Malaria is caused in humans by five species of single-celled eukaryotic Plasmodium parasites (mainly Plasmodium falciparum and Plasmodium vivax) that are transmitted by the bite of Anopheles spp. mosquitoes. Malaria remains one of the most serious infectious diseases; it threatens nearly half of the world's population and led to hundreds of thousands of deaths in 2015, predominantly among children in Africa. Malaria is managed through a combination of vector control approaches (such as insecticide spraying and the use of insecticide-treated bed nets) and drugs for both treatment and prevention. The widespread use of artemisinin-based combination therapies has contributed to substantial declines in the number of malaria-related deaths; however, the emergence of drug resistance threatens to reverse this progress. Advances in our understanding of the underlying molecular basis of pathogenesis have fuelled the development of new diagnostics, drugs and insecticides. Several new combination therapies are in clinical development that have efficacy against drug-resistant parasites and the potential to be used in single-dose regimens to improve compliance. This ambitious programme to eliminate malaria also includes new approaches that could yield malaria vaccines or novel vector control strategies. However, despite these achievements, a well-coordinated global effort on multiple fronts is needed if malaria elimination is to be achieved.

Malaria caused by Plasmodium falciparum is a catastrophic disease worldwide (880,000 deaths yearly). Vaccine development has proved difficult and resistance has emerged for most antimalarials. In order to discover new antimalarial chemotypes, we have employed a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library, many of which exhibited potent in vitro activity against drug resistant strains, and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in multiple organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Overall, our findings provide the scientific community with new starting points for malaria drug discovery.

A new drug screening method was devised utilizing Trypanosoma cruzi cells that express the Escherichia coli ␤-galactosidase gene. Transfected parasites catalyze a colorimetric reaction with chlorophenol red ␤-D-galactopyranoside as substrate. Parasite growth in the presence of drugs in microtiter plates was quantitated with an enzyme-linked immunosorbent assay reader. The assay was performed with the mammalian form of T. cruzi that requires intracellular growth on a monolayer of fibroblast cells. To determine if selective toxicity to the parasites was occurring, the viability of the host cells in the drug was assayed with AlamarBlue. The drugs benznidazole, fluconazole, and amphotericin B were shown to inhibit the parasites at concentrations similar to those previously reported. Several compounds were tested that are inhibitors of glyceraldehyde-3-phosphate dehydrogenase of the related organisms Leishmania mexicana and Trypanosoma brucei. One of these compounds, 2-guanidino-benzimidazole, had an 50% inhibitory concentration of 10 M in our assay. Two derivatives of this compound were identified with in vitro activity at even lower concentrations. In addition, the assay was modified for testing compounds for lytic activity against the bloodstream form of the parasite under conditions used for storing blood products. Thus, an assay with ␤-galactosidase-expressing T. cruzi greatly simplifies screening drugs for selective anti-T. cruzi activity, and three promising new compounds have been identified.

Despite substantial scientific progress over the past two decades, malaria remains a worldwide burden that causes hundreds of thousands of deaths every year. New, affordable and safe drugs are required to overcome increasing resistance against artemisinin-based treatments, treat vulnerable populations, interrupt the parasite life cycle by blocking transmission to the vectors, prevent infection and target malaria species that transiently remain dormant in the liver. In this Review, we discuss how the antimalarial drug discovery pipeline has changed over the past 10 years, grouped by the various target compound or product profiles, to assess progress and gaps, and to recommend priorities.

A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429), originally detected in California, carries spike glycoprotein mutations S13I in the signal peptide, W152C in the N-terminal domain (NTD), and L452R in the receptor-binding domain (RBD). Plasma from individuals vaccinated with a Wuhan-1 isolate-based mRNA vaccine or convalescent individuals exhibited neutralizing titers, which were reduced 2-3.5 fold against the B.1.427/B.1.429 variant relative to wildtype pseudoviruses. The L452R mutation reduced neutralizing activity of 14 out of 34 RBD-specific monoclonal antibodies (mAbs). The S13I and W152C mutations resulted in total loss of neutralization for 10 out of 10 NTD-specific mAbs since the NTD antigenic supersite was remodeled by a shift of the signal peptide cleavage site and formation of a new disulphide bond, as revealed by mass spectrometry and structural studies.

We have obtained four monoclonal antibodies, IB4, OKM1, OKM9, and OKM1O, all directed against the C3bi receptor of human monocytes and macrophages (M4). Two criteria were used to determine the specificity of these antibodies. First, culture surfaces coated with the antireceptor antibodies caused specific down modulation of C3bi receptor activity on MO adherent to these substrates. Second, receptor protein purified by using IB4 or OKM1 retained the ability to bind selectively to C3bi-coated erythrocytes. Each of the antibodies recognizes a distinct epitope on the C3bi receptor; they do not compete with one another for binding to monocytes. Further, when immobilized on a solid support, each of the antibodies binds a molecule from MO lysates that can simultaneously bind one of the other monoclonal anti-C3bi receptor antibodies. OKM1O binds and masks the ligand-binding site of the C3bi receptor, while IB4, OKM1, and OKM9 bind to sites remote from the C3bi binding site. All four antibodies immunoprecipitated polypeptides of Mr 185,000 and 105,000 from '251-surface-labeled M4. IB4 also precipitates polypeptides of Mr 185,000, 153,000, and 105,000. We conclude that the C3bi receptor of human MO is a complex composed of two polypeptides, Mr 185,000 and 105,000. We have identified monoclonal antibodies reacting with four distinct antigenic determinants of this complex. The determinant recognized by antibody OKM10 is at or near the ligand-binding site of the receptor. The determinant recognized by antibody IB4 is shared by at least two other leukocyte surface proteins.The third component of complement, C3, binds covalently to cell surfaces (1) yielding a species, C3b, that is recognized by receptors on leukocytes (2). Surface-bound C3b is rapidly cleaved by the serum enzyme, I, to yield an altered form, C3bi, that is also recognized by receptors on leukocytes (3, 4). There are separate receptors for C3b and C3bi on human monocytes, and it has been shown that each type of receptor can independently mediate phagocytosis of C3b-or C3bi-coated particles (5). We are particularly interested in the receptors for C3 because their ability to promote phagocytosis is regulated: human macrophages (MO) bind but do not ingest C3b-or C3bi-coated erythrocytes, but MO readily ingest both C3b-and C3bi-coated erythrocytes after a brief treatment with the tumor-priomoting compound phorbol myristate acetate (5).Fearon (6) (8). Monoclonal antibodies against the human C3b receptor (antiC3bR) have been described (9), as have monoclonal antibodies, OKM1, OKM9, OKM10, and OKM5 (10). Fab fragments of 1B4 were prepared by papain digestion (11).Cells. Human blood monocytes were purified and cultured for 5-8 days in Teflon beakers as described (5). Cultured monocytes (MO) were surface labeled with 1"I by the lactoperoxidase-glucose oxidase procedure (12).Sheep erythrocytes bearing C3b (EC3b) or C3bi (EC3bi) were prepared as described (5). The attachment of EC3b or EC3bi to monolayers of phagocytes was scored visually in duplicate wells (5). T...

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds designed to be inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site at residue 128. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii cells expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to this class of selective kinase inhibitors. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable.The food-borne apicomplexan protozoan Toxoplasma gondii is the causative agent of toxoplasmosis and may be the most common infectious eukaryotic parasite of humans, based Correspondence should be addressed to W.C.V.V. (wesley@u.washington.edu) or E.A.M. (merritt@u.washington.edu). Accession codes. Protein Data Bank: Atomic coordinates and structure factors have been deposited with accession numbers 3I79 (apo), 3I7c (NA-PP2 complex) and 3I7b (NM-PP1 complex).Note: Supplementary information is available on the Nature Structural & Molecular Biology website. AUTHOR CONTRIBUTIONSK.K.O., K.R.K., K.K.I. and W.C.V.V. were involved in the biochemical characterization and testing of inhibitors of TgCDPK1; L.J.C., K.K.O., K.R.K., A.J.N., C.L.M.J.V., F.S.B. and W.C.V.V. selected, cloned and purified the recombinant wild-type and mutant TgCDPK1 protein; E.T.L., J.E.K., T.L.A., L.Z., W.G.J.H. and E.A.M. crystallized and solved the structure of TgCDPK1; R.M. and D.J.M. synthesized the inhibitors; A.E.D. and M.P. performed the cellular T. gondii experiments; K.K.O., E.T.L., A.E.D., D.J.M., M.P., E.A.M. and W.C.V.V. wrote the paper; all authors reviewed and edited the paper. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/. 10 . These drugs are problematic in that they can cause rash, leucopenia and nephrotoxicity11, and sulfadiazine and pyrimethamine can result in complications during pregnancy. New therapeutics against T. gondii are needed. NIH Public AccessCalcium levels have long been associated with T. gondii's interrelated processes of invasion, gliding motility and secretion 12 . The intracellular Ca 2+ level oscillates during gliding motility and is promptly dampened upon cell invasion, preventing T. gondii from immediately gliding out of cells 13 . Calcium oscillations control many targets in the cell, and the mediation of invasion, micronemal secretion and gliding motility is thought to be largely due to T. gondii calcium-dependent protein kinases (TgCDPKs) 12,14 .Protein ...