A decade of discovery and development of new anti-malarial medicines has led to a renewed focus on malaria elimination and eradication. Changes in the way new anti-malarial drugs are discovered and developed have led to a dramatic increase in the number and diversity of new molecules presently in pre-clinical and early clinical development. The twin challenges faced can be summarized by multi-drug resistant malaria from the Greater Mekong Sub-region, and the need to provide simplified medicines. This review lists changes in anti-malarial target candidate and target product profiles over the last 4 years. As well as new medicines to treat disease and prevent transmission, there has been increased focus on the longer term goal of finding new medicines for chemoprotection, potentially with long-acting molecules, or parenteral formulations. Other gaps in the malaria armamentarium, such as drugs to treat severe malaria and endectocides (that kill mosquitoes which feed on people who have taken the drug), are defined here. Ultimately the elimination of malaria requires medicines that are safe and well-tolerated to be used in vulnerable populations: in pregnancy, especially the first trimester, and in those suffering from malnutrition or co-infection with other pathogens. These updates reflect the maturing of an understanding of the key challenges in producing the next generation of medicines to control, eliminate and ultimately eradicate malaria.

We previously reported that NF-KB and a complex we referred to as NF-ELAM1 play a central role in cytokine-induced expression of the E-selectin gene. In this study we identify cyclic AMP (cAMP)-independent members of the ATF family binding specifically to the NF-ELAM1 promoter element. The NF-ELAM1 element (TGACATCA) differs by a single nucleotide substitution from the cAMP-responsive element consensus sequence. We demonstrate that this sequence operates in a cAMP-independent manner to induce transcription and thus define it as a non-cAMP-responsive element (NCRE). We show that ATFa is a component of the NF-ELAM1 complex and its overexpression activates the E-selectin promoter. In addition, ATFa, ATF2, and ATF3 interact directly with NF-cB in vitro, linking two unrelated families of transcription factors in a novel protein-protein interaction. Furthermore, we demonstrate that the ability of overexpressed NF-KB to transactivate the E-selectin promoter in vivo is dependent on the NF-ELAM1 complex. Our results suggest that a direct interaction between ATFs and NF-cB is, at least in part, the mechanism by which these factors specifically regulate E-selectin promoter activity.

Cytokine-induced expression of the E-selectin gene requires the promoter binding and interaction of the transcription factors NF-KB and ATF. Here we have further analyzed the E-selectin promoter and revealed an additional region (nucleotides -140 to -105 [-1401-105]) which is essential in controlling promoter activation by cytokines. We identified high-mobility-group protein I(Y) [HMG-I(Y)] interacting specifically at two sites within this region. We noted that one of the HMG-I(Y)-binding sites overlaps a sequence element (-127/-118) diverging at only one position from the NF-KB consensus binding sequence. This led us to ask whether the -127/-118 element represents a second functional NF-KB-binding site within the E-selectin promoter. Using specific antisera, we show that p50, p65, and, interestingly, RelB are components of the complex interacting at this site. Mutational analysis of the -127/-118 NF-KB site indicates that both NF-KB and HMG-I(Y) binding at this site are essential for interleukin-1 induction of the promoter. We demonstrate that the binding affinity of the p50 subunit of NF-KB to both NF-KB sites within the E-selectin promoter is significantly enhanced by HMG-I(Y). In addition, an essential role for cooperative interaction between the two NF-KB complexes is shown by the requirement for both NF-KB sites to mediate E-selectin promoter activation by interleukin-1 and p5O/p65 expression. We conclude that HMG-I(Y) mediates binding of a distinct NF-KB complex at two sites within the E-selectin promoter. Furthermore, a unique cooperativity between these NF-KB complexes is essential for induced E-selectin expression. These results suggest mechanisms by which NF-KB complexes are involved in specific gene activation.When activated under physiological or pathological conditions, leukocytes transiently adhere to the vascular endothelium. This is an important step in leukocyte migration from the vascular compartment to extravascular tissues. Leukocyte adhesion is thus critical in the development of immune and inflammatory responses. This process requires the expression of adhesion proteins on the endothelial cell surface (for reviews, see references 7 and 46). E-selectin, a member of the selectin family of cell surface glycoproteins, is one of the important endothelial cell adhesion proteins (for a review, see reference 29). The E-selectin protein is rapidly and transiently expressed on the endothelial cell surface following cytokine induction and plays a central role in the extravasation of both neutrophils and a subset of T lymphocytes (5, 38). Transcription of the gene encoding E-selectin is tightly regulated in a cell-specific manner, being expressed exclusively on endothelial cells following exposure to either interleukin-113 (IL-1,B) or tumor necrosis factor alpha (5, 17, 51).Our work has focused on understanding the mechanisms regulating induced E-selectin expression at the level of gene transcription. In particular, we have focused on identifying the transcription factors involved and have examined ho...

No abstract

A powerful chemical modification procedure has been developed to define determinants of DNA recognition by the p50 subunit of NF-kappa B. Differential labelling with [14C] iodoacetate has identified a conserved cysteine residue, Cys62, that was protected from modification by the presence of an oligonucleotide containing the specific recognition site of the protein. To determine the importance of this cysteine residue, each of the conserved cysteines in p50 was changed to serine and the DNA binding properties of the mutant proteins determined. Scatchard analysis indicated that the C62S mutant bound to its DNA recognition site with a 10-fold larger dissociation constant than the wild type protein, while the other two mutants bound with an intermediate affinity. Dissociation rate constant measurements correlated well with the dissociation constants for the wild type, C119S, and C273S p50 proteins, whereas the p50 C62S-DNA complex dissociated anomalously quickly. Competition analyses with oligonucleotide variants of the DNA recognition site and nonspecific E. coli DNA revealed that the C62S p50 mutant had an altered DNA binding site specificity and was impaired in its ability to discriminate between specific and non-specific DNA. Thus the sulphydryl group of Cys62 is an important determinant of DNA recognition by the p50 subunit of NF-kappa B.