Mycothione reductase as a potential target in the fight against Mycobacterium abscessus infections

Abstract: While infections caused by Mycobacterium abscessus complex (MABC) are rising worldwide, the current treatment of these infections is far from ideal due to its numerous shortcomings thereby increasing the urge for novel drug targets. In this study, mycothione reductase (Mtr) was evaluated for its potential as a drug target for MABC infections since it is a key enzyme needed in the recycling of mycothiol, the main low-molecular-weight thiol protecting the bacteria against reactive oxygen species and other reactive intermediates. First, a Mab increment mtr mutant strain was generated, lacking mtr expression. Next, the in vitro sensitivity of Mab increment mtr to oxidative stress and antimycobacterial drugs was determined. Finally, we evaluated the intramacrophage survival and the virulence of Mab increment mtr in Galleria mellonella larvae. Mab increment mtr demonstrated a 39.5-fold reduction in IC90 when exposed to bedaquiline in vitro. Furthermore, the Mab increment mtr mutant showed a decreased ability to proliferate inside macrophages and larvae, suggesting that Mtr plays an important role during MABC infection. Altogether, these findings support the assumption of Mtr being a potential target for antimycobacterial drugs

An echistatin-like arg-gly-asp (rgd)-containing sequence in the heavy-chain cdr3 of a murine monoclonal-antibody that inhibits human platelet glycoprotein iib/iiia function

We describe the production and biochemical characterization of the first GPIIb/IIIa-inhibiting monoclonal antibody that contains an RGD sequence in the CDR3 region of the heavy chain. Monoclonal antibodies obtained by immunizing mice with human platelets were screened using consecutive ELISAs based on human platelets and immuno-affinity-purified glycoprotein (GP) IIb/IIIa coated on microtitre plates. Out of 30 monoclonal antibodies reacting with GPIIb/IIIa, one, MA-16N7C2, potently inhibited platelet aggregation induced by ADP, thrombin, arachidonic acid, collagen, U46619, adrenaline and platelet-activating factor, whereas ristocetin-induced aggregation was unaffected. MA-16N7C2 (IgG2a) bound approximately 4 times faster to activated than to resting platelets, with a Kdcalc of 6.6nM and of 17.5nM, respectively. Equilibrium binding studies to non-activated platelets showed a Kd of 18.2nM with 41 x 10(3) binding sites per platelet. The antibody recognized GPIIb/IIIa only as a Ca(2+)-dependent complex. MA-16N7C2 blocked fibrinogen and von Willebrand factor binding to GPIIb/IIIa in a competitive manner with a Ki of 8.5nM and 13.2nM, respectively. Sequence analysis revealed a RGD-containing sequence with homology to disintegrins, in the CDR3 region of the heavy chain. That this RGD-containing sequence could be involved in the interaction of the antibody to GPIIb/IIIa was finally indicated by showing that the binding is completely and competitively inhibited by echistatin.status: publishe