Diversity-oriented synthesis yields novel multistage antimalarial inhibitors

Abstract

Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

Extended Data Figure 1. Three screening-hit series yield new compound scaffolds against known targets

a–d, BRD0026 exhibits the same mode of action as NITD609 and showed moderate in vitro potency against asexual (EC₅₀ = 0.346 µ M) and late-sexual (EC₅₀ = 1.98 µM) blood stages of the parasites and exhibited reduced potency against P. falciparum NITD609^R (EC₅₀ = 1.77 µM), a transgenic strain carrying a point mutation in P. falciparum ATPase4 (ref. 9). P. falciparum ATPase4 is the presumed molecular target of NITD609 (ref. 9). a, b, Three of the eight possible stereoisomers (R,S,R; S,S,S; and R,S,S) of BRD0026 have activity. c, Initial characterization of BRD0026 showed good solubility in PBS and low cytotoxicity. d, Treatment with BRD0026 resulted in a rapid increase in the parasite cytosolic Na⁺ concentration, while artesunate- or mefloquine-treated parasites maintained a constant cytosolic Na⁺ concentration. This result suggests that parasites treated with BRD0026 are not able to counter the influx of Na⁺ by actively extruding the cation, similar to the proposed mechanism for NITD609 (data are mean ± s.d.; two biological and two technical replicates). e–h, BRD7539 targets and inhibits P. falciparum DHODH. BRD7539 showed excellent in vitro potency against liver-stages (EC₅₀ = 0.015 µM) and asexual blood-stages (EC₅₀ = 0.010 µM) of the parasite, conferring markedly reduced potency against PfscDHODH. This strain heterologously expresses the cytosolic S. cerevisiae DHODH, which does not require ubiquinone as an electron acceptor. Thus, this transgenic strain is resistant to inhibitors of mitochondrial electron transport chain functions. BRD7539 was tested against three different P. falciparum strains with mutations in mitochondrial genes targeted by other antimalarial agents: (i) TM90C6B strain, containing a point mutation in the quinol oxidase domain of P. falciparum cytochrome b (Q_o site) and resistant to atovaquone; (ii) a P. falciparum CYTb^G33V mutant strain, selected against IDI5994 and containing a point mutation in the quinone reductase site of P. falciparum cytochrome b (Qi site); and (iii) a P. falciparum DHODH^E182D mutant strain, selected against Genz-666136 and containing a point mutation in the P. falciparum DHODH gene. BRD7539 exhibited an approximately 59-fold shift in potency against the P. falciparum DHODH^E182D strain, whereas potency was unaffected in the TM90C6B and P. falciparum CYTb^G33V strains. BRD7539 also inhibits recombinant P. falciparum DHODH in an in vitro biochemical assay (IC₅₀ = 0.033 µ M) but not the human orthologue. Altogether, these results demonstrate that BRD7539 targets P. falciparum DHODH. e, f, Only two (S,S,S and R,S,S) of eight possible stereoisomers of BRD7539 showed activity. g. In vitro growth inhibition assays showed no change in activity in P. falciparum CYTb^G33V and TM90C6B strains but exhibited a tenfold change in potency in P. falciparum DHODH^E182D strain, indicating that BRD7539 targets P. falciparum DHODH but not P. falciparum cytochrome bc1h, BRD7539 inhibited recombinant P. falciparum DHODH in vitro with an IC₅₀ of 33 nM; no inhibition of the human orthologues was observed (data are mean ± s.d. for two biological and two technical replicates). i–m, BRD73842 targets and inhibits P. falciparum PI4K. BRD73842 showed excellent in vitro activity against asexual (EC₅₀ = 0.069 µM), late-sexual blood-stage (EC₅₀ = 0.643 µM) and liver-stage (EC₅₀ = 0.459 µM) parasites. i, j, The structure of BRD73842 indicates the required stereochemistry for activity (R stereoisomer). k, Initial characterization of BRD73842 showed good solubility and limited cytotoxicity. To gain insight into the mechanism of action of BRD73842, two resistant P. falciparum lines were evolved against BRD73842 from four independent cultures (a total of over 4 × 10⁹ inocula, see Extended Data Fig. 2a). After more than 3 months of drug pressure, the EC₅₀ values increased approximately 10- to 20-fold. Two clones were obtained from each culture. Sequence analyses revealed that all clones contain non-synonymous SNVs in PF3D7_0509800, the locus that encodes P. falciparum PI4K (Supplementary Table 3). l, To confirm that PI4K is the molecular target of BRD73842, the compound was assayed against purified recombinant P. vivax PI4K protein. BRD73842 selectively inhibits the kinase activity of P. vivax PI4K (IC₅₀ = 21 nM), but not human PI4K. P. falciparum PI4K has been identified as the molecular target of two recently described antimalarial compounds, KAI407 (ref. 20) and MMV048 (ref. 21).(data are mean ± s.d.; two biological and two technical replicates). m, The biphasic dose–response curve is a signature of P. falciparum PI4K inhibitors (data are mean ± s.d.; three biological and three technical replicates). The EC₅₀ values reported in this study are derived from the first transition of the dose–response curves (indicated by arrow).

Extended Data Figure 2. Resistance selection of BRD38427 and BRD1095

a, Over 3 months of intermittent and increasing resistance selection pressure of BRD73842 starting at 150 nM (EC_99.9) or 0.5 µM (10× EC₅₀) yielded two cultures showing a 13- to 16-fold EC₅₀ shift. Two clonal lines from each culture were developed and subjected to whole-genome sequencing. b, Over 3 months of intermittent pressure of BRD1095 at 60 nM (EC_99.9) or 150 nM (10 ×EC₅₀) yielded three cultures showing a 3- to 67-fold EC₅₀ shift. Two clonal lines from each culture were developed and subjected to whole-genome sequencing.

Extended Data Figure 3. In vivo blood-stage efficacy study of BRD7929

a, BRD7929 shows single-dose in vivo efficacy in a P. berghei model of malaria. CD-1 mice were inoculated intravenously with approximately 2 × 10⁷ P. berghei (ANKA GFP-luc) blood-stage parasites intravenously 24 h before treatment and BRD7929 was administered as a single 50, 25, or 12.5 mg kg⁻¹ dose orally at 0 h (n = 4 for each group, this study was conducted once). Infections were monitored using IVIS. A single 100 mg kg⁻¹ dose of artesunate results in rapid suppression of parasites, but owing to its short half-life, the parasites re-emerge very quickly. A single 25 mg kg⁻¹ dose of BRD7929 resulted in 100% cure of the infected animals. One in four animals treated with a single oral dose of 12.5 mg kg⁻¹ showed recrudescence at 6 days after treatment, but all other animals administered with 12.5 mg kg⁻¹ were also completely parasite-free for 30 days. To ensure that no viable parasites remained, approximately 100 µl of combined blood samples from the four animals treated with 25 mg kg⁻¹ of BRD7929 was intravenously injected into two naive mice and parasitaemia was monitored for an additional 30 days. No parasites were detected, suggesting that BRD7929 achieved a sterile cure for P. berghei with a single oral dose of as low as 25 mg kg⁻¹. The same colour scale is used for the all images; not all time-point images are shown here. b, Bioluminescent intensity was quantified from each mouse and plotted against time. The dotted horizontal line represents the mean bioluminescence intensity level obtained from all the animals before the parasite inoculation. c, BRD7929 shows single-dose in vivo efficacy in a P. falciparum huRBC NSG mouse blood-stage model. huRBC NSG mice were inoculated intravenously with approximately 1 × 10⁷ P. falciparum 3D7^HLH/BRD blood-stage parasites 48 h before treatment and BRD7929 was administered as a single 50, 25, 12.5 or 6.12 mg kg⁻¹ dose orally at 0 h (n = 2 for each group, this study was conducted once). Infections were monitored using the IVIS. No recrudescence was observed at doses as low as a single 12.5 mg kg⁻¹ of BRD7929 in the infected animals. To ensure that no viable parasites remained, approximately 350 µl of combined blood samples from the two animals treated with 12.5 mg kg⁻¹ of BRD7929 was cultured in vitro and monitored for an additional 30 days. No parasites were detected, suggesting that BRD7929 achieved a sterile cure for P. falciparum 3D7^HLH/BRD with a single oral dose as low as 12.5 mg kg⁻¹ (see Fig. 4a). The same colour scale is used for the all images; not all time point images are shown. Images of mice treated with vehicle on days 11 and 20 are not shown, because the bioluminescent signal was too high to show in the same colour scale as other images.

Extended Data Figure 4. In vivo liver-stage efficacy study of BRD7929 in a mouse malaria model

a, BRD7929 shows single-dose causal prophylaxis in a P. berghei liver-stage model. CD-1 mice were inoculated intravenously with approximately 1 × 10⁵ freshly dissected P. berghei ANKA luc-GFP sporozoites freshly dissected from A. stephensi salivary glands and immediately treated with a single oral dose of BRD7929 (25, 5, 1 or 0.2 mg kg⁻¹). Infections were monitored using IVIS; mice were monitored until day 30 to ensure complete cure. No recrudescence was observed at doses as low as a single 5 mg kg⁻¹ of BRD7929 in the infected animals (n = 4 for each group, study conducted once). The same colour scale is used for the all images. Not all time point images are shown. b, Bioluminescent intensity was quantified from each mouse and plotted against time. c, BRD7929 shows single-dose causal prophylaxis in a P. berghei liver-stage model up to 3 days before infection and two days after infection. CD-1 mice were infected with P. berghei and infections were monitored as described in a. Single oral doses of BRD7929 (10 mg kg⁻¹) were administered at days 5, 3, and 1 before infection (days −5, −3 and − 1), on day 0, and on days 1 and 2 after infection (n = 4 for each group, this study was conducted once). All dosing regimens except for the day − 5 dose offered complete protection from infection for 32 days, indicating that BRD7929 has potent causal prophylaxis activity. The same colour scale is used for all images. Not all time-point images are shown.

Extended Data Figure 5. In vivo liver-stage efficacy study of BRD7929 in a humanized mouse model

a, BRD7929 shows single-dose in vivo efficacy in a P. falciparum huHep FRG-knockout mouse liver-stage model. huHep FRG knockout mice were inoculated intravenously with approximately 1 × 10⁵ P. falciparum (NF54HT-GFP-luc) sporozoites and BRD7929 was administered as a single 10 mg kg⁻¹ oral dose 1 day after inoculation (n = 2 for each group, this study was conducted once). Infections were monitored using IVIS. The same colour scale is used for all images. No increase in bioluminescence intensity level was observed from the mice treated with BRD7929 (see Fig. 4b). b, Blood samples were also collected from each mouse 7 days after inoculation (the first day of the blood stage) and analysed for the presence of the blood-stage transcripts PF3D7_0812600 (P. falciparum UCE) using qRT–PCR (two biological replicates for each group and three technical replicates for each biological sample). Each dot represents a technical replicate of a sample and each horizontal line represents a mean of technical replicates from each mouse. The presence of the blood-stage parasite specific transcripts was detected from the control (vehicle) mice, while no amplification of the marker was detected after 40 amplification cycles (C_t value = 40) from the mice treated with BRD7929. Primer efficiency and sensitivity of the primer pairs for P. falciparum UCE have a detection limit ranging between 10 and 100 transcript copies. Approximately 110 µl of combined blood samples from the two treated animals was also cultured in vitro and monitored for an additional 30 days but viable parasites were not detected.

Extended Data Figure 6. In vivo transmission-stage efficacy study of BRD7929

a, Oral doses of BRD7929 2 days before feeding mosquitoes upon infected mice resulted in complete blocking of transmission at 5 mg kg⁻¹, and reduced transmission activity at 1.25 mg kg⁻¹ and 0.31 mg kg⁻¹ (n = 2 for each group, this study was conducted once). b, Mosquitoes fed on vehicle-treated mice showed heavy infection 1 week after feeding, while mosquitoes fed on treated mice showed no or very few oocysts in the midguts. Representative images are shown; scale bars, 100 µm. c, To confirm that BRD7929 eliminates mature gametocytes in the host circulation rather than killing gametes, zygotes or ookinetes in the mosquito midgut, CD-1 mice infected with P. berghei (parasitaemia between 11 to 19%) were first treated with BRD7929 (oral, 25 mg kg⁻¹). Infected mice were then exposed to female A. stephensi mosquitoes for blood feeding 1, 4 or 10 days after the treatment. Blood samples were also obtained before the blood feedings to measure the plasma concentration of remaining BRD7929 (n = 2 for each group, this study was conducted once). No oocysts were found in midguts dissected from mosquitoes from all time points, whereas 896.5, 170.5 and 8.6 ng ml⁻¹ of the compound remained in the circulation 1, 4 and 10 days after respectively treatment, respectively, suggesting that BRD7929 eliminated mature gametocytes in the mice. d–f. In vivo transmission-stage efficacy study of BRD7929 (humanized mouse model). huRBC NSG mice were infected with the blood-stage P. falciparum 3D7^HLH/BRD for 2 weeks to allow the gametocytes to mature fully and were treated with a single oral dose of BRD7929 (12.5 mg kg⁻¹). n = 2 for each group, this study was conducted once. Blood samples collected from vehicle- and BRD7929-treated mice were tested for the presence of gametocyte-specific transcripts using mature gametocyte marker (PF3D7_1438800; d) and immature gametocyte marker (PF3D7_1477700; e). PF3D7_1120200 (P. falciparum UCE), a constitutively expressed gene, was used as a positive control marker for parasite detection (f). Data are mean ± s.d.; three technical replicates for each biological sample.

Extended Data Figure 7. Safety and resistance propensity profiling of the bicyclic azetidine series

a, Results of in vitro cytotoxicity, phototoxicity and CYP inhibition assays. * Phototoxicity was assessed using the NIH 3T3 neutral red assay at Cyprotec; †CYP1A, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A; ‡CYP1A, CYP2C9, CYP2D6, CYP3A. b, Histopathology analysis of mice treated with a high dose (100 mg kg⁻¹) of BRD7929. CD-1 mice were orally treated with 100 mg kg⁻¹ BRD7929 and organs were collected 10 days after treatment. No significant tissue damage was detected. Representative images are shown here. Scale bars, 200 µm. c, d, Measurement of the minimal inoculum for resistance of BRD7929. Cultures containing various numbers of inoculum (1 × 10⁵–1 × 10⁹) were exposed to a constant level of drug pressure (EC₉₀). Parasites developed resistance to atovaquone at the lowest inoculum of 1 × 10⁷ but not to BRD7929.

Figure 1. Cascading triage strategy reveals targets for some of the hit compounds and highlights potential novel mechanisms of action for others

a–e, A total of 468 compounds (‘positives’ in the growth inhibition primary assay) were tested in dose against P. falciparum Dd2, a transgenic P. falciparum line expressing Saccharomyces cerevisiae DHODH (PfscDHODH), a P. falciparum strain resistant to NITD609 (Pf NITD609^R) and a mammalian cell line (HepG2). P. falciparum ATPase4 is the presumed molecular target of NITD609 (ref. 9). a, Compounds were clustered across the horizontal axis by structural similarity. Colours represent compound potency (EC₅₀). Two compound clusters, exemplified by BRD0026 (b) and BRD7539 (c), showed selectively reduced potency against the Pf NITD609^R and PfscDHODH strains, respectively, while BRD73842 (d) and BRD3444 (e) were equipotent across the three P. falciparum strains. Pb, P. berghei; Pf, P. falciparum; Pv, P, vivax; PheRS, phenylalanyl-tRNA synthetase.

Figure 2. Structures of key compounds, SSAR study of BRD3444 and X-ray crystal structure of BRD7929

a, Structures of four bicyclic azetidine compounds. b, SSAR of BRD3444 showing that stereoisomers at the C₂ position are equipotent, which suggests that this position is not necessary for activity. c, X-ray crystal structure of BRD7929 showing 3D conformation (BRD7929 was crystallized as a salt with two equivalents of l-tartaric acid; only the structure of BRD7929 is shown for clarity).

Figure 3. The bicyclic azetidine series targets the cytoplasmic Pf PheRS

a, P. falciparum Dd2 clones resistant to BRD1095, a derivative of BRD3444 with increased aqueous solubility, were selected in vitro and non-synonymous SNVs were identified via whole-genome sequencing. All clones from three individual flasks contained non-synonymous SNVs within the PF3D7_0109800 locus, which encodes the alpha subunit of the cytoplasmic PheRS. b, The non-synonymous SNVs identified in clones from flask 1 (red), flask 2 (blue), and flask 3 (green) are shown overlaid on a homology model based on the human cytoplasmic PheRS (PDB accession 3L4G) generated in PyMol. c, BRD1095 was assayed against purified recombinant proteins of wild-type cytosolic Pf PheRS and a mutant containing a SNV (giving a L550V substitution), identified from the resistant strain. IC₅₀ value of the wild-type PheRS was 0.045 µM, whereas the IC₅₀ value for BRD1095^L550V was 1.30 µM (data are mean ± s.d. for two biological and two technical replicates). d, The bicyclic azetidine series showed a strong correlation between blood-stage growth inhibition and biochemical inhibition of cytosolic Pf PheRS activity. We assayed 15 bicyclic azetidine analogues with varying potency against blood-stage parasites (Dd2 strain) against purified recombinant Pf PheRS. The biochemically derived IC₅₀ values correlate strongly (r² = 0.89) with the EC₅₀ values determined using the blood-stage growth inhibition assay (see Extended Data Table 2 for structure-activity relationship study and chemical structures).

Figure 4. In vivo efficacy studies of BRD7929 using P. falciparum and humanized mouse models

a, huRBC NSG mice were inoculated with P. falciparum (3D7^HLH/BRD) blood-stage parasites 48 h before treatment and BRD7929 was administered as a single 50, 25, 12.5 or 6.25 mg kg⁻¹ oral dose at 0 h (n = 2 for each group, this study was conducted once). Infections were monitored using the in vivo imaging system (IVIS). Bioluminescent intensity was quantified from each mouse and plotted against time. The dotted horizontal line represents the mean bioluminescence intensity level obtained from all the animals before the parasite inoculation. No recrudescence was observed as low as a single 25 mg kg⁻¹ dose of BRD7929 in the infected animals (see Extended Data Fig. 3b). b, huHep FRG-knockout mice were inoculated intravenously with P. falciparum (NF54HT-GFP-luc) sporozoites. BRD7929 was administered as a single 10 mg kg⁻¹ oral dose 1 day after inoculation, and daily engraftment of human erythrocytes was initiated 5 days after inoculation (n = 2 for each group, this study was conducted once). Infections were monitored using IVIS. The dotted horizontal line represents the mean bioluminescence intensity level obtained from all the animals before the sporozoite inoculation. No increase in bioluminescence intensity level was observed from the mice treated with BRD7929 (see Extended Data Fig. 5a). c, huRBC NSG mice were infected with blood-stage P. falciparum (3D7^HLH/BRD) parasites for 2 weeks (allowing the gametocytes to mature fully) and were treated with a single oral dose of BRD7929 (12.5 mg kg⁻¹). Blood samples were collected for 11 days and analysed for the presence of the asexual marker SBP1 and the mature gametocyte marker Pfs25 using qRT-PCR (n = 2 for each group, this study was conducted once). The transcription of both SBP1 and Pfs25 decreased to undetectable levels 7 days after treatment, strongly suggesting that BRD7929 eliminates both asexual and gametocyte stages and is capable of preventing parasite transmission to the mosquito (data are mean ± s.d. for three technical replicates for each biological sample).