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. 2026 Jul 30;11(31):46143–46153. doi: 10.1021/acsomega.6c02719

Identification of a Novel 1,3,4-Thiadiazole-Based Scaffold as a Plasmepsin V Inhibitor

Diana Zelencova-Gopejenko †,‡, Jekaterina Bolsakova †, Laura Rudusa †,§, Kristine Kitoka †, Atis Jekabsons †, Marija Skvorcova †, Aigars Jirgensons †, Raitis Bobrovs †,*
PMCID: PMC13470848  PMID: 42597946

Abstract

The growing resistance to existing antimalarial drugs, along with the expanding distribution of mosquito vectors transmitting Plasmodium species, underscores the urgent need for new therapeutic agents. Plasmepsins (PMs), a family of aspartic proteases in Plasmodium, among which plasmepsin V (PMV) is the most structurally distinct enzyme compared to other Plasmodium and human aspartic proteases, allowing for the achievement of selectivity against nontarget proteases. The current literature demonstrates that depletion of PMV leads to the immediate cessation of parasite growth after its invasion into red blood cells, establishing PMV as a compelling target for drug discovery. Here, we performed grating-coupled interferometry (GCI) and fluorescence resonance energy transfer (FRET) enzymatic assay screening of a fragment library to identify novel nonpeptidomimetic PMV inhibitors. A 1,3,4-thiadiazole-based compound 11b (LE = 0.45) was identified as a promising hit and subjected to a preliminary structure–activity relationship (SAR) exploration. A total of more than 30 hit analogues were synthesized or obtained commercially and evaluated. A structure–activity relationship exploration led to compounds 11ad and 11ae with IC50 (PvPMV) values of 22.0 and 20.7 μM, respectively. The resulting SAR proved largely flat, with modifications failing to break the sub-10 μM potency threshold. This plateau suggests that the current 1,3,4-thiadiazole core may be approaching its potency ceiling. Although these compounds display suboptimal inhibitory potency, they map out the structural boundaries of this novel nonpeptidomimetic chemotype against PMV.

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Introduction

Malaria is a severe vector-borne disease transmitted by Plasmodium-infected female Anopheles mosquitoes. In 2024, the World Health Organization (WHO) reported 610,000 deaths attributable to malaria, with children and pregnant women representing the most affected demographic groups. Among the five Plasmodium species infecting humans (P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi), P. falciparum and P. vivax are responsible for the majority of cases and fatalities. , The widespread emergence of resistance to nearly all clinically used antimalarial drugs, combined with declining efficacy of insecticide-treated nets and drug resistance, highlights the urgent need for therapeutics with novel mechanisms of action. −

Plasmepsins (Plasmodium pepsins, PMs) are a family of aspartic proteases of the Plasmodium parasite that represent promising targets for antimalarial drug development. Experimental studies have demonstrated that inhibition of PMV, IX, and X enzymes disrupts parasite growth in red blood cells. ,, To date, ten PMs have been identified in the P. falciparum genome. Among these, PMV is the most distinct member of the plasmepsins family. This essential protease is involved in the cleavage of the highly conserved Plasmodium Export Element (PEXEL, RxL↓xE/Q/D) motif of exported proteins. , Depletion of PMV results in immediate arrest of parasite growth after invasion into red blood cells. ,

Although PMV plays a critical biological role, relatively few inhibitors targeting this enzyme have been identified. The transition-state mimetic WEHI-916 (1), which structurally resembles the native PEXEL substrate, disrupts protein export in P. falciparum-infected erythrocytes and validates PMV as an antimalarial target. The peptidomimetic analogue WEHI-842 (2) inhibits PMV from both P. falciparum and P. vivax, with the half-maximal inhibitory concentrations (IC50) of 0.8 and 1.6 nM, respectively. Human immunodeficiency virus (HIV) protease inhibitors, such as lopinavir, inhibit PfPMV and suppress parasite proliferation at 20 μM. In addition, only a small number of nonpeptidomimetic inhibitors have demonstrated activity against PMV (Figure ). Aminohydantoins are potent nanomolar inhibitors of the Plasmodium aspartic proteases PMII and PMIV, and derivative 3 has also been reported to inhibit PfPMV moderately (IC50 = 0.98 μM). Naphthoquinone compounds 4 and 5 inhibit PfPMV with IC50 values of 22.3 and 68.9 μM, respectively. Bobrovs et al. identified potential PMV binders in a high-throughput virtual screening (HTVS), and subsequent experimental validation using a fluorescence resonance energy transfer (FRET) assay confirmed that compounds 6 and 7 had IC50 values of 16.1 and 23.7 μM, respectively. Hodder et al. identified the imino-pyrimidinone-fused pyrrolidine S-isomer 8, which potently inhibits in vitro protease activity of PMV (IC50 = 0.096 μM) and obtained its high-resolution X-ray crystallographic structure in complex with PvPMV. More recently, amodiaquine 9 has been shown to efficiently inhibit PfPMV catalytic activity through competitive inhibition, with an IC50 of 0.42 μM.

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Representative inhibitors 1–9 of PMV.

Results and Discussion

Preparation of PMV and Enzymatic Testing

Both PfPMV and PvPMV have been characterized as targets for inhibitor development. Recombinant expression performed under identical conditions demonstrated that PvPMV consistently yielded higher protein quantities and exhibited improved chromatographic behavior relative to PfPMV, which was more prone to aggregation under the tested conditions. The catalytic aspartyl protease core is highly conserved between the two orthologs, including preservation of the active-site architecture, substrate-binding cleft, and flap loop topology. Both enzymes contain a conserved unpaired cysteine residue located within the flap loop region (Cys140 in PvPMV and Cys178 in PfPMV), positioned in proximity to the catalytic dyad and accessible from the substrate-binding cleft. Considering the strong structural conservation of the catalytic machinery together with the superior recombinant yield and biochemical stability of PvPMV, the P. vivax enzyme was selected as a robust and primary model for structure-based inhibitor design and evaluation.

Recombinant PvPMV (residues R35-R476) was produced in High Five (Hi5) insect cells using the baculovirus expression system. Initial intracellular expression constructs, containing MBP, GST, or SUMO fusion tags, yielded detectable protein. However, the protein was predominantly found in the aggregated form (data not shown). These observations suggested suboptimal folding under cytosolic conditions.

PMV is localized in the parasite endoplasmic reticulum (ER), , where oxidative folding promotes proper disulfide bond formation. To mimic this environment, secretion-based constructs containing the gp67 signal peptide were used to prepare the protein (see Methods section). Enzymatic activity of recombinant PvPMV was evaluated using a FRET-based enzymatic assay under optimized buffer conditions. Secreted SUMO-PvPMV showed improved solubility and approximately 150-fold higher enzymatic activity compared to the corresponding intracellular construct. Size-exclusion chromatography resolved monomeric and dimeric forms of PvPMV (Figure S1). The monomeric fraction showed higher enzymatic activity and was therefore used for subsequent enzymatic assays. Comparison of the fusion and cleaved forms demonstrated that, although the SUMO domain is relatively small, its presence reduced catalytic activity by approximately 2.5-fold (Figure S2). Full substrate conversion was achieved in 25–35 min for cleaved PvPMV and in 50–60 min for fusion SUMO-PvPMV. Both reactions were conducted using identical protein concentrations. These data indicate that removal of fusion elements enhances access to the active site or improves conformational flexibility.

Screening for PMV-Binding Fragments

Maybridge Rule of Three (Ro3) Diversity Fragment Library (840 compounds, Thermo Scientific) was screened against monomeric PvPMV in two assays: Grating-coupled interferometry (GCI) and FRET enzymatic assay. The first one shows protein–ligand physicochemical binding and kinetic parameters, while the second one shows functional effects/inhibition of the enzyme. A combination of both methods allows to reduce the number of false positives and better characterization of compound binding profiles.

For GCI experiments, PvPMV was immobilized on the 4PCZ sensor chip surface using an amine-coupling reaction. The functional integrity of the immobilized enzyme was confirmed using WEHI-916 (1), a previously reported peptidomimetic inhibitor of PMV. The measured dissociation constant (K D = 424 nM) was approximately 1 order of magnitude weaker than the value reported by Surface Plasmon Resonance (K D = 42.0 nM) (Figure S3A). Enzymatic inhibition was further evaluated using a FRET-based assay, yielding an IC50 value of 0.55 μM (Figure S4). This value is approximately 17–23-fold higher than literature data (IC50 = 24–32 nM). , Differences in assay configuration, including enzyme immobilization strategy, substrate composition, and buffer conditions, may account for the observed variation. However, measured dissociation constant was consistent with the inhibitory potency determined in the FRET assay, supporting preservation of enzymatic activity upon immobilization and agreement between the two screening methods. Primary GCI screening identified 94 fragments exhibiting binding responses greater than 100 pg/mm2 at the screening concentration of 200 μM (Figure S5), indicating detectable interaction with immobilized PvPMV. These compounds were used in the WaveRAPID kinetics assay at 50 μM ligand concentration to determine their binding affinities and kinetic parameters (Figure S6). To reduce the possibility of identifying false positives and cross-validate the data, the same fragment library was screened using the FRET enzymatic assay. A known limitation of fluorescence-based assays is susceptibility to optical interference from intrinsically colored or fluorescent compounds. Here, potential data distortion could be observed when yellow-to-orange-colored compounds are screened, as the wavelengths for fluorescence measurements (λEm = 490 nm and λEx = 336 nm) overlap with the intrinsic absorbance spectra of such chromophores. Therefore, fragments exhibiting substantial spectral overlap under assay conditions were excluded from analysis. FRET assay allowed the identification of 37 fragments that inhibited PvPMV activity by more than 50% at the screening concentration. The representative top-scoring hits are shown in Figure (full table available in Supporting Information (SI)).

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Representative examples of scaffolds from GCI and FRET fragment library screening.

The 1,3,4-thiadiazole-based compound 11a was selected for further optimization due to its structural simplicity, novelty, synthetic accessibility, and the availability of commercial analogues. To verify the potency of the commercially obtained hit, it was resynthesized, quality checked, and tested in the FRET enzymatic assay. The resynthesized compound was simplified by removing the trifluoromethyl group, which was not expected to interact with the catalytic dyad. Compound 11b showed good ligand efficiency (LE = 0.45) due to minor potency changes upon trifluoromethyl group removal (Figure ).

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Modification of amino-1,3,4-thiadiazole-2­(3H)-thione core.

Mode of Action

In solution, the 1,3,4-thiadiazole-2­(3H)-thione scaffold exists in tautomeric equilibrium between thione and thiol forms, with the distribution governed by substituents, solvent, and temperature. Spectroscopic and computational data indicate that the thione tautomer is thermodynamically favored. Nevertheless, even as a minor species, the thiol tautomer may retain sufficient reactivity to undergo covalent bond formation with accessible cysteine residues. PMV is unique among aspartic proteases in possessing a nonconserved, unpaired cysteine within the flap loop adjacent to the catalytic site (Cys140 or Cys178 in P. vivax or P. falciparum, respectively). This structural feature provides a potential opportunity for covalent inhibition. Accordingly, the hit compound may function as a covalent inhibitor by targeting the flap-loop cysteine, potentially through nucleophilic aromatic substitution (SNAr) or disulfide bond formation. To assess whether PMV inhibition by 11b proceeds through a covalent or noncovalent mechanism, MALDI-TOF mass spectrometry and incubation time-dependent inhibition assay analysis of PMV–11b adduct formation were performed.

MALDI-TOF mass spectrometry experiments were performed on the untreated PMV and the 11b-treated PMV. Due to the large protein size (51.8 kDa), intact MALDI-TOF mass spectrometry analysis was not feasible; therefore, peptide mass fingerprinting following proteolytic digestion with trypsin or Glu-C was performed. Trypsin digestion of the samples under reducing conditions led to the detection of the peptide Cys133-Arg163 (m/z 3706.5 Da) in both the reference and 11b-preincubated PMV samples. No mass increase consistent with covalently bound 11b was detected in the treated sample (Figure A), indicating that 11b does not react with C140 via an SNAr mechanism. To test whether 11b forms a disulfide bond with C140, Glu-C peptide fingerprinting under nonreducing conditions was performed (Figure B). In addition to 11b, a validated covalent PMV inhibitor (structure not disclosed) was included as a positive control. In Glu-C digests, the peptide Tyr135-Glu141 was detected in negative ion mode. The peptide was detected in both native (m/z 922.3 Da) and oxidized (m/z 938.3 Da) form in the reference and 11b-preincubated PMV samples, while significantly lower levels of peptide, mostly in its oxidized form, were detected in the positive control sample. No mass increase consistent with a disulfide-bound 11b was detected (Figure B middle panel), whereas the peptide adduct corresponding to covalent modification by the positive control inhibitor was readily observed (Figure B right panel).

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MALDI-TOF mass spectra of PMV (black line), PMV treated with 11b (red line), or PMV treated with verified covalent inhibitor (yellow line) after proteolytic digestion with (A) trypsin and (B) Glu-C. The reactive cysteine Cys140-containing peptides are Cys133-Arg163 (m/z 3706.5 Da) in panel A, and Tyr135-Glu141 (m/z 922.3 Da in native form; m/z 938.3 Da in oxidized form) in panel B. The peptide mass increase after treatment with a verified covalent inhibitor is 405.4 Da. C The fluorescence increases during the PMV enzymatic reaction after treatment with 11b without preincubation (gray line), 10 min preincubation (blue line), and 120 min preincubation (purple line).

The noncovalent binding mode of 11b was further supported by FRET enzymatic assays conducted after different preincubation periods (Figure C). Preincubation of PMV with 11b in the absence of fluorogenic substrate did not result in a time-dependent decrease in enzymatic activity, as no progressive inactivation of PMV was observed. Moreover, the IC50 values determined after 10 min and 2 h preincubation were comparable to those obtained without preincubation. These findings are consistent with a reversible, noncovalent mode of inhibition.

Modification of 1,3,4-Thiadiazole Core Scaffold

To assess the feasibility of simplifying the 5-amino-1,3,4-thiadiazole-2­(3H)-thione core, a series of analogues (Figure ) was synthesized. Substitution of the thione group with a keto group (17) resulted in a complete loss of activity, while the replacement with a methyl group (18) or the removal of this group (19) yielded PMV inhibitors with slightly reduced potency. Elimination of the nitrogen-atom bridge in compound 20 led to reduced inhibitory activity compared to the original structure.

Oxidized analogues of 11b were also examined. Among these, sulfine 21 maintained substantial inhibitory potency with a slightly reduced ligand efficiency (LE = 0.39). Nitrogen- and sulfur-methylated analogues (23–25) showed lower PMV inhibitor potency than 11b (Figure ). This, most likely, is a result of a disrupted hydrogen bonding network, as installation of the methyl group at either position reduces the number of hydrogen bond donors from two to one. While there is no crystallographic information available on the 1,3,4-thiadiazole scaffold binding mode, the initial rationale for the potential scaffold optimization strategy relied on a computationally generated docking model, which must be interpreted with caution. The docking model suggests that the protonated 1,3,4-thiadiazole ring nitrogen atom (3N) is acting as a hydrogen bond donor, interacting with Asp313, either directly or via a water bridge, as demonstrated by other aspartic protease inhibitors. , At the same time, the deprotonated nitrogen atom of the 1,3,4-thiadiazole ring (4N) acts as a hydrogen-bond acceptor, forming a hydrogen bond with the protonated Asp80 residue. The linker amino group is believed to interact with a flap loop Ser138 main chain carbonyl group (Figure ). Methylation of the linker amino group or the terminal thione group seems to have less severe effects on potency, as these modifications do not directly interfere with a catalytic dyad binding. Installation of the methyl group at the 3N position removes the hydrogen bond donor believed to directly engage with a catalytic dyad; therefore, the impact on the binding potency is more pronounced. Interestingly, this does not render the compound completely inactive. This might be due to an alternative binding mode available for this compound. Replacement of the benzene ring at the amino-1,3,4-thiadiazole core with aliphatic groups, such as cyclohexyl (26) or isopropyl (27), led to a significant reduction in inhibitory activity. We speculate that this is due to the disruption of the stacking interaction between the ligand benzene ring and aromatic residues under the flap loop (Tyr135, Tyr139, His173, and/or Phe180). In light of the initial experimentally obtained SAR data, compound 11b was selected for further hit-to-lead optimization, focusing on the introduction of substituents on the benzene ring.

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Modification of amino-1,3,4-thiadiazole-2­(3H)-thione core.

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Compound 11b modeled in the active site of the PvPMV structure 8TYG. Compound (teal) and key PMV binding site residues (gray) are shown as sticks. The flap loop is shown in salmon. Yellow dashed lines indicate hydrogen bonds. Nonpolar hydrogen atoms are omitted for clarity.

Structure–Activity Relationships of Aniline Substitution

A total of more than 30 compounds, featuring substituents at the ortho, para, and meta positions of the benzene ring, were synthesized or obtained commercially and subsequently evaluated (Table ). The IC50 values were determined only for compounds that inhibited PvPMV by more than 75% at 100 μM concentration. Incorporation of small substituents in the ortho and meta positions (11e,g-i) resulted in either negligible changes or modest improvements in potency, whereas installation of sterically bulky substituents, such as isopropyl or phenyl (11c,d,f), reduced inhibition potency. Computational modeling suggests that the benzene ring of the hit compound binds under the flap loop, where spatial constraints restrict accommodation of substituents at the ortho and meta positions. In contrast, a more spacious cavity, also known as a flap pocket, , is accessible for substituents installed in the para position. Taking this into account, small substituents most likely do not clash with the binding site residues, whereas more bulky substituents cannot bind under the flap.

1. SAR Analysis of Benzene Substituents.

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a

Compound potency was determined by FRET assay using fluorogenic substrate Dabcyl- LNKRLLHETQ-Edans. The assay was performed in duplicate (n = 2).

b

IC50 was determined at five concentrations of the inhibitor (0.01–100 μM), and each data point was the result of two determinations (n = 2). WEHI-916, a known PMV inhibitor, was used as a positive control.

The minor subpocket, deeper under the flap loop, was explored by installing various substituents in the para position. The para-substituted compounds had inhibition potencies in the range from 24 to 87% at a concentration of 100 μM. The most potent compounds were 11ab-ae. Introduction of a cyano group at the para position of either the benzene ring (11ad) or the pyridine heterocycle (11ae) of the initial hit 11b enhanced inhibitory potency, with corresponding IC50(PvPMV) values of 22 and 20.7 μM, respectively. These data suggest that the steric profile and electrostatic characteristics of the cyano group are well matched to the depth and polarity of the flap pocket. In the proposed binding mode, the cyano group is positioned to engage in a hydrogen-bonding interaction with His173. Among the poly substituted derivatives, only the 2-chloro-4-cyanophenyl group-containing derivative 11aj showed comparable activity, with an IC50 value of 32 μM.

The selectivity of the most potent derivatives was assessed by determining their inhibitory activity against human cathepsin D (CatD). Compound 11ad displayed a 7-fold reduction in potency against CatD (IC50 = 153 ± 22 μM) relative to its activity against PMV. In contrast, compounds 11ae and 11ac inhibited the host protease with potencies nearly equipotent to their PMV inhibition (IC50 = 26 ± 7 μM and IC50 = 34 ± 8 μM, respectively). These findings highlight that further optimization is required for this scaffold not only to improve its potency, but also selectivity.

Conclusions

The synthesized 1,3,4-thiadiazole-based compounds expand the currently limited repertoire of known PMV inhibitors. These molecules are readily synthesized, possess low molecular weight, and are amenable to further derivatization. Incorporation of a cyano group at the para position of either the benzene ring (11ad) or pyridine heterocycle (11ae) on the amino-1,3,4-thiadiazole-2­(3H)-thione core produces inhibitors with corresponding IC50 (PvPMV) values of 22.0 and 20.7 μM, respectively. These results suggest that these derivatives represent a promising source of aspartic protease inhibitors, although further chemical modifications are required.

Methods

Synthesis of 1,3,4-Thiadiazole-Based Compounds

N-Methylated analogues 23, 24 and compounds 18, 19 were synthesized according to the literature, while compounds 20, 26–27, 11a, 11n, and 11ai were obtained commercially. A range of 1,3,4-thiadiazolethione compounds 11 with various benzene substituents was synthesized according to the general procedures outlined in the scheme (Scheme ). Noncommercially available isothiocyanates 29 were prepared from the corresponding aniline 28 derivatives using a thiocarbonyl transfer reagent, such as thiocarbonyldiimidazole. The synthesis of unavailable aniline derivatives 28 is provided in the Supporting Information. The resulting isothiocyanates 29 were subsequently reacted with hydrazine hydrate to produce thiosemicarbazides 30, which were then cyclized with CS2 to obtain the target products 11 (yields are given in Table ).

1. Synthesis of 1,3,4-Thiadiazolethione Compounds 11 .

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a Reagents and conditions: (a) 1,1′-thiocarbonyldiimidazole (1.2 equiv), THF (0.1 M), 0 °C to rt; (b) hydrazine monohydrate (1.2 equiv), iPrOH (0.1 M), 0 °C to rt; (c) CS2 (1.2 equiv), DMF (0.3 M), 80 °C.

2. Yields of Products 29, 30, 11 .

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Commercially available isothiocyanate 29 was used.

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Isothiocyanate 29 was synthesized using the conditions described in Scheme .

The conventional thiocarbonyldiimidazole-mediated synthesis of isothiocyanates proved ineffective for electron-deficient amines, such as 6-aminonicotinonitrile (28ae) and 4-aminobenzonitrile (28aj). Alternatively, the dithiocarbamate salts were generated in situ by treating the amine derivatives 28ae,aj with NaH, followed by the addition of CS2 (Scheme ). Subsequent desulfurization using FeCl3 yielded the desired isothiocyanate products 29ae,aj.

2. Synthesis of Electron-Deficient Isothiocyanates 29ae,aj .

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a Reagents and conditions: (a) NaH (1.2 equiv), DMF (1 M), CS2 (1.2 equiv), 0 °C to rt, 6 h then TEA (1.0 equiv), FeCl3·6H2O (2.0 equiv), H2O (0.5 M), rt, 1 h.

A series of thiadiazole compounds containing thiole, S-methyl sulfide, sulfinyl, and sulfonyl functional groups was synthesized from commercially available phenyl-3-thiosemicarbazide (30b) (Scheme ). The synthetic route began with the reaction of phenyl-3-thiosemicarbazide (30b) with CS2 in the presence of NaOH to give compound 11b. Alkylation of the thiol group with MeI using TEA base yielded the S-methyl sulfide 25. Selective oxidation of the sulfide derivative 25 with 1 equiv of Oxone salt (potassium peroxysulfate, potassium sulfate, potassium bisulfate) afforded the sulfoxide derivative 21, whereas oxidation with 2 equiv. Oxone produced the sulfone derivative 22.

3. Synthesis of Compounds 11b, 21-22, 25 .

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a Reagents and conditions: (a) CS2 (1.2 equiv), NaOH (1.0 equiv), EtOH (0.3 M), 80 °C; (b) MeI (1.2 equiv), TEA (4.0 equiv), DMF (0.5 M), 80 °C; (c) 2KHSO5·KHSO4·K2SO4 (1.0 equiv), EtOH/H2O = 2/1, 0 °C to rt; (d) 2KHSO5·KHSO4·K2SO4 (2.0 equiv), EtOH/H2O = 1.5/1, rt.

The thiadiazolone compound 17 was synthesized using phenyl isothiocyanate as the starting material (Scheme ). Initially, isothiocyanate 29b was reacted with formylhydrazine, followed by a cyclization reaction with triphosgene. Subsequent acidic cleavage of the formamide bond yielded the target compound 17.

4. Synthesis of Compound 17 .

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a Reagents and conditions: (a) formylhydrazine (1.0 equiv), EtOH (0.4 M), 55 °C, 51%; (b) triphosgene (0.3 equiv), Tol/Acetone = 3/1, 0 °C to rt, 32%; (c) 4 M HCl in 1,4- dioxane, Acetone/MeOH = 5/1, rt, 46%.

Protein Expression and Purification

Expression and purification of PMV were performed as previously described by Hodder et al., 2015 with minor modifications. The coding sequence of PvPMV (residues R35-R476, strain Salvador I) was cloned into a baculovirus transfer vector containing an N-terminal gp67 signal peptide, followed by a SUMO fusion domain and tobacco etch virus (TEV) protease-cleavage site. The construct additionally included a C-terminal human rhinovirus 3C (HRV-3C) protease cleavage site and a His12-tag.

Recombinant bacmid DNA was generated by transforming the transfer plasmid into DH10Bac competent cells containing a helper plasmid and a bacmid for Tn7-mediated transposition. Positive colonies were identified by blue/white screening and verified by colony PCR. Bacmid DNA was isolated and used to transfect Sf9 cells cultured in Sf-900 II SFM (Gibco) at 27 °C to produce viral stocks. The amplified virus was subsequently used to infect Hi5 cells (Gibco) for protein expression at 27 °C in Express Five SFM (Gibco). Cells were harvested 72 h postinfection by centrifugation at 800g.

The secreted protein was captured from the clarified supernatant by binding to Ni-NTA resin (Thermo Scientific) in a buffer containing 20 mM Tris-Cl (pH 8.0), 200 mM NaCl, and 20 mM imidazole. Bound protein was eluted with 500 mM imidazole. Eluted fractions were immediately buffer-exchanged by desalting into cleavage buffer (20 mM Tris-Cl, pH 8.0, 100 mM NaCl, 5 mM DTT). Sequential cleavage was performed first with HRV-3C protease (Sigma-Aldrich), followed by TEV protease at 4 °C overnight. DTT was included in the cleavage buffer to maintain reducing conditions required for optimal activity of HRV-3C and TEV proteases, both of which are cysteine proteases whose catalytic cysteine residues are sensitive to oxidation. In addition, DTT helps to prevent oxidation of exposed cysteine residues in recombinant Pv_PMV, thereby reducing non-native disulfide formation, aggregation, and loss of protein recovery during purification.

Cleavage products were separated by reverse immobilized metal affinity chromatography to remove affinity tags and proteases. Final purification was performed by size-exclusion chromatography (SEC) on a HiLoad Superdex 200pg 16/600 (Cytiva) column equilibrated in SEC buffer [20 mM HEPES (pH 7.2), 100 mM NaCl, and 1 mM DTT] (Figure S1). Eluted protein was concentrated, aliquoted, flash-frozen, and stored at −80 °C. A lower concentration of DTT was maintained during SEC to preserve the protein in a reduced state while minimizing excessive reducing-agent exposure during storage. Protein concentrations were determined using a NanoDrop 2000c UV–vis spectrometer by measuring the absorbance at 280 nm, applying the theoretical molar extinction coefficient (full length SUMO-PvPMV-3C-C-His: ε280 = 66,085 M–1 cm–1 and MW = 71.3 kDa; cleaved PvPMV: ε280 = 60,125 M–1 cm–1 and MW = 51.8 kDa).

Enzymatic Assay

The enzymatic activity of PvPMV was measured using a fluorescence resonance energy transfer (FRET) cleavage assay based on the cleavage of fluorogenic PEXEL peptide (Dabcyl-LNKRLLHETQ-Edans). Black 384-well microplates with top-optics detection were used to minimize background fluorescence and well-to-well optical cross-talk during FRET measurements. Assays were performed at 37 °C in a final volume of 40 μL per well. Activity was evaluated in SEC buffer and in phosphate–citrate (PC, pH 6.5) buffer as described previously. Unless otherwise stated, data are reported for the SEC buffer. Each 40 μL reaction contained 20 μL of 2× buffer, 1 μL of DMSO or compound solution, enzyme (final concentration 0.02–0.08 mg/mL), and FRET substrate (AnaSpec, #AS-64939) added last. The enzyme was diluted in an assay buffer and preincubated with DMSO or inhibitor prior to reaction initiation. Reactions were initiated by the addition of FRET substrate (prepared as a 1 mM stock in DMSO-d 6) to a final substrate concentration of 10 μM. Substrate hydrolysis was monitored as an increase in fluorescence (λex = 336 nm, λem = 490 nm) at 37 °C for 60 min using a plate reader (CLARIOstar Plus, BMG Labtech) equipped for top-optics detection. Unless stated otherwise, reactions were monitored kinetically, and initial rates were determined from the linear portion of the progress curves. Control wells included buffer + DMSO (blank), substrate without enzyme (reference), and enzyme + substrate (activity control). DTT was prepared fresh and added immediately before testing.

The potential for artifactual inhibition, compound aggregation, and nonspecific pan-assay interference by the follow-up compounds was evaluated using computational, structural, and experimental evidence. None of the follow-up compounds triggered PAINS alerts associated with chemically reactive functionalities, redox-active motifs, or known promiscuous metal-chelating substructures. Throughout the study, compound stock solutions remained stable under the storage and assay conditions employed. In addition, concentration–response curves obtained in the FRET assay exhibited conventional sigmoidal behavior and did not display unusually steep slopes that can be indicative of aggregate-mediated inhibition. The detergent-supplemented counter-screens were not performed.

The Maybridge library was screened at 0.04 mg/mL PvPMV and 250 μM fragment concentration to facilitate identification of weak, high-micromolar binders. Selected lower-affinity compounds from the GCI screen were further evaluated at 0.02 mg/mL PvPMV and 100 μM fragment concentration to assess concentration-dependent effects.

Grating-Coupled Interferometry (GCI) Analysis

GCI experiments were carried out on the Creoptix WAVEdelta instrument (Malvern Panalytical) using Creoptix WAVE control software (4.5.14 version). A 4PCZ sensor chip with a zwitterionic polymer bearing carboxylic acids and tertiary amines in similar densities was selected. Amine coupling was performed using the default setup at pH 7.5 in 10 mM HEPES buffer. The first channel was used as a reference, and the second channel was used for protein immobilization. Chip surface in both channels was activated by a freshly prepared 1:1 mixture of 200 mM EDC and 50 mM NHS (final concentration) that was injected for 420 s on the chip surface at a flow rate of 10 μL/min with a target immobilization level of 10,000. Next, PvPMV was injected at a 60 μg/mL concentration in the second channel in three impulses at 10 μL/min for 420 s to reach the selected immobilization level on one of the chip channels. The first channel was left intact. Afterward, both channels were blocked by injection of 1 M ethanolamine pH 8.0, for 420 s at a flow rate of 10 μL/min. Immobilized protein level: 9376.6 pg/mm2.

The running buffer for screening was composed of 10 mM HEPES pH 7.2, 150 mM NaCl, 0.005% (v/v) Tween-20, 2% (v/v) DMSO-d 6. A constant chip temperature of 25 °C was applied. WaveRAPID (Repeated Analyte Pulses of Increasing Duration) kinetics assay was performed to evaluate the activity of immobilized PvPMV. Two compounds were used: WEHI-916 and in-house synthesized aspartic protease inhibitor - LK-400 (Figure S3).

Maybridge Ro3 Diverse Fragment Screening was performed by injecting ligands at a 200 μM concentration. Compounds were allowed to associate and dissociate for 60 s. LK-400 was used as a positive control, and running buffer was used as a negative control (Figure S5). 94 compounds with the highest response rates were used further in the WaveRAPID kinetics assay to determine their binding affinities toward PvPMV at 50 μM concentration each (Figure S6).

Sensorgrams were processed by the Creoptix WAVEControl software (4.8.4 version) using 1:1 kinetics mode and the standard evaluation tool.

In-Solution Digestion and Peptide Extraction for MS

PMV (1 mg/mL) was preincubated with compounds for 10 min or 2 h at 37 °C in a 5-molar excess. For the reference sample, PMV was incubated under the same conditions without compounds. For tryptic digestions under reducing conditions, 15 μL of digestion buffer (50 mM ammonium bicarbonate) and 1.5 μL of reducing buffer (100 mM DTT) were added to 10.5 μL of sample. The samples were incubated at 95 °C for 5 min. After cooling, 3 μL of alkylation buffer (100 mM iodoacetamide) was added, and the tubes were incubated in the dark at RT for 30 min. Then, 2 μL of activated trypsin was added, and the tubes were incubated overnight at 30 °C. For Glu-C digestions under nonreducing conditions, 10 μL of 5X digestion buffer (500 mM ammonium bicarbonate) and 29 μL of MQ were added to 10 μL of the sample. Next, 0.5 μL of Glu-C (1 mg/mL) was added to the tubes, and the tubes were incubated overnight at 30 °C.

The following day, the digestion mixtures were acidified using TFA. The samples were desalted and concentrated using Pierce C18 10 μL pipet tips. The samples were mixed with a CHCA matrix and analyzed using a Bruker MALDI TOF/TOF mass spectrometer. To identify trypsin-resistant peptides, the average masses were searched against a sequence database generated using the ProteinProspector Tool MS-digest. Trypsin peptides were detected in positive mode, while Glu-C peptides were detected in both positive and negative modes.

Computational Modeling

The docking model for the molecular modeling was created using the PMV cocrystal structure in complex with nonpeptidomimetic inhibitor WM08 (PDB ID: 8TYG) as a template. The structure for docking was prepared using the Maestro Protein Preparation Wizard, side chain protonation states were adjusted at pH 7.0, the Asp80 carboxylic group was protonated, and the complex was minimized using Prime, allowing heavy atoms convergence up to 0.30 Å. Molecular docking was performed using Glide, with scaling of the van der Waals radii set to 0.9 for protein and ligand heavy atoms, and docking compounds flexibly. The designed compounds were prepared for docking using LigPrep by generating possible tautomers and ionization states at pH 7.0 ± 2.0. Docked poses were visualized using PyMOL.

Supplementary Material

ao6c02719_si_001.pdf (13.9MB, pdf)

Acknowledgments

This work was supported by RRF grant No. 67/OSI/PG (Project No. 5.2.1.1.i.0/2/24/I/CFLA/001). The recombinant p. vivax plasmepsin V expression plasmid was kindly provided by Alan Cowman’s group. Chrislaine Withers-Martinez is acknowledged for guidance on protein expression, and Enamine for providing a fluorinated fragment library, designed for NMR screening. R.B. acknowledges RRF grant No. 34/OSI/ZG (Project No. 5.2.1.1.i.0/2/24/I/CFLA/001) for financial support.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c02719.

  • Additional experimental details, figures, methods and fragment screening hits (PDF)

The authors declare no competing financial interest.

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Supplementary Materials

ao6c02719_si_001.pdf (13.9MB, pdf)

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