very challenging to find a potent NDM-1 inhibitor for clinical use. In our study, we used a metal-binding pharmacophore (MBP) enriched virtual fragment library to screen NDM-1 hits. SPR screening helped to verify the MBP virtual hits and identified a new NDM-1 binder and weak inhibitor A1. A solution NMR study of ¹⁵N-labeled NDM-1 showed that A1 disturbed all three residues coordinating the second zinc ion (Zn2) in the active pocket of NDM-1. The perturbation only happened in the presence of zinc ion, indicating that A1 bound to Zn2. Based on the scaffold of A1, we designed and synthesized a series of NDM-1 inhibitors. Several compounds showed synergistic antibacterial activity with meropenem against NDM-1 producing K. pneumoniae.     

Changing water use and adaptive strategies along rainfall gradients in Mediterranean lupins

• There is growing interest in harnessing the genetic and adaptive diversity of crop wild relatives to improve drought resilience of elite cultivars. Rainfall gradients exert strong selection pressure on both natural and agricultural ecosystems. Understanding plant responses to these facilitates crop improvement.
• Wild and domesticated narrow‐leafed lupin (NLL) collected along Mediterranean terminal drought stress gradients was evaluated under contrasting reproductive phase water supply in controlled field, glasshouse and cabinet studies. Plant phenology, growth and productivity, water use and stress responses were measured over time.
• There is an integrated suite of adaptive changes along rainfall gradients in NLL. Low rainfall ecotypes flower earlier, accumulate lower seed numbers, biomass and leaf area, and have larger root:shoot ratios than high rainfall ecotypes. Water‐use is lower and stress onset slower in low compared to high rainfall ecotypes. Water‐use rates and ecotypic differences in stress response (Ψleaf decline, leaf loss) are lower in NLL than yellow lupin (YL). To mitigate the effects of profligate water use, high rainfall YL ecotypes maintain higher leaf water content over declining leaf water potential than low rainfall ecotypes. There is no evidence for such specific adaptation in NLL.
• The data suggests that appropriate phenology is the key adaptive trait to rainfall gradients in NLL because of the flow‐on effects on biomass production, fitness, transpiration and stress onset, and the lack of physiological adaptations as in YL. Accordingly, it is essential to match phenology with target environment in order to minimize risk and maximize yield potential.

     

Downstream separation and purification of bio-based alpha-ketoglutaric acid from post-fermentation broth using a multi-stage membrane process

In this study, a multi-stage membrane process, assisted by vacuum evaporation and crystallization, for recovery of bio-based alpha-ketoglutaric acid from the actual post-fermentation broth was designed and investigated. In the first part of this study, pre-treatment of crude fermentation broth (centrifugation-ultrafiltration-nanofiltration) was carried out to remove biomass, proteins, sugars, part of inorganic ions and color compounds. The commercial ceramic UF membrane (15 kDa) and nanofiltration ceramic membrane (200 Da or 450 Da) were applied. Electrodialysis with a bipolar membrane was proposed for separation of ionic compounds and simultaneous electro-acidification to the acid form. During bipolar membrane electrodialysis carried out under acidic conditions, it was possible to remove close to 90 % of alpha-ketoglutaric acid. Moreover, the migration of other acids present in the fermentation broth (lactic and acetic) was significantly limited. The calculated specific energy consumption was low and equal to 0.6 kW h/kg. The final purification using crystallization assisted vacuum evaporation allowed obtaining alpha-ketoglutaric acid in solid form. Analysis of the final product showed that the proposed method of alpha-ketoglutaric acid recovery gives the acid of high purity equal to 94.8 %. Furthermore, the presented results have practical relevance and may in future be the basis for the development of separation technologies of alpha-ketoglutaric acid from the fermentation broth on industrial scale.     

Adapter Protein Shc Regulates Janus Kinase 3 Phosphorylation

Although constitutive activation of Janus kinase 3 (Jak3) leads to different cancers, the mechanism of trans-molecular regulation of Jak3 activation is not known. Previously we reported that Jak3 interactions with adapter protein p52ShcA (Shc) facilitate mucosal homeostasis. In this study, we characterize the structural determinants that regulate the interactions between Jak3 and Shc and demonstrate the trans-molecular mechanism of regulation of Jak3 activation by Shc. We show that Jak3 autophosphorylation was the rate-limiting step during Jak3 trans-phosphorylation of Shc where Jak3 directly phosphorylated two tyrosine residues in Src homology 2 (SH2) domain and one tyrosine residue each in calponin homology 1 (CH1) domain and phosphotyrosine interaction domain (PID) of Shc. Direct interactions between mutants of Jak3 and Shc showed that although FERM domain of Jak3 was sufficient for binding to Shc, CH1 and PID domains of Shc were responsible for binding to Jak3. Functionally Jak3 was autophosphorylated under IL-2 stimulation in epithelial cells. However, Shc recruited tyrosine phosphatases SHP2 and PTP1B to Jak3 and thereby dephosphorylated Jak3. Thus we not only characterize Jak3 interaction with Shc, but also demonstrate the molecular mechanism of intracellular regulation of Jak3 activation where Jak3 interactions with Shc acted as regulators of Jak3 dephosphorylation through direct interactions of Shc with both Jak3 and tyrosine phosphatases.     

TMTC1 and TMTC2 Are Novel Endoplasmic Reticulum Tetratricopeptide Repeat-containing Adapter Proteins Involved in Calcium Homeostasis

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.     

Shear Stress-induced Redistribution of Vascular Endothelial-Protein-tyrosine Phosphatase (VE-PTP) in Endothelial Cells and Its Role in Cell Elongation

Vascular endothelial cells (ECs) are continuously exposed to shear stress (SS) generated by blood flow. Such stress plays a key role in regulation of various aspects of EC function including cell proliferation and motility as well as changes in cell morphology. Vascular endothelial-protein-tyrosine phosphatase (VE-PTP) is an R3-subtype PTP that possesses multiple fibronectin type III-like domains in its extracellular region and is expressed specifically in ECs. The role of VE-PTP in EC responses to SS has remained unknown, however. Here we show that VE-PTP is diffusely localized in ECs maintained under static culture conditions, whereas it undergoes rapid accumulation at the downstream edge of the cells relative to the direction of flow in response to SS. This redistribution of VE-PTP triggered by SS was found to require its extracellular and transmembrane regions and was promoted by integrin engagement of extracellular matrix ligands. Inhibition of actin polymerization or of Cdc42, Rab5, or Arf6 activities attenuated the SS-induced redistribution of VE-PTP. VE-PTP also underwent endocytosis in the static and SS conditions. SS induced the polarized distribution of internalized VE-PTP. Such an effect was promoted by integrin engagement of fibronectin but prevented by inhibition of Cdc42 activity or of actin polymerization. In addition, depletion of VE-PTP by RNA interference in human umbilical vein ECs blocked cell elongation in the direction of flow induced by SS. Our results suggest that the polarized redistribution of VE-PTP in response to SS plays an important role in the regulation of EC function by blood flow.     

Hydroxysafflor yellow A (HYSA) inhibited the proliferation and differentiation of 3T3-L1 preadipocytes

Hydroxysafflor yellow A (HSYA), a main component of safflor yellow, has been demonstrated to prevent steroid-induced avascular necrosis of femoral head by inhibiting primary bone marrow-derived mesenchymal stromal cells adipogenic differentiation induced by steroid. In this study, we investigate the effect of HSYA on the proliferation and adipogenesis of mouse 3T3-L1 preadipocytes. The effects of HSYA on proliferation and differentiation of 3T3-L1 cells and its possible mechanism were studied by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide spectrophotometry, Oil Red O staining, intracellular triglyceride assays, real-time quantitative RT-PCR, transient transfection and dual luciferase reporter gene methods. HSYA inhibited the proliferation of 3T3-L1 preadipocytes and cell viability greatly decreased in a dose and time dependent manner. HSYA (1 mg/l) notably reduced the amount of intracellular lipid and triglyceride content in adipocytes by 21.3 % (2.13 ± 0.36 vs 2.71 ± 0.40, P < 0.01) and 22.6 % (1.33 ± 0.07 vs 1.72 ± 0.07, P < 0.01) on days 8 following the differentiation, respectively. HSYA (1 mg/l) significantly increased hormone-sensitive lipase (HSL) mRNA expression and promoter activities by 2.4- and 1.55-fold, respectively (P < 0.01), in differentiated 3T3-L1 adipocytes. HSYA inhibits the proliferation and adipogenesis of 3T3-L1 preadipocytes. The inhibitory action of HYSA on adipogenesis may be due to the promotion of lipolytic-specific enzyme HSL expression by increasing HSL promoter activity.     

Ascorbic acid prolongs the viability and stability of isolated perfused lungs: A mechanistic study using 31P and hyperpolarized 13C nuclear magnetic resonance

Ex vivo lung perfusion (EVLP) has recently shown promise as a means of more accurately gauging the health of lung grafts and improving graft performance post-transplant. However, reperfusion of ischemic lung promotes the depletion of high-energy compounds and a progressive loss of normal mitochondrial function, and it remains unclear how and to what extent the EVLP approach contributes to this metabolic decline. Although ascorbate has been used to mitigate the effects of ischemia–reperfusion injury, the nature of its effects during EVLP are also not clear. To address these uncertainties, this study monitored the energy status of lungs during EVLP and after the administration of ascorbate using ³¹P and hyperpolarized ¹³C NMR (nuclear magnetic resonance). Our experiments demonstrated that the oxidative phosphorylation capacity and pyruvate dehydrogenase flux of lungs decline during ex vivo perfusion. The addition of ascorbate to the perfusate prolonged lung viability by 80% and increased the hyperpolarized ¹³C bicarbonate signal by a factor of 2.7. The effect of ascorbate is apparently due not to its antioxidant quality but rather to its ability to energize cellular respiration given that it increased the lung’s energy charge significantly, whereas other antioxidants (glutathione and α-lipoic acid) did not alter energy metabolism. During ascorbate administration, inhibition of mitochondrial complex I with rotenone depressed energy charge and shifted the metabolic state of the lung toward glycolysis; reenergizing the electron transport chain with TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) recovered metabolic activity. This indicates that ascorbate slows the decline of the ex vivo perfused lung’s mitochondrial activity through an independent interaction with the electron transport chain complexes.