Synthesis and Characterization of Novel Quinazoline Type Inhibitors for Mutant and Wild-Type EGFR and RICK Kinases

The development of selective protein kinase inhibitors has become an important area of drug discovery for the treatment of different diseases. We report the synthesis and characterization of a series of novel quinazoline derivatives against three therapeutically important and pharmacologically related kinases: 1) epidermal growth factor receptor (EGFR; wild type and mutant) in the field of cancer, 2) receptor-interacting caspase-like apoptosis-regulatory kinase (RICK) in the field of inflammation, and 3) pUL97 of human cytomegalovirus (HCMV). For reference purpose we have synthesized the four clinically relevant quinazolines, including the lead compounds, which we previously identified for RICK and pUL97. A total of 52 quinazoline derivatives were synthesized and tested on the basis of these leads to specifically target the hydrophobic pocket of the ATP-binding site. Selected compounds were tested on wild-type and mutant forms of EGFR, RICK, and pUL97 kinases; their logP and logS values for assessing suitability as drugs were calculated and hit or lead compounds identified.     

Optimization of physical factors affecting the production of thermo-stable organic solvent-tolerant protease from a newly isolated halo tolerant Bacillus subtilis strain Rand

Background  

Many researchers have reported on the optimization of protease production; nevertheless, only a few have reported on the optimization of the production of organic solvent-tolerant proteases. Ironically, none has reported on thermostable organic solvent-tolerant protease to date. The aim of this study was to isolate the thermostable organic solvent-tolerant protease and identify the culture conditions which support its production. The bacteria of genus Bacillus are active producers of extra-cellular proteases, and the thermostability of enzyme production by Bacillus species has been well-studied by a number of researchers. In the present study, the Bacillus subtilis strain Rand was isolated from the contaminated soil found in Port Dickson, Malaysia.     

Kininase I-type carboxypeptidases enhance nitric oxide production in endothelial cells by generating bradykinin B1 receptor agonists

Kininase I-type carboxypeptidases convert native kinin agonists for B(2) receptors into B(1) receptor agonists by specifically removing the COOH-terminal Arg residue. The membrane localization of carboxypeptidase M (CPM) and carboxypeptidase D (CPD) make them ideally situated to regulate kinin activity. Nitric oxide (NO) release from human lung microvascular endothelial cells (HLMVEC) was measured directly in real time with a porphyrinic microsensor. Bradykinin (1-100 nM) elicited a transient (5 min) peak of generation of NO that was blocked by the B(2) antagonist HOE 140, whereas B(1) agonist des-Arg(10)-kallidin caused a small linear increase in NO over 20 min. Treatment of HLMVEC with 5 ng/ml interleukin-1beta and 200 U/ml interferon-gamma for 16 h upregulated B(1) receptors as shown by an approximately fourfold increase in prolonged (>20 min) output of NO in response to des-Arg(10)-kallidin, which was blocked by the B(1) antagonist des-Arg(10)-Leu(9)-kallidin. B(2) receptor agonists bradykinin or kallidin also generated prolonged NO production in treated HLMVEC, which was significantly reduced by either a B(1) antagonist or carboxypeptidase inhibitor, and completely abolished with a combination of B(1) and B(2) receptor antagonists. Furthermore, CPM and CPD activities were increased about twofold in membrane fractions of HLMVEC treated with interleukin-1beta and interferon-gamma compared with control cells. Immunostaining localized CPD primarily in a perinuclear/Golgi region, whereas CPM was on the cell membrane. These data show that cellular kininase I-type carboxypeptidases can enhance kinin signaling and NO production by converting B(2) agonists to B(1) agonists, especially in inflammatory conditions.     

<i>Trichoderma</i> spp. fostering growth on <i>Capsicum chinense</i> Jacq. seedlings and antagonistic against <i>Meloidogyne incognita</i>

Fourteen native strains of Trichoderma spp. from wildand agricultural pathosystems in the state of Yucatan, Mexico, with growth-promoting ability of Capsicum chinense Jacq. seedlings were evaluated and antagonistic effect of their filtrate against second-stage juveniles (J2) of Meloidogyne incognita. The strains Th05-02 and Th27-08 showed the best significant effects on plant hight variable increments 55.57 and 47.62%, theTh07-04 with 29.48% more root length, theTh02-01 and Th07-04 isolates increased from 48.71 to 84.61% in volume radical and 53.40% of total dry biomass. Statistical analysis (p≤0.001) of Th43 and Th43-13-14 filtrates caused 100% mortality at 24 and 48h. In the test of reversibility to 24 h after replacing the filtrates Th43-13, Th43-14, TH09-06 and TH20-07 by sterile distilled water, the J2 did not recover their viability, so they were considered as the best potential strains of Trichoderma spp. with antagonistic capacity in J2 of M.incognita.     

Replacement of the transmembrane anchor in angiotensin I-converting enzyme (ACE) with a glycosylphosphatidylinositol tail affects activation of the B2 bradykinin receptor by ACE inhibitors

To investigate further the relationship of angiotensin I-converting enzyme (ACE) inhibitors to activation of the B(2) bradykinin (BK) receptor, we transfected Chinese hamster ovary cells to stably express the human receptor and either wild-type ACE (WT-ACE), an ACE construct with most of the cytosolic portion deleted (Cyt-del-ACE), or ACE with a glycosylphosphatidylinositol (GPI) anchor replacing the transmembrane and cytosolic domains (GPI-ACE). BK or its ACE-resistant analogue were the agonists. All activities (arachidonic acid release and calcium mobilization) were blocked by the B(2) antagonist HOE 140. B(2) was desensitized by repeated administration of BK but resensitized to agonist by ACE inhibitors in the cells expressing both B(2) and either WT-ACE or Cyt-del-ACE. In GPI-ACE expressing cells, the B(2) receptor was still activated by the agonists, but ACE inhibitors did not resensitize. Pretreatment with filipin returned the sensitivity to inhibitors. In immunocytochemistry, GPI-ACE showed patchy, uneven distribution on the plasma membrane that was restored by filipin. Thus, ACE inhibitors were inactive as long as GPI-ACE was sequestered in cholesterol-rich membrane domains. WT-ACE and B(2) receptor in Chinese hamster ovary cells co-immunoprecipitated with antibody to receptor, suggesting an interaction on the cell membrane. ACE inhibitors augment BK effects on receptors indirectly only when enzyme and receptor molecules are sterically close, possibly forming a heterodimer.     

Crystal structure of the human carboxypeptidase N (kininase I) catalytic domain

Human carboxypeptidase N (CPN), a member of the CPN/E subfamily of "regulatory" metallo-carboxypeptidases, is an extracellular glycoprotein synthesized in the liver and secreted into the blood, where it controls the activity of vasoactive peptide hormones, growth factors and cytokines by specifically removing C-terminal basic residues. Normally, CPN circulates in blood plasma as a hetero-tetramer consisting of two 83 kDa (CPN2) domains each flanked by a 48 to 55 kDa catalytic (CPN1) domain. We have prepared and crystallized the recombinant C-terminally truncated catalytic domain of human CPN1, and have determined and refined its 2.1 A crystal structure. The structural analysis reveals that CPN1 has a pear-like shape, consisting of a 319 residue N-terminal catalytic domain and an abutting, cylindrically shaped 79 residue C-terminal beta-sandwich transthyretin (TT) domain, more resembling CPD-2 than CPM. Like these other CPN/E members, two surface loops surrounding the active-site groove restrict access to the catalytic center, offering an explanation for why some larger protein carboxypeptidase inhibitors do not inhibit CPN. Modeling of the Pro-Phe-Arg C-terminal end of the natural substrate bradykinin into the active site shows that the S1' pocket of CPN1 might better accommodate P1'-Lys than Arg residues, in agreement with CPN's preference for cleaving off C-terminal Lys residues. Three Thr residues at the distal TT edge of CPN1 are O-linked to N-acetyl glucosamine sugars; equivalent sites in the membrane-anchored CPM are occupied by basic residues probably involved in membrane interaction. In tetrameric CPN, each CPN1 subunit might interact with the central leucine-rich repeat tandem of the cognate CPN2 subunit via a unique hydrophobic surface patch wrapping around the catalytic domain-TT interface, exposing the two active centers.     

Carboxypeptidase M in Brain and Peripheral Nerves

Carboxypeptidase M (CPM), a plasma membrane-bound enzyme, cleaves C-terminal basic amino acids with a neutral pH optimum. We studied its distribution in human, baboon, and dog brain and in dog peripheral nerves. Areas were dissected, homogenized, centrifuged, and assayed for activity with dansyl-Ala-Arg. The corpus callosum and the pyramidal and optic tract were especially rich in CPM, whereas basal ganglia and cortex had low activity. The identity of the basic carboxypeptidase activity with CPM was shown by similarities in subcellular localization, membrane attachment, substrate hydrolysis, inhibition by a specific basic carboxypeptidase inhibitor, and cross-reaction with anti-human CPM antiserum. This antiserum immunoprecipitated an average of 85% of the activity in human and baboon brain and approximately 66% in dog brain. CPM co-purified with myelin extracted from the brain. Consistent with results obtained in placenta and cultured kidney cells, CPM in the brain appears to be membrane-bound via a phosphatidylinositol glycan anchor. In the peripheral nerves, the specific activity in dog sciatic nerve and in vagus was high (98 and 149 nmol/h/mg of protein, respectively). In immunohistochemical studies, glia in the brain, which appear to be oligodendrocytes or astrocytes, and the outer aspects of myelin sheaths and Schwann cells in sciatic and vagus nerves were stained. We conclude that in some areas of the CNS and the PNS, CPM is closely associated with myelin and myelin-forming cells. Northern blot analysis revealed the presence of mRNA coding for CPM in the brain, showing that the enzyme is indeed synthesized there.     

A basal sphenodontian (Lepidosauria) from the Jurassic of Patagonia: new insights on the phylogeny and biogeography of Gondwanan rhynchocephalians

Herein we describe a new rhynchocephalian taxon from the Middle Jurassic of Patagonia, Argentina, representing the first Jurassic record of the group in South America. The new taxon, consisting of a complete dentary, is ascribed to Sphenodontia based on the presence of a deep and wide Meckelian groove, long posterior process, well‐developed coronoid process, and acrodont teeth showing dental regionalization including successional, alternate hatchling, and additional series. This allocation is reinforced by a phylogenetic analysis that places the new taxon in a basal position within a clade of sphenodontians that excludes Diphydontosaurus and Planocephalosaurus. Additionally, the new taxon clusters within a Gondwanan clade with the Indian Godavarisaurus from the Jurassic Kota Formation, sharing the presence of recurved and relatively large posterior successional teeth that are ribbed and bear a peculiar anterolingual groove. This sister‐group relationship is intriguing from a palaeobiogeographical viewpoint, as it suggests some degree of endemism during the initial stages of the breakup of Pangaea. We also discuss the ontogenetic stage of the new taxon and provide insights on the evolution of successional dentition in rhynchocephalians. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166 , 342–360.