The discovery and initial optimisation of pyrrole-2-carboxamides as inhibitors of p38α MAP kinase

A novel pyrrole-2-carboxamide series of p38α inhibitors, discovered through the application of virtual screening, is presented. Following evaluation of activity, selectivity and developability properties of commercially available analogues, a synthesis program enabled rapid assessment of the series’ suitability for further lead optimisation studies.     

Membrane damage by hemolytic viruses, toxins, complement, and other cytotoxic agents. A common mechanism blocked by divalent cations

Hemolytic viruses, bacterial and animal toxins, the components of activated complement, cationic proteins, and detergents induce a sequence of permeability changes at the plasma membrane that are in every case sensitive to changes in ionic strength and to divalent cations. Individually, each agent exhibits positive cooperativity; when two agents are present together, they show synergy. It is concluded that such cytotoxic agents damage membranes by a common mechanism. Hence permeability changes are unlikely to depend on the formation of specific, protein-lined channels, as previously envisaged in the case of activated complement or certain bacterial toxins.     

Randomised Controlled Double-Blind Non-Inferiority Trial of Two Antivenoms for Saw-Scaled or Carpet Viper (Echis ocellatus) Envenoming in Nigeria

Background

In West Africa, envenoming by saw-scaled or carpet vipers (Echis ocellatus) causes great morbidity and mortality, but there is a crisis in supply of effective and affordable antivenom (ISRCTN01257358).

Methods

In a randomised, double-blind, controlled, non-inferiority trial, “EchiTAb Plus-ICP” (ET-Plus) equine antivenom made by Instituto Clodomiro Picado was compared to “EchiTAb G” (ET-G) ovine antivenom made by MicroPharm, which is the standard of care in Nigeria and was developed from the original EchiTAb-Fab introduced in 1998. Both are caprylic acid purified whole IgG antivenoms. ET-G is monospecific for Echis ocellatus antivenom (initial dose 1 vial) and ET-Plus is polyspecific for E. ocellatus, Naja nigricollis and Bitis arietans (initial dose 3 vials). Both had been screened by pre-clinical and preliminary clinical dose-finding and safety studies. Patients who presented with incoagulable blood, indicative of systemic envenoming by E. ocellatus, were recruited in Kaltungo, north-eastern Nigeria. Those eligible and consenting were randomly allocated with equal probability to receive ET-Plus or ET-G. The primary outcome was permanent restoration of blood coagulability 6 hours after the start of treatment, assessed by a simple whole blood clotting test repeated 6, 12, 18, 24 and 48 hr after treatment. Secondary (safety) outcomes were the incidences of anaphylactic, pyrogenic and late serum sickness-type antivenom reactions.

Findings

Initial doses permanently restored blood coagulability at 6 hours in 161/194 (83.0%) of ET-Plus and 156/206 (75.7%) of ET-G treated patients (Relative Risk [RR] 1.10 one-sided 95% CI lower limit 1.01; P = 0.05). ET-Plus caused early reactions on more occasions than did ET-G [50/194 (25.8%) and 39/206 (18.9%) respectively RR (1.36 one-sided 95% CI 1.86 upper limit; P = 0.06). These reactions were classified as severe in 21 (10.8%) and 11 (5.3%) of patients, respectively.

Conclusion

At these doses, ET-Plus was slightly more effective but ET-G was slightly safer. Both are recommended for treating E. ocellatus envenoming in Nigeria.

Trial Registration

Current Controlled Trials ISRCTN01257358     

Tyrosine phosphorylation as a conformational switch: a case study of integrin β3 cytoplasmic tail

Reversible protein phosphorylation is vital for many fundamental cellular processes. The actual impact of adding and removing phosphate group(s) is 3-fold: changes in the local/global geometry, alterations in the electrostatic potential and, as the result of both, modified protein-target interactions. Here we present a comprehensive structural investigation of the effects of phosphorylation on the conformational as well as functional states of a crucial cell surface receptor, α(IIb)β(3) integrin. We have analyzed phosphorylated (Tyr(747) and Tyr(759)) β(3) integrin cytoplasmic tail (CT) primarily by NMR, and our data demonstrate that under both aqueous and membrane-mimetic conditions, phosphorylation causes substantial conformational rearrangements. These changes originate from novel ionic interactions and revised phospholipid binding. Under aqueous conditions, the critical Tyr(747) phosphorylation prevents β(3)CT from binding to its heterodimer partner α(IIb)CT, thus likely maintaining an activated state of the receptor. This conclusion was tested in vivo and confirmed by integrin-dependent endothelial cells adhesion assay. Under membrane-mimetic conditions, phosphorylation results in a modified membrane embedding characterized by significant changes in the secondary structure pattern and the overall fold of β(3)CT. Collectively these data provide unique molecular insights into multiple regulatory roles of phosphorylation.