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The function of the Staphylococcus aureus eukaryotic-like serine/threonine protein kinase PknB was investigated by performing transcriptome analysis using DNA microarray technology and biochemical assays. The transcriptional profile revealed a strong regulatory impact of PknB on the expression of genes encoding proteins which are involved in purine and pyrimidine biosynthesis, cell wall metabolism, autolysis, and glutamine synthesis. Functional activity of overexpressed and purified PknB kinase was demonstrated using the myelin basic protein as a surrogate substrate. Phosphorylation occurred in a time-dependent manner with Mn 2؉ as a preferred cofactor. Furthermore, biochemical characterization revealed regulation of adenylosuccinate synthase (PurA) activity by phosphorylation. Phosphorylated PurA showed a 1.8-fold decrease in enzymatic activity compared to unphosphorylated PurA. Loss of PknB led to formation of larger cell clusters, and a pknB deletion strain showed 32-fold-higher sensitivity to the cell wall-active antibiotic tunicamycin. The results of this study strongly indicate that PknB has a role in regulation of purine biosynthesis, autolysis, and central metabolic processes in S. aureus.The phosphorylation of proteins is a key regulatory mechanism in the signal transduction pathways of both prokaryotes and eukaryotes. Typically, extracellular signals are translated into cellular responses. The phosphorylation of proteins is carried out by specific protein kinases and is coupled to dephosphorylation reactions catalyzed by protein phosphatases. In prokaryotes sensing of extracellular signals and transduction of information are usually mediated by two-component signal transduction systems consisting of histidine kinase sensors and their associated response regulators (42). In contrast, signal transduction in eukaryotes occurs via phosphorylation of serine, threonine, and tyrosine residues. Serine/threonine and tyrosine kinases and phosphatases control reversible phosphorylation of target proteins in eukaryotes and are essential for cell cycle control and differentiation (17,19).It has recently been shown in a number of studies that eukaryotic-type serine/threonine protein kinases (STPKs) and phosphatases are also expressed in many prokaryotes (2). Prokaryotic STPKs regulate various cellular functions, such as stress responses, biofilm formation, sporulation, and metabolic and developmental processes (20,23,30,34,37,39,46). STPKs also play a role in the virulence of many bacterial pathogens, such as streptococci, Mycobacterium tuberculosis, Yersinia pseudotuberculosis, and Pseudomonas aeruginosa (11,16,21,36,47). Although the functional roles of protein kinases have been described in previous studies, only a small number of target substrates have been identified so far. Moreover, the impact of phosphorylation and dephosphorylation of target protein functions has been investigated in only some cases (33,38).A single STPK has been found to be conserved in all sequenced strains of Staphylococcus aureus. Origi...

The coronavirus main protease (M(pro)) represents an attractive drug target for antiviral therapy of coronavirus (CoV) infections, including severe acute respiratory syndrome (SARS). The SARS-CoV M(pro) and related CoV proteases have several distinct features, such as an uncharged Cys-His catalytic dyad embedded in a chymotrypsin-like protease fold, that clearly separate these enzymes from archetypical cysteine proteases. To further characterize the catalytic system of CoV main proteases and to obtain information about improved inhibitors, we performed comprehensive simulations of the proton-transfer reactions in the SARS-CoV M(pro) active site that lead to the Cys(-)/His(+) zwitterionic state required for efficient proteolytic activity. Our simulations, comprising the free enzyme as well as substrate-enzyme and inhibitor-enzyme complexes, lead us to predict that zwitterion formation is fostered by substrate binding but not inhibitor binding. This indicates that M(pro) employs a substrate-induced catalytic mechanism that further enhances its substrate specificity. Our computational data are in line with available experimental results, such as X-ray geometries, measured pKa values, mutagenesis experiments, and the measured differences between the kinetic parameters of substrates and inhibitors. The data also provide an atomistic picture of the formerly postulated electrostatic trigger involved in SARS-CoV M(pro) activity. Finally, they provide information on how a specific microenvironment may finely tune the activity of M(pro) toward specific viral protein substrates, which is known to be required for efficient viral replication. Our simulations also indicate that the low inhibition potencies of known covalently interacting inhibitors may, at least in part, be attributed to insufficient fostering of the proton-transfer reaction. These findings suggest ways to achieve improved inhibitors.

Improving the binding affinity of a chemical series by systematically probing one of its exit vectors is a medicinal chemistry activity that can benefit from molecular modeling input. Herein, we compare the effectiveness of four approaches in prioritizing building blocks with better potency: selection by a medicinal chemist, manual modeling, docking followed by manual filtering, and free energy calculations (FEP). Our study focused on identifying novel substituents for the apolar S2 pocket of cathepsin L and was conducted entirely in a prospective manner with synthesis and activity determination of 36 novel compounds. We found that FEP selected compounds with improved affinity for 8 out of 10 picks compared to 1 out of 10 for the other approaches. From this result and other additional analyses, we conclude that FEP can be a useful approach to guide this type of medicinal chemistry optimization once it has been validated for the system under consideration.

Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC 50 s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC 50 s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations. Dengue viruses (DENVs) are enveloped positive-strand RNA viruses and belong to the family Flaviviridae. DENV is the most important arthropod-borne viral infection. Over one-third of the world population lives in areas of DENV endemicity, and an estimated 390 million infections occur every year. In addition, the number of countries having experienced DENV epidemics has risen from 9 in 1970 to more than 100 today (1, 2). Furthermore, the number of diagnosed infections across America, Southeast Asia, and the Western Pacific nearly doubled from 1.2 million in 2008 to over 2.3 million in 2010 (2). Four different DENV serotypes have been identified so far. Recently, evidence for an additional subtype has been presented (3). Serotypes 1 to 4 are now prevalent in Asia, Africa, and America, and the regions where dengue is endemic are still increasing (4-6), with dengue endangering even Europe and the United States due to vector spread. DENV infections can be associated with dengue fever, but up to 88% of the infections remain inapparent (7). These nonpersistent infected patients serve besides persistently infected mosquitoes as a virus reservoir. Severe DENV infections and especially reinfections may lead to dengue hemorrhagic fever and dengue shock syndrome, with lethality up to 5% (2,8,9). There is neither a vaccination nor a specific treatment for DENV infections.The DENV genome contains a single open reading frame, which encodes the structural proteins capsid, membrane precursor (prM), and envelope and the nonstructural proteins NS1, NS2, NS3, NS4, and NS5 (10). Cellular proteases and the viral serine protease (PR) are responsible for cleaving the viral precursor polyprotein into functional proteins. The DENV PR consists of the amino-terminal domain of the NS3 protein and requires NS2B, a 14-kDa protein, as a cofactor to form a stable complex. This heterodimeric PR cleaves at the capsid-prM, NS2A/NS2...

Lewis diagrams and the octet rule [1] are central concepts in chemistry. Hypervalent molecules break the octet rule because they contain atoms with more than four electron pairs in their valence shell. [2] To describe them with the Lewis model requires hybridization schemes involving d orbitals (sp 3 d or sp 3 d 2 hybrids). [3, 4] The problem is that the formation of these hybrid orbitals requires large promotion energies. [5] Therefore, the significance of ionic resonance diagrams, which obviate the need for hypervalency, has long been discussed. [4] In this context, the electronic structure of SO 2 has been controversial. SO 2 can be described as a hypervalent molecule (Figure 1, left). Apart from d-orbital hybridization, multiple covalent bonding in SO 2 may be explained by three-center p pp p interactions of the sulfur 3p p orbital with non-bonding oxygen p p electrons (this interpretation goes back to Ref. [6]).However, other non-hypervalent ionic resonance structures can be formulated that preserve the octet rule ( Figure 1).The neutral Lewis structure is thought to be dominant, as the SÀO bond length in SO 2 is shorter than that in sulfur monoxide, SO (1.4299(3) in SO 2 from this study, compared to 1.481 for SO [7] ). The bond dissociation energy is also higher in SO 2 than in SO (547.3(8) kJ mol À1 vs. 517.1(8) kJ mol À1 [8] ). Furthermore, O 3 and O 2 , which are valence-isoelectronic with SO 2 and SO, have no available d orbitals, so they cannot be hypervalent, implying bond orders no higher than 1.5 and 2.0, respectively. This is consistent with the fact that the O À O distance in O 3 (1.2717(2) [9] ) is longer than in O 2 (1.15(8) , [10] 1.207 [7] ), in direct contrast with SO 2 relative to SO. This could support the notion that multiple covalent bonding is significant in SO 2 . Indeed, a large number of textbooks [11,12] adhere to this conclusion; for example, in ref. [12] it is stated that the S À O bond order is "at least 2".The simple empirical analysis above is in sharp contrast to the fact that computational studies have found significant ionic contributions to the SÀO bond, and very little sulfur dorbital participation. [13] Today, there is agreement among theoreticians that the role of d orbitals in the formation of bonds involving second and higher row elements is predominantly one of polarization functions, not of hybridization involving d orbitals. [5,14] In fact, the shorter and stronger bonds in SO 2 compared to SO (which is formally a double bond) support the conclusion that there are significant noncovalent contributions to the bonding. Indeed, calculations employing the electron localization function have shown that the polarity of a bond only depends on the electronegativity differences of the bonded atoms, so that molecules formerly classified as being hypervalent can be readily described with various ionic resonance structures. [15] So from a computational viewpoint, hypervalency is avoided by introducing ionic bonds.Experimentally, it has hitherto been difficult to obtain i...

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