Novel antimalarial therapeutics that target multiple stages of the parasite lifecycle are urgently required to tackle the emerging problem of resistance with current drugs. Here, we describe the optimization of the 2-anilino quinazoline class as antimalarial agents. The class, identified from publicly available antimalarial screening data, was optimized to generate lead compounds that possess potent antimalarial activity against P. falciparum parasites comparable to the known antimalarials, chloroquine and mefloquine. During the optimization process, we defined the functionality necessary for activity and improved in vitro metabolism and solubility. The resultant lead compounds possess potent activity against a multidrug resistant strain of P. falciparum and arrest parasites at the ring phase of the asexual stage and also gametocytogensis. Finally, we show that the lead compounds are orally efficacious in a 4 day murine model of malaria disease burden.

Suppressor of cytokine signaling (SOCS)2 protein is a key negative regulator of the growth hormone (GH) and Janus kinase (JAK)-Signal Transducers and Activators of Transcription (STAT) signaling cascade. The central SOCS2-Src homology 2 (SH2) domain is characteristic of the SOCS family proteins and is an important module that facilitates recognition of targets bearing phosphorylated tyrosine (pTyr) residues. Here we identify an exosite on the SOCS2-SH2 domain which, when bound to a non-phosphorylated peptide (F3), enhances SH2 affinity for canonical phosphorylated ligands. Solution of the SOCS2/F3 crystal structure reveals F3 as an α-helix which binds on the opposite side of the SH2 domain to the phosphopeptide binding site. F3:exosite binding appears to stabilise the SOCS2-SH2 domain, resulting in slower dissociation of phosphorylated ligands and consequently, enhances binding affinity. This biophysical enhancement of SH2:pTyr binding affinity translates to increase SOCS2 inhibition of GH signaling.

The membrane-associated RING-CH (MARCH) family of membrane-bound E3 ubiquitin ligases regulates the levels of cell-surface membrane proteins, many of which are involved in immune responses. Although their role in ubiquitin-dependent endocytosis and degradation of cell-surface proteins is extensively documented, the features of MARCH proteins and their substrates that drive the molecular recognition events leading to ubiquitin transfer remain poorly defined. In this study, we sought to determine the features of human MARCH9 that are required for regulating the surface levels of its substrate proteins. Consistent with previous studies of other MARCH proteins, we found that susceptibility to MARCH9 activity is encoded in the transmembrane (TM) domains of its substrates. Accordingly, substitutions at specific residues and motifs within MARCH9's TM domains resulted in varying degrees of functional impairment. Most notably, a single serine-to-alanine substitution in the first of its two TM domains rendered MARCH9 completely unable to alter the surface levels of two different substrates: the major histocompatibility class I molecule HLA-A2 and the T-cell coreceptor CD4. Solution NMR analysis of a MARCH9 fragment encompassing the two TM domains and extracellular connecting loop revealed that the residues contributing most to MARCH9 activity are located in the ␣-helical portions of TM1 and TM2 that are closest to the extracellular face of the lipid bilayer. This observation defines a key region required for substrate regulation. In summary, our biochemical and structural findings demonstrate that specific sequences in the ␣-helical MARCH9 TM domains make crucial contributions to its ability to down-regulate its protein substrates.The membrane-associated RING-CH (MARCH) 5 proteins are a family of E3 ubiquitin ligases that regulate the cell-surface expression of proteins with important roles in immunity (1-3). Six mammalian MARCH proteins (MARCH1-4, MARCH8, and MARCH9) share a common structural organization featuring an N-terminal RING-CH domain that facilitates ubiquitin transfer, two predicted transmembrane (TM) domains connected by a short extracellular loop, and a C-terminal cytosolic tail of variable length (1). Biologically, the best characterized member of this family is MARCH1, whose biological roles include targeting major histocompatibility class II (MHC-II) complexes and the T-cell co-stimulatory molecule CD86 (B7.2) to endocytotic vesicles in antigen-presenting cells via ubiquitin-dependent pathways (4 -9). In dendritic cells, when maturation is triggered by the uptake of antigenic cargo and/or exposure to Toll-like receptor ligands, MARCH1 activity is opposed by CD83 expression (10) and MARCH1 transcription stops (9, 11), resulting in MHC-II and CD86 up-regulation at the cell surface and increased CD4 ϩ T-cell stimulatory capacity. A similar role for MARCH8 has recently been identified in thymic epithelial cells (12,13), where it plays a key role in the development of CD4 ϩ T-cells. The dynamic control of MARCH...