Survivin, an apoptosis inhibitor/cell-cycle regulator, is critically required for suppression of apoptosis and ensuring normal cell division in the G2/M phase of the cell cycle. It is highly expressed in a cell cycle-regulated manner and localizes together with caspase-3 on microtubules within centrosomes. Whether survivin is a physiologically relevant caspase inhibitor has been unclear due to the difficulties with obtaining correctly folded survivin and finding the right conditions for inhibition assay. In this study, recombinant, active human survivin was expressed in Escherichia coli and purified to homogeneity. The protein, existing as a homodimer in solution, binds caspase-3 and -7 tightly with dissociation constants of 20.9 and 11.5 nM, respectively, when evaluated by surface plasmon resonance spectroscopy. Consistently, survivin potently inhibits the cleavage of a physiological substrate poly(ADP-ribose) polymerase and an artificial tetrapeptide by caspase-3 and -7 in vitro with apparent inhibition constants of 36.0 and 16.5 nM, respectively. The data suggest that sequestering caspase-3 and -7 in inhibited states on microtubules is at least one mechanism of survivin in the suppression of default apoptosis in the G2/M phase. The localization of survivin on microtubules, which is essential for its function, should increase the protective activity at the action site.

SEDL is an evolutionarily highly conserved protein in eukaryotic organisms. Deletions or point mutations in the SEDL gene are responsible for the genetic disease spondyloepiphyseal dysplasia tarda (SEDT), an X-linked skeletal disorder. SEDL has been identified as a component of the transport protein particle (TRAPP), critically involved in endoplasmic reticulum-to-Golgi vesicle transport. Herein, we report the 2.4 Å resolution structure of SEDL, which reveals an unexpected similarity to the structures of the N-terminal regulatory domain of two SNAREs, Ykt6p and Sec22b, despite no sequence homology to these proteins. The similarity and the presence of unusually many solvent-exposed apolar residues of SEDL suggest that it serves regulatory and/or adaptor functions through multiple protein-protein interactions. Of the four known missense mutations responsible for SEDT, three mutations (S73L, F83S, V130D) map to the protein interior, where the mutations would disrupt the structure, and the fourth (D47Y) on a surface at which the mutation may abrogate functional interactions with a partner protein.Intracellular targeting and fusion of transport vesicles in eukaryotes are tightly regulated to avoid inappropriate mixing of the contents in different compartments. Central components of the membrane fusion are the proteins denoted as SNAREs 1 (soluble N-ethylmaleimide-sensitive factor attachment receptor proteins). SNAREs constitute a superfamily of proteins that share a highly conserved sequence motif, the SNARE motif composed of 60 -70 amino acids (1). Most SNAREs are membrane proteins anchored on vesicular carriers (v-SNARE) and target organelles (t-SNARE) (1). Association of the SNARE domains between v-and t-SNAREs to form a helical bundle, termed the core complex (2), is believed to be the prime event that drives membrane fusion (3, 4). While the specific pairing of v-and t-SNAREs is one mechanism of providing the fidelity of membrane fusion, other proteins or protein complexes such as the transport protein particle (TRAPP) are known to provide further specificity by controlling the tethering process, in which a transport vesicle is properly docked on target membrane prior to pairing of SNAREs (5). TRAPP is localized to an early Golgi compartment (6) and is able to exchange the nucleotide of Ypt1p GTPase, which is an upstream event of v-and t-SNARE interactions (7,8). Recent in vitro transport studies showed that yeast TRAPP I binds COPII, the vesicle coat derived from the endoplasmic reticulum (ER), indicating that TRAPP I is the receptor for tethering COPII vesicles to Golgi membranes (6). The TRAPP complexes (TRAPP I and TRAPP II) are composed of 7-10 different polypeptides, which are highly conserved in evolution, with the yeast subunits sharing between 29 and 54% sequence identity with their human counterparts (9). The biochemical function of any of the constituent proteins is virtually unknown, although the TRAPP complex was shown to stimulate nucleotide exchange on the Ypt1p and the Ypt31/32 GTPases (10).Spond...

The Michaelis-Menten equation is one of the best-known models describing the enzyme kinetics of in vitro drug elimination experiments, and takes a form of equation relating reaction rate (V) to the substrate concentration ([S]) via the maximum reaction rate (V max ) and the Michaelis constant (K m ). The current study was conducted to compare the accuracy and precision of the parameter estimates in the Michaelis-Menten equation from various estimation methods using simulated data. One thousand replicates of simulated [S] over serial time data were generated using the results of a previous study, incorporating additive or combined error models as a source of random variables in the Monte-Carlo simulation using R. From each replicate of simulated data, V max and K m were estimated by five different methods, including traditional linearization methods and nonlinear ones without linearization using NONMEM. The relative accuracy and precision of the estimated parameters were compared by the median values and their 90% confidence intervals. Overall, V max and K m estimation by nonlinear methods (NM) provided the most accurate and precise results from the tested 5 estimation methods. The superiority of parameter estimation by NM was even more evident in the simulated data incorporating the combined error model. The current simulation study suggests that NMs using a program such as NONMEM provide more reliable and accurate parameter estimates of the Michaelis-Menten equation than traditional linearization methods in in vitro drug elimination kinetic experiments.

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LCB01-0371 is a novel oxazolidinone with broad-spectrum activity against Gram-positive pathogens in both studies and animal infection models. The objectives of this study were to evaluate its safety, tolerability, pharmacokinetics, and pharmacodynamics following single ascending doses. Single oral doses of 600 mg linezolid, a placebo, or LCB01-0371 of between 50 mg and 3,200 mg were tested in 69 healthy male subjects. Blood and urine were sampled, LCB01-0371 concentrations were measured, and the serum inhibitory and bactericidal titers of LCB01-0371 and linezolid were determined. LCB01-0371 was well tolerated up to 2,400 mg. The most common drug-related clinical and laboratory adverse events were nausea with or without vomiting, decreased neutrophil counts, and increased total bilirubin levels. The frequency of adverse events and drug-related adverse events was similar among the treatment groups. The systemic exposure was approximately dose proportional over the range of 50 mg to 800 mg, which includes the anticipated clinical dose. The mean clearance, renal clearance, and volume of distribution were significantly decreased at higher doses (above 800 mg). LCB01-0371 exhibited early bacteriostatic activity against all tested strains except for strains, and the potency of LCB01-0371 at 800 mg was similar to that of linezolid at the therapeutic dose (600 mg). However, LCB01-0371 had less bactericidal activity than linezolid. Taken together, LCB01-0371 was well tolerated, exhibited approximate dose proportionality within the anticipated clinically relevant dose range, and showed bacteriostatic and bactericidal activity comparable to that of linezolid. These results support the further clinical development of LCB01-0371. (This study has been registered at ClinicalTrials.gov under registration no. NCT01554995.).

The wide variability of isoniazid (INH) pharmacokinetics is mainly attributed to the trimodal N‐acetyltransferase 2 (NAT2) acetylator phenotype, that is, rapid, intermediate, and slow. Consequently, a uniform INH dose in current clinical practice may lead to treatment failure and emergence of drug resistance. There is a lack of studies on specific doses of INH for different NAT2 acetylator phenotypes among tuberculosis (TB) patients. Therefore, we aimed to provide insight into the optimal dosing of INH for each NAT2 acetylator phenotype with respect to the probability of achieving a pharmacokinetic (PK)/pharmacodynamic target. PK, the NAT2 genotype, and clinical data were collected in a multicenter prospective cohort study conducted at 13 clinical centers in Korea. Population PK modeling and simulation were carried out. Data from 454 TB patients were divided into a training data set and a test data set at a ratio of 4 to 1. The PK of the training data were best described by a 2‐compartment model with allometric scaling for body size effect. Importantly, NAT2 acetylator phenotypes significantly affected the apparent clearance. Our model, which provided better predictive performance compared with previously published models, was evaluated by external validation using the test set. The simulation for assessing target efficacy and toxicity indicated that the best INH dosing regimens for Korean tuberculosis patients were once‐daily doses of 400, 300, and 200 mg for rapid, intermediate, and slow acetylators, respectively. In conclusion, our study provides a step forward in precision dosing for antituberculosis management.

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Cdc7 is a serine/threonine kinase conserved from yeasts to human and is known to play a key role in the regulation of initiation at each replication origin. Its catalytic function is activated via association with the activation subunit Dbf4/activator of S phase kinase (ASK). It is known that two conserved motifs of Dbf4/ASK are involved in binding to Cdc7, and both are required for maximum activation of Cdc7 kinase. Cdc7 kinases possess unique kinase insert sequences (kinase insert I-III) that are inserted at defined locations among the conserved kinase domains. However, precise mechanisms of Cdc7 kinase activation are largely unknown. We have identified two segments on Cdc7, DAM-1 (Dbf4/ASK interacting motif-1; amino acids 448 -457 near the N terminus of kinase insert III) and DAM-2 (C-terminal 10-amino acid segment), that interact with motif-M and motif-C of ASK, respectively, and are essential for kinase activation by ASK. The C-terminal 143-amino acid polypeptide (432-574) containing DAM-1 and DAM-2 can interact with Dbf4/ASK. Characterization of the purified ASK-free Cdc7 and Cdc7-ASK complex shows that ATP binding of the Cdc7 catalytic subunit requires Dbf4/ASK. However, the "minimum" Cdc7, lacking the entire kinase insert II and half of kinase insert III, binds to ATP and shows autophosphorylation activity in the absence of ASK. However, ASK is still required for phosphorylation of exogenous substrates by the minimum Cdc7. These results indicate bipartite interaction between Cdc7 and Dbf4/ ASK subunits facilitates ATP binding and substrate recognition by the Cdc7 kinase.