We have identified a class of molecules, known as 2-aminothiazoles (2-ATs), as frequent-hitting fragments in biophysical binding assays. This was exemplified by 4-phenylthiazol-2-amine being identified as a hit in 14/14 screens against a diverse range of protein targets, suggesting that this scaffold is a poor starting point for fragment-based drug discovery. This prompted us to analyze this scaffold in the context of an academic fragment library used for fragment-based drug discovery (FBDD) and two larger compound libraries used for high-throughput screening (HTS). This analysis revealed that such "promiscuous 2-aminothiazoles" (PrATs) behaved as frequent hitters under both FBDD and HTS settings, although the problem was more pronounced in the fragment-based studies. As 2-ATs are present in known drugs, they cannot necessarily be deemed undesirable, but the combination of their promiscuity and difficulties associated with optimizing them into a lead compound makes them, in our opinion, poor scaffolds for fragment libraries.

Apical membrane antigen 1 (AMA1) is an essential component of the moving junction complex used by Plasmodium falciparum to invade human red blood cells. AMA1 has a conserved hydrophobic cleft that is the site of key interactions with the rhoptry neck protein complex. Our goal is to develop small molecule inhibitors of AMA1 with broad strain specificity, which we are pursuing using a fragment-based approach. In our screening campaign, we identified fragments that bind to the hydrophobic cleft with a hit rate of 5 %. The high hit rate observed strongly suggests that a druggable pocket is present within the cleft.

No abstract

Interferon epsilon (IFNɛ) is a type I IFN that is expressed constitutively in the female reproductive tract (FRT), and contributes to protection in models of sexually transmitted infections. Using multiple cell systems, including reporter cell lines and activated peripheral blood lymphocytes (PBLs), we show that recombinant IFNɛ impairs HIV infection at stage(s) post HIV entry and up to the translation of viral proteins. Consistent with this, IFNɛ upregulated a number of host cell restriction factors that block HIV at these stages of the replication cycle. The potency of IFNɛ induction of these HIV restriction factors was comparable to conventional type I IFNs, namely IFNα and IFNβ. IFNɛ also significantly reduced the infectivity of progeny virion particles likely by inducing expression of HIV restriction factors, such as IFITM3, which act at that stage of infection. Thus, our data demonstrate that human IFNɛ suppresses HIV replication at multiple stages of infection.

No abstract

Apical membrane antigen 1 (AMA1) interacts with RON2 to form a protein complex that plays a key role in the invasion of host cells by malaria parasites. Blocking this protein-protein interaction represents a potential route to controlling malaria and related parasitic diseases, but the polymorphic nature of AMA1 has proven to be a major challenge to vaccine-induced antibodies and peptide inhibitors exerting strain-transcending inhibitory effects. Here we present the X-ray crystal structure of AMA1 domains I and II from Plasmodium falciparum strain FVO. We compare our new structure to those of AMA1 from P. falciparum 3D7 and Plasmodium vivax. A combination of normalized B factor analysis and computational methods has been used to investigate the flexibility of the domain I loops and how this correlates with their roles in determining the strain specificity of human antibody responses and inhibitory peptides. We also investigated the domain II loop, a key region involved in inhibitor binding, by comparison of multiple AMA1 crystal structures. Collectively, these results provide valuable insights that should contribute to the design of strain-transcending agents targeting P. falciparum AMA1.

Edited by Luke O'NeillThe interaction of IFN-␤ with its receptor IFNAR1 (interferon ␣/␤ receptor subunit 1) is vital for host-protective anti-viral and anti-proliferative responses, but signaling via this interaction can be detrimental if dysregulated. Whereas it is established that IFNAR1 is an essential component of the IFNAR signaling complex, the key residues underpinning the IFN-␤-IFNAR1 interaction are unknown. Guided by the crystal structure of the IFN-␤-IFNAR1 complex, we used truncation variants and site-directed mutagenesis to investigate domains and residues enabling complexation of IFN-␤ to IFNAR1. We have identified an interface on IFNAR1-subdomain-3 that is differentially utilized by IFN-␤ and IFN-␣ for signal transduction. We used surface plasmon resonance and cell-based assays to investigate this important IFN-␤ binding interface that is centered on IFNAR1 residues Tyr 240 and Tyr The type I IFNs, including 14 IFN-␣ and lone IFN-␤, IFN-⑀, and IFN-, have critical roles in response to viral and bacterial infections and cancers (1, 2). They are also applied clinically for the treatment of hepatitis virus B and C (1), cancers including melanoma (3), and multiple sclerosis (4). Although they show clinical efficacy, their use is restricted by dose-limiting toxicities, including leukopenia, nausea, fatigue, neurological disorders (3), and localized cutaneous effects (5). All type I IFNs engage their cognate receptors, IFNAR1 and IFNAR2, to activate the canonical JAK-STAT signaling pathway, but ligand engagement can also activate alternative signaling pathways (6). Despite sharing these receptor components, there are IFN subtype-specific elements to signaling; compared with IFN-␣, IFN-␤ has specific roles in osteoclastogenesis (7), control of chronic viral infection (8), the potent induction of apoptotic pathways required for control of tumor cell growth, and the development of B cells and myelopoiesis (9).Structural insight into the IFN-IFNAR 5 interactions has been gleaned from the crystal structures of a human IFN-␣2 variant and human IFN-in complex with both IFNAR1 and IFNAR2 (10). Furthermore, specific insight into the mode of IFN-␤-mediated activation of IFNAR1 was obtained from the crystal structure of the murine IFN-␤-IFNAR1 complex (11). Comparison of these structures and evidence from the literature (12) suggests that the minimal ligand binding domains for human and mouse IFNAR1 are similar and sit broadly across the three membrane distal SDs (SD1-3) of the receptor with limited involvement of the membrane proximal subdomain, SD4 (Fig. 1A). It has also been shown that key residues discriminate between ligands and that there is potential for ligand-specific interaction interfaces (10, 11).

No abstract