Repositioning and Characterization of 1-(Pyridin-4-yl)pyrrolidin-2-one Derivatives as Plasmodium Cytoplasmic Prolyl-tRNA Synthetase Inhibitors.

Prolyl-tRNA synthetase (PRS) is a clinically validated antimalarial target. Screening of a set of PRS ATP-site binders, initially designed for human indications, led to identification of 1-(pyridin-4-yl)pyrrolidin-2-one derivatives representing a novel antimalarial scaffold. Evidence designates cytoplasmic PRS as the drug target. The frontrunner 1 and its active enantiomer 1-S exhibited low-double-digit nanomolar activity against resistant Plasmodium falciparum (Pf) laboratory strains and development of liver schizonts. No cross-resistance with strains resistant to other known antimalarials was noted. In addition, a similar level of growth inhibition was observed against clinical field isolates of Pf and P. vivax. The slow killing profile and the relative high propensity to develop resistance in vitro (minimum inoculum resistance of 8 × 105 parasites at a selection pressure of 3 × IC50) constitute unfavorable features for treatment of malaria. However, potent blood stage and antischizontal activity are compelling for causal prophylaxis which does not require fast onset of action. Achieving sufficient on-target selectivity appears to be particularly challenging and should be the primary focus during the next steps of optimization of this chemical series. Encouraging preliminary off-target profile and oral efficacy in a humanized murine model of Pf malaria allowed us to conclude that 1-(pyridin-4-yl)pyrrolidin-2-one derivatives represent a promising starting point for the identification of novel antimalarial prophylactic agents that selectively target Plasmodium PRS.

Malaria remains a global scourge with 229 million cases of malaria and 409 000 deaths reported in 2019.[1] Human malaria is caused by five different species of parasites belonging to the Plasmodium genus. Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are the most prevalent species, the former being the most lethal. Increasing concerns of resistance to current drugs, including artemisinin combination therapies,[2,3] underscore the urgent need for novel antimalarials.[4,5] In that regard, focused target candidate and product profiles have been recently defined.[6]

The natural product febrifugine, with proven antimalarial activity in human,[7,8] has been shown to kill parasites through cytoplasmic prolyl-tRNA synthetase (cPRS) inhibition, demonstrating its clinical validation as an antimalarial target.[9] Febrifugine and its synthetic analogues, such as halofuginone,[10] (Figure ) had unacceptable side effects precluding their further development but not detracting from the clinical relevance of this antimalarial target.[8,11] In addition, this chemical class was found to act through a proline-competitive binding mode[9] associated with a deleterious phenotypic drug resistance mechanism via accumulation of L-proline;[12] hence, it is necessary to explore alternative approaches to inhibit this enzyme. Allosteric inhibitors of PfPRS were reported to provide high selectivity against the human orthologue (HsPRS), but in vitro potency remained modest.[13] Recently, Takeda reported three novel chemotypes as HsPRS inhibitors that were shown to act through competitive binding in the ATP site.[14−16] High structural homology between HsPRS and PfPRS[17,18] offers an avenue to reposition HsPRS inhibitors for malaria but poses challenges with respect to selectivity over the human orthologue. In that regard, N-(2,3-dihydro-1H-inden-2-yl)-3-(piperidine-1-carboxamido)pyrazine-2-carboxamide, the most potent antiplasmodium molecule (Dd2 IC50 63 nM) from a recent patent application,[19] is a very close urea analogue of T-3767758, previously reported as HsPRS ATP-site binder[14] (Figure ). This compound was shown to exert its activity in a proline-uncompetitive fashion with no indication regarding selectivity over HsPRS.

Figure 1

Chemical structures of febrifugine, halofuginone, 2,3-dihydro-1H-inden-2-yl)-3-(piperidine-1-carboxamido)pyrazine-2-carboxamide and T-3767758.

Herein, we report the identification of 1-(pyridin-4-yl)pyrrolidin-2-one derivatives, a novel antimalarial class, through phenotypic screening of a set of HsPRS ATP-site binders. cPRS appears as the likely antimalarial target of this chemotype. The frontrunner 1 and its active enantiomer 1- exhibited low double-digit nanomolar activity against all laboratory strains tested, Pf/Pv clinical field isolates, and developing Pf liver schizonts. In addition, in vitro Pf killing rate and resistance profile associated with this chemotype were obtained. Finally, both compounds demonstrated in vivo oral efficacy in a humanized SCID malaria model.

Results

Identification of Novel Potent Antimalarials with Dual-Stage Activity

Approximately 200 HsPRS ATP-site binders from a Takeda proprietary library[20,21] were screened in a phenotypic Pf growth inhibition assay[22] and against HEK293 (human embryonic kidney) cell line to assess intraerythrocytic antiplasmodium activity and mammalian cytotoxicity, respectively. Results highlighted 1-(pyridin-4-yl)pyrrolidin-2-one derivatives as demonstrating attractive potency and acceptable selectivity index (SI > 50) against HEK293 cells.

Four representative compounds were nominated for resynthesis (Figure ). As one of the most potent molecules presenting a SI > 50, compound 1 was selected as the frontrunner. Its attractive profile was confirmed from fresh solid material (Pf3D7 IC50 = 10 nM, HEK293 CC50 = 769 nM). The screening set provided by Takeda was composed of racemates and enantiopure compounds, the latter presenting exclusively the S stereochemistry. Although this information was not available initially, enantiopreference was suspected. To confirm this suspicion, chiral separation was performed on compound 1. The respective potencies of the two enantiomeric forms 1- (+) and 1- (−) revealed that the antiplasmodium activity was associated almost entirely with the S enantiomer (Figure ). To maximize the chemical diversity within the selected set of molecules, 2 was chosen as a close structural analogue of 1. Polarity was introduced by replacing the alkylnitrile with a primary alcohol to alter the physicochemical properties. Compound 3- highlights the scope for modifications at the heterocyclic moiety attached to the benzylic group. Compound 4- harbors an anilide instead of the carboxamide present in the other molecules and has no substituents on the benzylic group. These modifications were well tolerated without any deleterious impact on selectivity against mammalian cells. This small set of compounds can only provide very preliminary observations. Bioassays guided structural–activity relationships on a larger number of compounds initially designed as HsPRS inhibitors, and interactive design of new molecules is required to expand upon these provisional observations.

Figure 2

Representative compounds of the 1-(pyridin-4-yl)pyrrolidin-2-one scaffold. 72 h DAPI growth inhibition assay, N = 1, duplicate points (same biological replicate). Pyrimethamine, chloroquine, artesunate, dihydroartemisinin, and puromycin were used as internal controls (mean IC50 = 0.005; 0.023; 0.0005; 0.0002; 0.091 μM, respectively). 72 h assay, N = 1, duplicate points. T-REx 293 cells transfected with plasmid encoding proline-rich peptide, N = 2, duplicate points; ND = not determined. = 2, duplicate points. Mean values, N = 4, duplicate points.

The most potent compound 1- was selected for Pf life-cycle profiling. 1- potently inhibited the formation of Pf liver schizonts[23] (IC50s of 13 and 9 nM against NF54 and NF175 strains, respectively, Figure S1) In contrast, no activity was evident against stage V gametocytes nor gamete formation based on a 1 μM incubation in the Pf male/female gamete formation assay.[24]

Evidence Supports Cytoplasmic PRS as the Antimalarial Target

Pf expresses two PRS enzymes: a cytoplasmic prolyl-tRNA synthetase (cPRS) and an apicoplast prolyl-tRNA synthetase (aPRS).[25] Compounds 2, 3-, and 4- were tested against Pf lines with an anhydrotetracycline (aTc)-inducible gene expression system to conditionally knockdown (cKD)[26] expression of cPRS or aPRS, and an unrelated dispensable reference target, tRNA dihydrouridine synthase (duRS; PF3D7_1408900). Using this inducible system, the regulated target protein level is highest at 50 nM aTc and reduced upon lowering aTc concentration. This allows profiling changes in parasite sensitivity to compounds after knockdown of putative target protein expression. When a target protein’s function is inhibited by a test compound, reducing its expression level is expected to result in hypersensitivity to the compound. No such hypersensitization is expected, however, if a nontarget protein’s expression is similarly titrated. Halofuginone, a known cPRS inhibitor,[9] was used as a positive control to benchmark relative responses of the cPRS, aPRS, and duRS cKD lines (Figure S2 and Table S1). Halofuginone inhibited the aPRS and duRS cKD lines with similar IC50 values at all aTc concentrations. In contrast, at 50 nM aTc (maximum possible cPRS expression), the cPRS cKD line was more sensitive to halofuginone than the aPRS and duRS cKD lines. This suggests that modification of the cPRS locus to achieve regulated expression most likely resulted in reduced maximal cPRS expression[26] in the cPRS cKD line relative to the aPRS and duRS control lines, which express cPRS from its native locus. As cPRS expression levels were reduced further by lowering aTc concentration, the cPRS cKD line became even more sensitive to halofuginone (Figure and Table S1). The basal and increasing hypersensitivity to halofuginone seen as aTc concentration is lowered for the cPRS cKD line, but not for the aPRS and duRS cKD lines, are consistent with the known inhibition of cPRS by halofuginone. Specifically, as cPRS levels decrease, lower halofuginone concentrations are required to inhibit cPRS activity to cause parasite death. Having validated the cPRS cKD line, compounds 2, 3-, and 4- were evaluated. As with halofuginone, only the cPRS cKD line displayed basal and aTc-dependent hypersensitivity (Figures and S2, IC50 values reported in Table S1). Overall, these data support cytoplasmic PRS as the antimalarial target for this series of compounds.

Figure 3

Dose–response curves for compounds incubated for 72 h with cPRS cKD parasites at low or higher aTc concentrations. Values are mean ± SEM (error bars). (A) Halofuginone as positive control, (B) compound 2, (C) compound 3-, (D) compound 4-.

Human Cellular Protein Synthesis Inhibition Assay

After obtaining further confidence that this chemotype exerts its antiplasmodium activity through inhibition of PRS, we focused our attention on the selectivity against the human orthologue. A protein synthesis inhibition assay previously established to monitor the intracellular synthesis of artificial proline-rich protein[14] was used to assess the effect due to inhibition of HsPRS for compounds 1, 2, 3-, 4-. Data are included in Figure . As these compounds were initially designed as HsPRS inhibitors, the observed selectivity at the cellular level in favor of antimalarial activity is encouraging for 1 and 4- (36- and 15-fold, respectively). On the basis of the data gathered, 1 was selected for antimalarial profiling to further evaluate the potential of repositioning this chemical series for malaria.

Activity against Resistant Laboratory Strains of Malaria and Clinical Field Isolates

Minimizing the risk of resistance is critical for development of novel malaria therapeutic agents. In that regard, pre-existing mutations can cause lack of correlation with potencies observed in vitro against sensitive parasite strains. Consequently, 1 was tested against various laboratory-resistant Pf strains to ensure activity against parasites resistant to historical antimalarial drugs.[27] Encouragingly, full activity was maintained against all resistant strains tested, irrespective of their resistance profile and geographical origin, with respect to the sensitive strain NF54 (Table ). At the same time, no cross-resistance was observed with representative antimalarial agents currently in development[4] (Table S2).

Table 1

In Vitro Intraerythrocytic Antimalarial Activity of 1 against Laboratory Strains (Chloroquine and Artesunate as Controls)

laboratory strains	mutated loci	compound 1 IC50 (μM)a	chloroquine IC50 (μM)a	artesunate IC50 (μM)a
NF54b	-	0.018	0.008	0.004
Dd2	Pfcrt, Pfmdr1, Pfdhfr, Pfdhps	0.024	0.157	0.006
K1	Pfcrt, Pfmdr1, Pfdhfr, Pfdhps	0.020	0.202	0.002
7G8	Pfcrt, Pfmdr1, Pfdhfr, Pfdhps	0.018	0.079	0.002
TM90C2b	Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, PfcytbQ0	0.017	0.135	0.005
Cam3.I	Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, Pfkelch13	0.015	0.180	0.011

72 h [3H]hypoxanthine incorporation assay, mean values from two independent biological replicates (variations between individual values did not exceed 11%).

Sensitive strain.

In addition, the active enantiomer 1- was tested against Pf and Pv Brazilian clinical field isolates[28] and demonstrated potent parasiticidal activity with median IC50 values of 9 and 15 nM, respectively (Figure S3, Table S3). Although the number of isolates tested was modest, these data suggest equipotency against the two most prevalent species of human malaria.

In Vitro Killing Profile

To further characterize the antimalarial profile associated with this chemotype, the in vitro killing profile of 1 was determined in a parasite reduction ratio (PRR) assay. This assay uses a limiting dilution technique to quantify the number of Pf parasites that remain viable after drug treatment.[29] Data are depicted in Figure with comparison to antimalarial reference drugs covering a broad range of different speed of kill profiles. Compound 1 was shown to be parasiticidal with a very slow killing profile (slower than atovaquone).

Figure 4

In vitro killing profile of 1 compared with reference antimalarial drugs (all tested at 10× their respective IC50s).

In Vitro Resistance Profile

To assess the in vitro resistance profile of 1, minimum inoculum resistance (MIR) selections were set up with a selection pressure of 3 × IC50. The first selection had a starting inoculum of 2 × 105Pf parasites in 24 replicates to give a parasite inoculum range from 2 × 105 (single replicate) to 4.8 × 106 (cumulation of all replicates). A second separate selection in parallel was set up with a starting inoculum of 2 × 107Pf parasites in triplicate for a higher inoculum range of 2 × 107 to 6 × 107 parasites. Parasites recrudesced on day 21 in the first lower starting inoculum selection in 6 of the 24 wells and on day 18 in all three wells of the second higher inoculum selection. Therefore, the MIR of 1 was determined to be 8 × 105 (4.8 × 106 divided by the 6 positive replicates). In contrast, the positive control DSM265,[30] which inhibits DHODH, never recrudesced within the smaller selection size (2 × 105 parasites) and recrudesced within the larger size (2 × 107 parasites) on day 25 in all three wells, giving it a MIR value of 2 × 107.

To confirm resistance, resistant bulk cultures from both selections were assayed, revealing a small (3- to 4-fold) increase in IC50 values (Figure S4). Whole genome sequencing performed on four 1-resistant clones showed copy number variations (CNV) with a 2-fold amplification of a 63 kb region on chromosome 12 (Tables S4–S5). The position and range of the amplification between the selection at 105 parasites (A2, C4, D1) and the one at 107 (D4) varied slightly, yet in all four cases, the amplified region included cPRS (PF3D7_1213800) (Figure S5). This discovery reinforced confidence in cPRS as a resistance determinant and likely target of this compound. No single nucleotide polymorphisms (SNPs) were found in these selections.

In Vitro DMPK and Physicochemical Properties

The in vitro DMPK (drug metabolism and pharmacokinetics) and physicochemical properties of this chemical series were then explored. Representative 1-(pyridin-4-yl)pyrrolidin-2-one derivatives were tested for oxidative metabolism in human and rat liver microsomes, and aqueous solubility was measured (Table ). Compounds showed low to moderate microsomal stability in both species studied, explainable by several metabolic hot spots (vide infra metabolite identification experiments) and a concomitant contribution of a nonoptimal LogD. The latter is corroborated by the higher microsomal stability exhibited by 2. Poor aqueous solubility at pH 6.5 was obtained for all compounds and highlights another parameter to be improved for this chemotype.

Table 2

In Vitro DMPK and Physicochemical Properties for Selected Compounds

compound	CL h-LMa (μL/min/mg protein)	CL r-LMb (μL/min/mg protein)	solubilityc (μg/mL)	LogDd
1	55	39	<1.6	4.1
2	41	11	3.1–6.3	3.2
3-S	249	79	<1.6	4.2
4-S	106	25	6.3–12.5	3.5

Intrinsic clearance in human liver microsomes.

Intrinsic clearance in rat liver microsomes.

Aqueous kinetic solubility measured at pH = 6.5.

Distribution coefficient estimation at pH 7.4 using chromatography

On the basis of these data and antiplasmodium potency, 1 was chosen for further in vitro DMPK profiling (Table ). Given the very poor solubility of 1 under near neutral conditions, we cannot rule out the possibility of solubility-related issues in the different in vitro assays; however, the data provide a general indication of further liabilities that need to be addressed for the series. Metabolic stability data were collected from mouse liver microsomes prior to conducting an in vivo efficacy proof-of-principle in a murine malaria model. 1 was revealed to be more stable in mouse hepatocytes than in mouse microsomes. The basis for this result is not currently understood and would require further investigation. Studies using Caco-2 cell monolayers demonstrated relatively good permeability, with no evidence for efflux transport (apical to basolateral Papp of 22 × 106 cm/s). In spite of the potential for solubility-related issues in the CYP inhibition assay with 1, the inhibition of CYP2C9 and CYP3A4 indicates that further optimization might be necessary.

Table 3

Additional In Vitro DMPK Characterization of 1

CL m-LMa (μL/min/mg protein)	CL m-Hepsb (μL/min/106 cells)	Caco-2 Pappc (10–6 cm/s)	CYP1A2 IC50 (μM)	CYP2C9 IC50 (μM)	CYP2C19 IC50 (μM)	CYP2D6 IC50 (μM)	CYP3A4 IC50 (μM)
71	3	22	>20	3.4	>20	13.1	2.4

Intrinsic clearance in mouse liver microsomes.

Intrinsic clearance in mouse hepatocytes.

Caco-2 cell permeability assay. Papp, permeability coefficient in the apical to basolateral direction.

Metabolite Identification and Glutathione Trapping Experiments

To assess potential reactive metabolite formation and evaluate potential liabilities with respect to the alkylnitrile moiety, a comprehensive metabolite and glutathione (GSH) adduct search was conducted on 1 following incubation with human liver microsomes (Figure S6). N-Demethylation of the pyrazole was the predominant metabolite (based only on relative peak area as authentic metabolite standards were not available and therefore did not allow estimation of the percent contribution of the metabolites) followed by aliphatic hydroxylation at the pyrrolidinone moiety and N-dealkylation cleavage of the pyrazole-benzyl moiety. Remarkably, the nitrile moiety appeared to be particularly metabolically stable with no hydrolysis observed. Traces of a putative GSH conjugation product were observed; however, its MS/MS spectrum was too weak for structure confirmation.

Rat In Vivo Pharmacokinetics

To get a better understanding of the PK properties, the in vivo pharmacokinetic profile of 1 was assessed in rats (Figure , Table ). Following intravenous (IV) administration, plasma concentrations declined with a terminal half-life (t1/2) of ∼9 h. A relatively low blood clearance of 12 mL/min/kg and a volume of distribution of 3.2 L/kg were estimated from the plasma data (rat whole blood-to-plasma ratio = 0.61). Given that binding to plasma proteins and to the in vitro test matrices (microsomes and hepatocytes) were not assessed, no attempts were made to relate the in vitro and in vivo intrinsic clearance values. Unchanged compound was not detected in urine, suggesting that direct urinary excretion did not contribute significantly to elimination and hepatic metabolism was the most likely basis for the observed in vivo clearance.

Figure 5

Plasma concentration versus time profiles for 1 in rats (n = 3 animals per dose group). IV vehicle: 5% DMSO in 1% Solutol HS15 (in saline), 1 mL per rat; PO vehicle: 0.5% HPMC/0.4% Tween 80 aqueous suspension, 5 mL/kg.

Table 4

In Vivo Pharmacokinetic Profile in Male Sprague Dawley Rats (Mean ± SD; n = 3 per Route) for 1

route	dose (mg/kg)	AUC0-∞ (h·μM)	F (%)	Cmax (μM)	tmax (h)	t1/2 (h)	CLplasma (mL/min/kg)	Vss plasma (L/kg)
IV	0.9	4.3 ± 0.4	-	-	-	9.0 ± 3.1	7.2 ± 0.7	2.0 ± 0.2
PO	3	5.3 ± 1.6	37 ± 12	0.9 ± 0.2	1.5 ± 0.9	7.2 ± 2.4	-	-

Following oral administration (PO), the half-life was comparable to that observed after IV dosing. Oral bioavailability (F) ranged from 30 to 50%. Noting that the in vivo blood clearance was equivalent to <20% of the nominal rat hepatic blood flow, it seems unlikely that hepatic first-pass elimination is the only factor limiting oral bioavailability. It is also unlikely that absorption is limited by permeability since the compound exhibited good membrane permeability across Caco-2 cell monolayers. Because of poor aqueous solubility, incomplete dissolution within the gastrointestinal tract may be a contributing factor to the somewhat limited bioavailability.

In Vivo Efficacy

Given the encouraging rat PK profile of 1, in combination with the in vitro metabolic stability data in mouse, an in vivo proof-of-concept experiment was performed. The antimalarial efficacy of 1 was assessed in a Pf humanized severe combine immunodeficient (SCID) mouse model,[31] following oral administration of a 4-day treatment at 50 mg/kg given once daily. Blood concentration time profile after oral administration of 50 mg/kg of 1 is included in Figure S7. At day 7 postinfection, 99.8% reduction in parasitemia was observed in comparison to untreated control mice (Figure ), with no adverse events. Testing the active enantiomer 1- in the same murine model confirmed in vivo activity with 80% parasitemia inhibition achieved at day 7 postinfection following a 4-day treatment dosage at 10 mg/kg (Table S6, Figure S8).

Figure 6

Therapeutic efficacy of 1 in the in vivo Pf NODscidIL2Rγnull mouse model of malaria compared with chloroquine as a reference antimalarial drug (average parasitemia in peripheral blood, n = 2 mice/group). The arrows indicate the days of oral treatment at 50 mg/kg in the 4-day test.

Off-Target Profiling

Finally, the risks related to possible off-targets of 1- were evaluated. Data in Table illustrate the remarkable selectivity of 1- against a large 303 human kinase panel[32] obtained with 1 μM. A small-scale promiscuity panel[33] highlighted only adenosine A3, an important receptor extensively studied as a therapeutic target in cardiovascular disorder,[34] as a possible concern. This off-target activity is not surprising for an ATP-competitive inhibitor and would require further investigation through functional assays in the event this activity is not removed during the optimization process for this chemical series.

Table 5

In Vitro Pharmacological Profile of 1-

off-target binding assays	IC50 (μM)
panel of 303 kinases	all >1a
ROCK1	>10
PDE4D2	>10
GR	>10
PPARG	8.4
5-HT2B	>10
adenosine A3	1.8
M1	>10
GABAA	>10

All <18% of inhibition when tested at 1 μM.

Discussion

Although PRS is a clinically validated target, there are currently no antimalarial drugs acting through this mode of action.[18] Inhibitors binding to the ATP site of the PRS enzyme, in particular, appear attractive since they circumvent the phenotypic drug resistance associated with accumulation of l-proline, assuming ATP levels are more tightly regulated in the cell and thus resistance being less likely to occur by elevation of ATP levels. Screening of a set of HsPRS ATP-site binders led to identification of 1-(pyridin-4-yl)pyrrolidin-2-one derivatives as potential novel antimalarials. Targeting the Plasmodium PRS, without inhibiting the human counterpart, remains a challenge with previous experience demonstrating that the repositioning process requires significant medicinal chemistry efforts.[35] Low nanomolar potency and preliminary data obtained on 1 in a human protein synthesis inhibition assay established to monitor the intracellular synthesis of artificial proline-rich protein are encouraging when considering that these molecules were initially designed for human indications. The selectivity level against the human orthologue would require further investigation in relevant biochemical assays that have been established by several groups.[13,17] Co-crystallization with Pf cPRS has also been carried out[13,17] and could provide useful information to advance this chemical series.

In vitro activity against Pf resistant strains and no cross-resistance with molecules currently in development demonstrated by compound 1 are paramount features, as pre-existing resistance would preclude development of this chemical class. Furthermore, ex vivo testing against clinical field isolates not only confirmed the high potency observed in vitro but also suggested equivalent potency against Pf and Pv. Although Pf is the most lethal species, Pv is the most widespread human malaria parasite in the Americas and Asia. Moreover, this species is responsible for most malaria relapse cases; thus, it has major public health significance.[36] The other less-represented human Plasmodium species P. ovale, P. knowlesi, and P. malariae should not be forgotten, especially in the context of mixed infections, and are planned to be assessed at the preclinical candidate stage.

The Pf life-cycle fingerprint studies showed dual-stage activity consistent with inhibition of cytoplasmic PRS as previously described.[9] Inducible knock-down of the Plasmodium gene expression was revealed to be a valuable approach for target identification.[37,38] When applied to this chemotype, this pointed to cPRS as the likely target. This was further validated by the observation of increased copy number in a region containing cPRS in resistant mutants obtained in vitro. The low-grade resistant mutants and increased copy number variants were obtained at a drug pressure of 3 × IC50, which might not be optimal for such a slow killing profile. Additional experiments at higher inoculum and higher drug pressure would be required to assess fully the in vitro resistance profile associated with this chemotype and target.

The slow killing profile of 1 and its relative high propensity to develop resistance in vitro, under the conditions tested, require careful evaluation of the suitability to develop this chemical series for treatment of uncomplicated malaria. Although antimalarial drugs are intended to be used in combination therapy, which could mitigate the rapid evolution of resistance, such a slow onset of action (>48 h) would not decrease patient fever within 24 h as desired for antimalarial treatment. Fast parasite clearance would therefore need to be achieved through the partner drug. Moreover, molecules with slow onset of action need to demonstrate exceptionally long half-lives to achieve the desired parasite reduction ratio as monotherapy. This would impose an additional constraint to the combination partner since matched pharmacokinetic profiles are desirable to avoid resistance that could arise when a drug is exposed on its own to a large number of parasites. On the other hand, causal prophylaxis does not require rapid onset of action. In that regard, 1 benefits from a compelling profile with potent blood stage and antischizontal activity, the latter providing an extra 5–6 days of protection from a new infection. This is further supported by the smaller number of parasites in the liver leading to a lower anticipated risk of resistance selection[6] that would be further mitigated through combination therapy.

Ambitious goals have been set for the development of new antimalarials with the aspiration for single oral dose cure and weekly dosing for treatment of uncomplicated malaria and prophylaxis, respectively.[6] Clearance and volume of distribution are therefore two paramount PK parameters to be considered during the lead optimization process in order to achieve long half-lives. Representative compounds from this chemical series suffer from oxidative metabolism and solubility issues. Preliminary data such as metabolic identification experiments suggest reasonable scope for improvements of these aspects while maintaining potency. Despite nonoptimal metabolic stability and physicochemical properties, the frontrunner 1 exhibited acceptable PK properties in rats, which suggests the potential for delivery of a long-lasting antimalarial after optimization of this chemical series. Although the volume of distribution is acceptable (3.2 L/kg), its increase could result in extended half-life. While being aware of the potential off-target risks associated with lipophilic bases, chemical modifications such as the introduction of basic amine moieties could be considered, provided they do not have a deleterious impact on clearance. On the other hand, the observed oral in vivo efficacy with no associated adverse events in a murine model of human malaria clearly demonstrates the future potential of the series.

Special attention to the overall safety package is even more paramount when considering use as a prophylactic agent. Although it is debatable whether the observed toxicity previously seen with febrifugine and its analogues is related to HsPRS inhibition or off-targets, PRS is being explored as an anticancer target, and its inhibition is expected to impact cell growth. As the main target population for antimalarials comprises children and pregnant women, achieving a high level of selectivity against HsPRS should be the primary focus during the next steps of optimization of this chemical series. Greater than 1000-fold selectivity against HsPRS was arbitrarily set as the ultimate goal for a possible preclinical candidate to minimize the risk of on-target toxicity. High selectivity against mammalian PRS (e.g., rodents, dog) is also desirable to simplify the analysis of toxicity findings during preclinical studies. Despite a reassuring profile against a large kinase panel, off-targets such as adenosine-related targets and others will be assessed as optimization progresses.

Conclusions

We have identified a novel antimalarial scaffold exerting its dual-stage activity most likely through inhibition of the Plasmodium cytoplasmic PRS enzyme, a clinically validated antimalarial target. Extensive profiling of the frontrunner compound 1 and its active enantiomer 1- have revealed (i) low double-digit nanomolar potency against all malaria asexual blood stage strains tested, (ii) strong antischizontal activity, (iii) a slow killing profile, and (iv) a frequent propensity to develop moderate resistance in vitro. In addition, good permeability but poor aqueous solubility and moderate metabolic stability were noted for the four representative compounds of this chemical series. Metabolic identification experiments indicated the main soft spots to guide rational design of more metabolically stable analogues. Compound 1 demonstrated oral efficacy in a murine model of human malaria. Overall, 1-(pyridin-4-yl)pyrrolidin-2-one derivatives constitute a promising starting point as antimalarial PRS inhibitors but medicinal chemistry efforts are needed to optimize selectivity against HsPRS while further improving antimalarial potency, pharmacokinetics, and physicochemical properties in order to achieve the profile required for new antimalarial prophylactic drugs.

Methods

Chemistry

See Supporting Information, pages S15 and S28–S36).

Intraerythrocytic Pf3D7 Inhibition 72 h Growth Assay

The intraerythrocytic Pf3D7 inhibition 72 h growth assay involves incorporation of the DNA-intercalating dye DAPI (4′,6′,-diamidino-2-phenylindole) to monitor changes in the parasite number observed within infected erythrocytes utilizing high content confocal image analysis. This assay was performed as previously described.[22] IC50 values were calculated through a four-parameter logistic curve fitting in GraphPad Prism v.6.

HEK293 Viability Assay

See Supporting Information, page S16.

Human Cellular Protein Assay

See Supporting Information, page S16.

Pf Liver Stage Assay

See Supporting Information, pages S16–S17.

Gamete Formation Assay

See Supporting Information, pages S17–S18.

Conditional Knock-Down Experiments

See Supporting Information, page S19.

Pf Resistant Laboratory Strains and Cross-Resistance

Pf resistant laboratory strains and cross-resistance testing was performed with the modified [3H]-hypoxanthine incorporation assay as previously reported.[27] IC50s were calculated by linear interpolation.

Pf and Pv Clinical Field Isolates[28]

Pf and Pv clinical field isolates were collected in July 2018 from patients recruited at the Tropical Medicine Research Center – CEPEM in Porto Velho, state of Rondônia, in the Brazilian Western Amazon. The blood and the written consent were collected by a trained nurse. The study was approved by the CEPEM Ethics Committee (CAAE 61442416.7.0000.0011) (see Supporting Information pages S19–S20 for detailed protocols).

Parasite Reduction Ratio Assay.[29]

The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol (see Supporting Information pages S20–S21 for detailed protocols).

MIR Studies

MIR studies were performed on two selection groups: 105 and 107 parasites (see Supporting Information page S21 for detailed protocols).

Whole-Genome Sequencing

Whole-genome sequencing was performed on four compound 1-resistant clones from the two selection groups (see Supporting Information page S22 for detailed protocols).

In Vitro DMPK and Solubility Studies

See Supporting Information, pages S22–S25.

In Vivo Rat PK Studies

In vivo rat PK studies were conducted using established procedures in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and the study protocols for 1 were reviewed and approved by the Monash Institute of Pharmaceutical Sciences Animal Ethics Committee (see Supporting Information pages S25–S26 for detailed protocols).

SCID Mouse

SCID mouse efficacy studies were performed as previously described[31] using Pf3D70087/N9 strain. The animal experiments were adhering to local and national regulations of laboratory animal welfare in Switzerland (awarded permission n°2303). Protocols are regularly reviewed and revised following approval by the local authority (Veterinäramt Basel Stadt) (see Supporting Information page S26 for detailed protocols).

Kinase and Small Promiscuity Panels

See Supporting Information, pages S26–S27.