Selectivity of Inhibition of N-Succinyl-l,l-Diaminopimelic Acid Desuccinylase in Bacteria: The product of dapE-gene Is Not the Target of l-Captopril Antimicrobial Activity

The emergence of bacterial strains that are resistant to virtually all currently available antibiotics underscores the importance of developing new antimicrobial compounds. N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) is a metallohydrolase involved in the meso-diaminopimelate (mDAP)/lysine biosynthetic pathway necessary for lysine biosynthesis and for building the peptidoglycan cell wall. Because DapE is essential for Gram-negative and some Gram-positive bacteria, DapE has been proposed as a good target for antibiotic development. Recently, l-captopril has been suggested as a lead compound for inhibition of DapE, although its selectivity for this enzyme target in bacteria remains unclear (Gillner et al. (2009)). Here, we tested the selectivity of l-captopril against DapE in bacteria. Since DapE knockout strains of gram-negative bacteria are viable upon chemical supplementation with mDAP, we reasoned that the antimicrobial activity of compounds targeting DapE should be abolished in mDAP-containing media. Although l-captopril had modest antimicrobial activity in Escherichia coli and in Salmonella enterica, to our surprise, inhibition of bacterial growth was independent both of mDAP supplementation and DapE over-expression. We conclude that DapE is not the main target of l-captopril inhibition in these bacteria. The methods implemented here will be useful for screening DapE-selective antimicrobial compounds directly in bacterial cultures

Zinc-selective inhibition of the promiscuous bacterial amide-hydrolase DapE: implications of metal heterogeneity for evolution and antibiotic drug design

The development of resistance to virtually all current antibiotics makes the discovery of new antimicrobial compounds with novel protein targets an urgent challenge. The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) is an essential metallo-enzyme for growth and proliferation in many bacteria, acting in the desuccinylation of N-succinyl-l,l-diaminopimelic acid (SDAP) in a late stage of the anabolic pathway towards both lysine and a crucial building block of the peptidoglycan cell wall. l-Captopril, which has been shown to exhibit very promising inhibitory activity in vitro against DapE and has attractive drug-like properties, nevertheless does not target DapE in bacteria effectively. Here we show that l-captopril targets only the Zn(2+)-metallo-isoform of the enzyme, whereas the Mn(2+)-enzyme, which is also a physiologically relevant isoform in bacteria, is not inhibited. Our finding provides a rationale for the failure of this promising lead-compound to exhibit any significant antibiotic activity in bacteria and underlines the importance of addressing metallo-isoform heterogeneity in future drug design. Moreover, to our knowledge, this is the first example of metallo-isoform heterogeneity in vivo that provides an evolutionary advantage to bacteria upon drug-challenge

CRP/Albumin Ratio and Glasgow Prognostic Score Provide Prognostic Information in Myelofibrosis Independently of MIPSS70-A Retrospective Study.

In myelofibrosis, the C-reactive protein (CRP)/albumin ratio (CAR) and the Glasgow Prognostic Score (GPS) add prognostic information independently of the Dynamic International Prognostic Scoring System (DIPSS). Their prognostic impact, if molecular aberrations are considered, is currently unknown. We performed a retrospective chart review of 108 MF patients (prefibrotic MF n = 30; primary MF n = 56; secondary MF n = 22; median follow-up 42 months). In MF, both a CAR > 0.347 and a GPS > 0 were associated with a shorter median overall survival (21 [95% CI 0-62] vs. 80 months [95% CI 57-103], p 0.374 HR 3.53 [95% CI 1.36-9.17], p = 0.0095 and GPS > 0 HR 4.63 [95% CI 1.76-12.1], p = 0.0019. An analysis of serum samples from an independent cohort revealed a correlation of CRP with levels of interleukin-1β and albumin with TNF-α, and demonstrated that CRP was correlated to the variant allele frequency of the driver mutation, but not albumin. Albumin and CRP as parameters readily available in clinical routine at low costs deserve further evaluation as prognostic markers in MF, ideally by analyzing data from prospective and multi-institutional registries. Since both albumin and CRP levels reflect different aspects of MF-associated inflammation and metabolic changes, our study further highlights that combining both parameters seems potentially useful to improve prognostication in MF

Promiscuous behaviour of the bacterial metallohydrolase DapE : an evolutionary and mechanistic perspective

Enzyme promiscuity, defined as functional properties other than those for which they are evolved, is considered a key factor in the evolution of new enzyme functions. Many metalloproteins can be alternatively metallated, which may lead to metal-dependent promiscuity. The mechanisms and evolutionary implications of metal-mediated promiscuity appear to be underexplored, especially considering that approximately one-third of structurally characterized proteins are thought to be metalloproteins. Here, we investigated the bacterial binuclear metallohydrolase, N-Succinyl-L-LDiaminopimelic acid desuccinylase DapE (EC 3.5.1.18) of S.enterica. DapE is an essential enzyme in the late stage of the lysine biosynthetic pathway that also provides a crucial building block of the peptidoglycan cell wall. Since DapE is essential for most Gram-negative and many Gram-positive bacteria and it is not present in humans, it has been proposed as a very good target for antibiotic development. It was also reported that DapE has a metal dependent promiscuous aspartyl dipeptidase activity, in which incorporation of either zinc or manganese to the enzyme leads to activity with different substrates and this phenomenon occurs both in vivo and in vitro. We addressed the reaction mechanism of the native desuccinylase activity as well as the Mn2+-dependent aspartyl dipeptidase promiscuous activity of DapE, by investigating a series of substrate analogues and potential inhibitors. We postulated a plausible mechanism for metal-dependent promiscuity, based on subtle differences in coordination preferences between Mn2+ and Zn2+,which may be widely applicable to other enzymes. We revealed why a promising inhibitor of the enzyme in vitro, L-captopril, fails to exert antibiotic activity and propose broad practical implications of this discovery for drug-design, as well as fundamental evolutionary implications. We described kinetic cooperativity in DapE, which offers clues on structural rearrangements that occur during catalysis and is also of relevance to inhibitor design. Finally, we explored the evolutionary aspects of functional robustness of native activity over the promiscuous activity using DapE as a model enzyme and addressed the molecular mechanisms underlying the emergence of functional robustness through laboratory evolution

Varied Immune Response to FVIII: Presence of Proteolytic Antibodies Directed to Factor VIII in Different Human Pathologies

The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation of the complement and its activation, and activation of effector cells. In addition to this plethora of functions, antibodies are capable of expressing enzymatic activity. Antibodies with catalytic function are a result of the productive interplay between the highly evolved machinery of the immune system and the chemical framework used to induce them (antigens). Catalytic antibodies are immunoglobulins with an ability to catalyze the reactions involving the antigen for which they are specific. Catalytic immunoglobulins of the IgM and IgG isotypes have been detected in the serum of healthy donors. In addition, catalytic immunoglobulins of the IgA isotype have been detected in the milk of healthy mothers. Conversely, antigen-specific hydrolytic antibodies have been reported in a number of inflammatory, autoimmune, and neoplastic disorders. The pathophysiological occurrence and relevance of catalytic antibodies remains a debated issue. Through the description of the hydrolysis of coagulation factor VIII as model target antigen, we propose that catalytic antibodies directed to the coagulation factor VIII may play a beneficial or a deleterious role depending on the immuno-inflammatory condition under which they occur