Optimization of Tetrahydroindazoles as Inhibitors of Human Dihydroorotate Dehydrogenase and Evaluation of Their Activity and In Vitro Metabolic Stability.

Human dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo pyrimidine synthesis pathway, is a target for the treatment of rheumatoid arthritis and multiple sclerosis and is re-emerging as an attractive target for cancer therapy. Here we describe the optimization of recently identified tetrahydroindazoles (HZ) as DHODH inhibitors. Several of the HZ analogues synthesized in this study are highly potent inhibitors of DHODH in an enzymatic assay, while also inhibiting cancer cell growth and viability and activating p53-dependent transcription factor activity in a reporter cell assay. Furthermore, we demonstrate the specificity of the compounds toward the de novo pyrimidine synthesis pathway through supplementation with an excess of uridine. We also show that induction of the DNA damage marker γ-H2AX after DHODH inhibition is preventable by cotreatment with the pan-caspase inhibitor Z-VAD-FMK. Additional solubility and in vitro metabolic stability profiling revealed compound 51 as a favorable candidate for preclinical efficacy studies.

The quantification of antibody elements and receptors subunit expression using qPCR: The design of VH, VL, CH, CL, FcR subunits primers for a more holistic view of the immune system.

The expression levels of immunoglobulin elements and their receptors are important markers for health and disease. Within the immunoglobulin locus, the constant regions and the variable region families are associated with certain pathologies, yet a holistic view of the interaction between the expressions of the multiple genes remain to be fully characterized. There is thus an important need to quantify antibody elements, their receptors and the receptor subunits in blood (PBMC cDNA) for both screening and detailed studies of such associations. Leveraging on qPCR, we designed primers for all Vκ1-6, VH1-7, Vλ1-11, nine CH isotypes, Cκ, Cκ, Cλ1 &3, FcεRI α,ß, and γ subunits, all three FcγR and their subunits, and FcαR. Validating this on a volunteer PBMC cDNA, we report a qPCR primer set repertoire that can quantify the relative expression of all the above genes to the GAPDH housekeeping gene, with implications and uses in both clinical monitoring and research.

Simulations of mutant p53 DNA binding domains reveal a novel druggable pocket.

The DNA binding domain (DBD) of the tumor suppressor p53 is the site of several oncogenic mutations. A subset of these mutations lowers the unfolding temperature of the DBD. Unfolding leads to the exposure of a hydrophobic ß-strand and nucleates aggregation which results in pathologies through loss of function and dominant negative/gain of function effects. Inspired by the hypothesis that structural changes that are associated with events initiating unfolding in DBD are likely to present opportunities for inhibition, we investigate the dynamics of the wild type (WT) and some aggregating mutants through extensive all atom explicit solvent MD simulations. Simulations reveal differential conformational sampling between the WT and the mutants of a turn region (S6-S7) that is contiguous to a known aggregation-prone region (APR). The conformational properties of the S6-S7 turn appear to be modulated by a network of interacting residues. We speculate that changes that take place in this network as a result of the mutational stress result in the events that destabilize the DBD and initiate unfolding. These perturbations also result in the emergence of a novel pocket that appears to have druggable characteristics. FDA approved drugs are computationally screened against this pocket.

The Standardized Letter of Evaluation for Postgraduate Training: A Concept Whose Time Has Come?

A medical student's letter of recommendation for postgraduate training applications should provide a fair and accurate assessment of academic and clinical performance, as well as define character attributes pertinent to the practice of medicine. Since its inception in 1997, the emergency medicine (EM) standardized letter of evaluation (SLOE) has evolved into an instrument that provides just such an assessment. Concise, standardized, and discriminating in its assessment of performance relevant to the practice of EM, the SLOE is judged by program directors in EM as the most valuable component of a potential resident's application. Other specialties would benefit from such a specialty-specific perspective, which is currently lacking in most Electronic Residency Application Service application materials. Creation of specialty-specific SLOEs which define performance metrics or competencies and noncognitive personality traits critical to each unique specialty would add substantially to the holistic review of our graduating students. As a result, specialty-specific SLOEs would increase the likelihood that programs could effectively identify applicants who would not only be a "good fit" for their programs but also graduate to become successful physicians.

Effectiveness of combined acupuncture and manual therapy relative to no treatment for canine musculoskeletal pain.

Despite the rise in popularity of both acupuncture and manual therapy in veterinary medicine, and the increasing number of Canadian veterinarians practising these techniques, there is little research demonstrating their effectiveness. In this repeated measures, therapeutic trial, 47 client-owned dogs with naturally occurring lameness were assessed for clinical response to treatment. Owners were blinded to the treatment schedule and completed questionnaires to assess their dogs' comfort and mobility. Comparison between pre- and post-treatment results demonstrated that combined acupuncture and manual therapy provides immediate short-term improvement in comfort and mobility, as demonstrated by owner observed changes in play behavior (P = 0.015), walking (P < 0.001), trotting (P = 0.002), jumping (P < 0.001), descending stairs (P = 0.003), rising from a lying position (P < 0.001), and reduced stiffness after rest (P < 0.001) or following exercise (P < 0.001). Mood and attitude also improved, but did not attain statistical significance.

Efficacité de l'acupuncture et de la thérapie manuelle combinées par rapport à l'absence de traitement pour la douleur musculo-squelettique canine. Malgré la croissance de la popularité de l'acupuncture et de la thérapie manuelle en médecine vétérinaire et le nombre grandissant de vétérinaires canadiens qui pratiquent ces techniques, il existe peu de recherche démontrant leur efficacité. Dans cet essai thérapeutique à mesures répétées, 47 chiens appartenant à des clients atteints de boiterie naturelle ont été évalués pour leur réponse clinique au traitement. Les propriétaires n'ont pas été informés du calendrier de traitement et ils ont rempli des questionnaires afin d'évaluer le confort et la mobilité de leurs chiens. La comparaison entre les résultats avant et après le traitement ont démontré que l'acupuncture et la thérapie manuelle combinées offraient une amélioration immédiate à court terme pour le confort et la mobilité, comme le démontrent les observations faites par les propriétaires pour le comportement de jeu (P = 0,015), la marche (P < 0,001), le galop (P = 0,002), le saut (P < 0,001), la descente d'escaliers (P= 0,003), le lever d'une position couchée (P < 0,001) et une raideur réduite après le repos (P < 0,001) ou après de l'exercice (P < 0,001). L'humeur et l'attitude se sont aussi améliorées, mais sans atteindre une importance statistique.(Traduit par Isabelle Vallières).

Protective effects of non-anticoagulant activated protein C variant (D36A/L38D/A39V) in a murine model of ischaemic stroke.

Ischaemic stroke is caused by occlusive thrombi in the cerebral vasculature. Although tissue-plasminogen activator (tPA) can be administered as thrombolytic therapy, it has major limitations, which include disruption of the blood-brain barrier and an increased risk of bleeding. Treatments that prevent or limit such deleterious effects could be of major clinical importance. Activated protein C (APC) is a natural anticoagulant that regulates thrombin generation, but also confers endothelial cytoprotective effects and improved endothelial barrier function mediated through its cell signalling properties. In murine models of stroke, although APC can limit the deleterious effects of tPA due to its cell signalling function, its anticoagulant actions can further elevate the risk of bleeding. Thus, APC variants such as APC(5A), APC(Ca-ins) and APC(36-39) with reduced anticoagulant, but normal signalling function may have therapeutic benefit. Human and murine protein C (5A), (Ca-ins) and (36-39) variants were expressed and characterised. All protein C variants were secreted normally, but 5-20% of the protein C (Ca-ins) variants were secreted as disulphide-linked dimers. Thrombin generation assays suggested reductions in anticoagulant function of 50- to 57-fold for APC(36-39), 22- to 27-fold for APC(Ca-ins) and 14- to 17-fold for APC(5A). Interestingly, whereas human wt APC, APC(36-39) and APC(Ca-ins) were inhibited similarly by protein C inhibitor (t½ - 33 to 39 mins), APC(5A) was inactivated ~9-fold faster (t½ - 4 mins). Using the murine middle cerebral artery occlusion ischaemia/repurfusion injury model, in combination with tPA, APC(36-39), which cannot be enhanced by its cofactor protein S, significantly improved neurological scores, reduced cerebral infarct area by ~50% and reduced oedema ratio. APC(36-39) also significantly reduced bleeding in the brain induced by administration of tPA, whereas wt APC did not. If our data can be extrapolated to clinical settings, then APC(36-39) could represent a feasible adjunctive therapy for ischaemic stroke.

The role of the ADAMTS13 cysteine-rich domain in VWF binding and proteolysis.

ADAMTS13 proteolytically regulates the platelet-tethering function of von Willebrand factor (VWF). ADAMTS13 function is dependent upon multiple exosites that specifically bind the unraveled VWF A2 domain and enable proteolysis. We carried out a comprehensive functional analysis of the ADAMTS13 cysteine-rich (Cys-rich) domain using engineered glycans, sequence swaps, and single point mutations in this domain. Mutagenesis of Cys-rich domain-charged residues had no major effect on ADAMTS13 function, and 5 out of 6 engineered glycans on the Cys-rich domain also had no effect on ADAMTS13 function. However, a glycan attached at position 476 appreciably reduced both VWF binding and proteolysis. Substitution of Cys-rich sequences for the corresponding regions in ADAMTS1 identified a hydrophobic pocket involving residues Gly471-Val474 as being of critical importance for both VWF binding and proteolysis. Substitution of hydrophobic VWF A2 domain residues to serine in a region (residues 1642-1659) previously postulated to interact with the Cys-rich domain revealed the functional importance of VWF residues Ile1642, Trp1644, Ile1649, Leu1650, and Ile1651. Furthermore, the functional deficit of the ADAMTS13 Cys-rich Gly471-Val474 variant was dependent on these same hydrophobic VWF residues, suggesting that these regions form complementary binding sites that directly interact to enhance the efficiency of the proteolytic reaction.

TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain.

Protein S is a cofactor for tissue factor pathway inhibitor (TFPI), accelerating the inhibition of activated factor X (FXa). TFPI Kunitz domain 3 residue Glu226 is essential for enhancement of TFPI by protein S. To investigate the complementary functional interaction site on protein S, we screened 44 protein S point, composite or domain swap variants spanning the whole protein S molecule for their TFPI cofactor function using a thrombin generation assay. Of these variants, two protein S/growth arrest-specific 6 chimeras, with either the whole sex hormone-binding globulin (SHBG)-like domain (Val243-Ser635; chimera III) or the SHBG laminin G-type 1 subunit (Ser283-Val459; chimera I), respectively, substituted by the corresponding domain in growth arrest-specific 6, were unable to enhance TFPI. The importance of the protein S SHBG-like domain (and its laminin G-type 1 subunit) for binding and enhancement of TFPI was confirmed in FXa inhibition assays and using surface plasmon resonance. In addition, protein S bound to C4b binding protein showed greatly reduced enhancement of TFPI-mediated inhibition of FXa compared with free protein S. We show that binding of TFPI to the protein S SHBG-like domain enables TFPI to interact optimally with FXa on a phospholipid membrane.

The fluorescent two-hybrid assay to screen for protein-protein interaction inhibitors in live cells: targeting the interaction of p53 with Mdm2 and Mdm4.

Protein-protein interactions (PPIs) are attractive but challenging targets for drug discovery. To overcome numerous limitations of the currently available cell-based PPI assays, we have recently established a fully reversible microscopy-assisted fluorescent two-hybrid (F2H) assay. The F2H assay offers a fast and straightforward readout: an interaction-dependent co-localization of two distinguishable fluorescent signals at a defined spot in the nucleus of mammalian cells. We developed two reversible F2H assays for the interactions between the tumor suppressor p53 and its negative regulators, Mdm2 and Mdm4. We then performed a pilot F2H screen with a subset of compounds, including small molecules (such as Nutlin-3) and stapled peptides. We identified five cell-penetrating compounds as potent p53-Mdm2 inhibitors. However, none exhibited intracellular activity on p53-Mdm4. Live cell data generated by the F2H assays enable the characterization of stapled peptides based on their ability to penetrate cells and disrupt p53-Mdm2 interaction as well as p53-Mdm4 interaction. Here, we show that the F2H assays enable side-by-side analysis of substances' dual Mdm2-Mdm4 activity. In addition, they are suitable for testing various types of compounds (e.g., small molecules and peptidic inhibitors) and concurrently provide initial data on cellular toxicity. Furthermore, F2H assays readily allow real-time visualization of PPI dynamics in living cells.

Discovery and characterization of 2-anilino-4- (thiazol-5-yl)pyrimidine transcriptional CDK inhibitors as anticancer agents.

The main difficulty in the development of ATP antagonist kinase inhibitors is target specificity, since the ATP-binding motif is present in many proteins. We introduce a strategy that has allowed us to identify compounds from a kinase inhibitor library that block the cyclin-dependent kinases responsible for regulating transcription, i.e., CDK7 and especially CDK9. The screening cascade employs cellular phenotypic assays based on mitotic index and nuclear p53 protein accumulation. This permitted us to classify compounds into transcriptional, cell cycle, and mitotic inhibitor groups. We describe the characterization of the transcriptional inhibitor class in terms of kinase inhibition profile, cellular mode of action, and selectivity for transformed cells. A structural selectivity rationale was used to optimize potency and biopharmaceutical properties and led to the development of a transcriptional inhibitor, 3,4-dimethyl-5-[2-(4-piperazin-1-yl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one, with anticancer activity in animal models.

Semisubmersible oil platforms: understudied and potentially major vectors of biofouling-mediated invasions.

Biofouling has long been recognised as a major pathway for the introduction of non-indigenous species. This study records the decapods and stomatopod crustaceans fouling a semisubmersible oil platform dry docked for hull cleaning in Jurong Port, Singapore. Of the 25 species of decapods identified, 13 were non-indigenous and represent new records to Singapore waters. Of these, the crabs Glabropilumnus seminudus and Carupa tenuipes are known to be invasive in other parts of the world. The stomatopod, Gonodactylaceus randalli, is the first mantis shrimp recorded in a biofouling community. The richness and diversity of this fouling community, consisting of many vagile species, highlights the difference between platforms and ships. With the expansion of maritime oil and gas exploration, the threat posed by an expanded fleet of semisubmersible oil platforms translocating non-indigenous fouling communities across biogeographical boundaries is very serious. Scientists, policy-makers, and stakeholders should turn their attention to this growing problem.

Upland rice and lowland rice exhibited different PIP expression under water deficit and ABA treatment.

Aquaporins play a significant role in plant water relations. To further understand the aquaporin function in plants under water stress, the expression of a subgroup of aquaporins, plasma membrane intrinsic proteins (PIPs), was studied at both the protein and mRNA level in upland rice (Oryza sativa L. cv. Zhonghan 3) and lowland rice (Oryza sativa L. cv. Xiushui 63) when they were water stressed by treatment with 20% polyethylene glycol (PEG). Plants responded differently to 20% PEG treatment. Leaf water content of upland rice leaves was reduced rapidly. PIP protein level increased markedly in roots of both types, but only in leaves of upland rice after 10 h of PEG treatment. At the mRNA level, OsPIP1;2, OsPIP1;3, OsPIP2;1 and OsPIP2;5 in roots as well as OsPIP1;2 and OsPIP1;3 in leaves were significantly up-regulated in upland rice, whereas the corresponding genes remained unchanged or down-regulated in lowland rice. Meanwhile, we observed a significant increase in the endogenous abscisic acid (ABA) level in upland rice but not in lowland rice under water deficit. Treatment with 60 microM ABA enhanced the expression of OsPIP1;2, OsPIP2;5 and OsPIP2;6 in roots and OsPIP1;2, OsPIP2;4 and OsPIP2;6 in leaves of upland rice. The responsiveness of PIP genes to water stress and ABA were different, implying that the regulation of PIP genes involves both ABA-dependent and ABA-independent signaling pathways during water deficit.

Cassette dosing pharmacokinetics of a library of 2,6,9-trisubstituted purine cyclin-dependent kinase 2 inhibitors prepared by parallel synthesis.

Determination of pharmacokinetic properties in the intact animal remains a major bottleneck in drug discovery. Cassette dosing involves administration of a cocktail of drugs to individual animals. Here we describe the cassette dosing properties of a 107-membered library of 2,6,9-trisubstituted purine cyclin-dependent kinase 2 (CDK2) inhibitors. A three-step parallel synthesis approach produced compounds with purity ranging from 63% to 100%. Cassette dosing was validated by comparing the pharmacokinetic parameters obtained following i.v. administration of a mixture of olomoucine, R-roscovitine (CYC202), and bohemine, each at 16.6 mg/kg, with results for administration of single agents at 50 mg/kg. No significant difference was observed between the pharmacokinetic parameters of agents when dosed in combination compared with those of individual compounds. CYC202 showed the highest area under the curve (AUC) and the longest elimination half-life (t(1/2)). Further cassettes evaluated the library of trisubstituted purines with CYC202 and purvalanol A included as pharmacokinetic standards in a validated limited sampling strategy. The ratios of pharmacokinetic parameters to that of CYC202 [AUC, maximum concentration (C(max)), and t(1/2)] remained similar when compounds were tested in two different cassettes or as individual compounds. Following dosing of the same cassette on three different days, there was less than 20% variation in pharmacokinetic parameters between days. The structure-pharmacokinetics relationship showed that the favored purine substituents are benzylamine and veratrylamine at position 6, amino-2 propanol at position 2, and methylpropyl or hydroxyethyl at position 9. Without cassette dosing, this study would have used 3 times as many animals and would have taken 4 times longer, illustrating the power of this method in lead optimization.