Drug repositioning and pharmacophore identification in the discovery of hookworm MIF inhibitors

Abstract

The screening of bioactive compound libraries can be an effective approach for repositioning FDA-approved drugs or discovering new pharmacophores. Hookworms are blood-feeding, intestinal nematode parasites that infect up to 600 million people worldwide. Vaccination with recombinant Ancylostoma ceylanicum macrophage migration inhibitory factor (rAceMIF) provided partial protection from disease, thus establishing a "proof-of-concept" for targeting AceMIF to prevent or treat infection. A high-throughput screen (HTS) against rAceMIF identified six AceMIF-specific inhibitors. A nonsteroidal anti-inflammatory drug (NSAID), sodium meclofenamate, could be tested in an animal model to assess the therapeutic efficacy in treating hookworm disease. Furosemide, an FDA-approved diuretic, exhibited submicromolar inhibition of rAceMIF tautomerase activity. Structure-activity relationships of a pharmacophore based on furosemide included one analog that binds similarly to the active site, yet does not inhibit the Na-K-Cl symporter (NKCC1) responsible for diuretic activity.

Figure 1. Immunization of Hamsters with Recombinant AceMIF Followed by Challenge with Ancylostoma Ceylanicum Hookworms

Hamsters (5/group) were immunized subcutaneously with 100 mg rAceMIF in the adjuvant alum, whereas controls received alum only. After boosting twice with 50 mg of rAceMIF, animals were challenged with 100 A. ceylanicum infective larvae and monitored for 40 days. As shown in (A), immunization of hamsters with rAceMIF was associated with partial protection from hookworm-associated growth delay, with significantly higher body weights noted at days 26–40 post infection (p < 0.03) compared with controls. In (B), immunized animals also exhibited less severe anemia after challenge infection, with higher blood hemoglobin levels from days 23–33 postinfection (p < 0.05).

Figure 2. Inhibition Assay against the Enzymatic and Biological Activity of AceMIF

(A–C) Representative AceMIF hydroxyphenylpyruvate tautomerase inhibition of (A) compound 2 (competitive), (B) compound 5 (noncompetitive), and (C) compound 3 (mixed). Nonlinear regression of the initial velocity at various substrate and inhibitor concentrations is shown on the top panel, and Lineweaver-Burk plots are shown on the bottom panel for each inhibitor. (D) PBMC migration inhibition assay with 8 nM AceMIF in the presence of 10 mM compound concentration. Migrated cells were measured by relative fluorescence units (RFU) as described in Experimental Procedures. The numbers above the bars are K_i’s (in mM) obtained from the tautomerase inhibition assay. One-letter codes above the numbers represent the type of enzymatic inhibition: C (competitive), NC (noncompetitive), and M (mixed). (E) Inhibition of CD74-AceMIF interaction by compound 2. Percent of AceMIF bound to immobilized CD74 in the presence of various concentrations of compound 2 is plotted. Chemical structure of compound 2 is shown next to the plot. The IC₅₀ value was determined based on the plot. (F) Toxicity of AceMIF-specific inhibitors was determined by observing the survival of cultured A. ceylanicum adult worms. Only three of six compounds revealed worm-killing activity (>15% toxicity) at a dose of 100 mM. See also Figure S1.

Figure 3. NKCC1 Flux Assay with Compound 2 and Its Structural Analogs

Percent inhibition of ⁸⁶Rb influx into NKCC1 cotransporter-expressed HEK293 cells was measured in presence or absence of the compounds. Bumetanide was used as a positive control. The assay was performed at 10 mM and 100 nM compounds (n = 3). Most of the furosemide analogs exhibit negative inhibition and this is probably because of the low value of the untreated control cells located at the edge row of the assay plate.

Figure 4. Complex Crystal Structure with Compound 2

(A) Difference in electron density of compound 2 was generated at 3s omitting the inhibitor in the final structure model. (B) Interaction of compound 2 with active site residues. The residues involved in hydrogen-bond (ball and stick) and hydrophobic (spiked hemisphere) interactions are depicted. Hydrogen-bond distances are present on the green dashed lines. (C) Compound 2 is shown on the electrostatic surface of AceMIF in an orientation pointing into the active site. (D) A 90° rotation of panel (C). In (C) and (D), the two protomers I and II forming the active site are represented in ribbons. The dotted area represents a protein site that could be used by novel analogs of the parent molecule to form new interactions, increasing the affinity for AceMIF and decreasing the affinity for carbonic anhydrase and the Na-K-Cl cotransporter. See also Figures S2–S4.

Figure 5. Complex Crystal Structure with Compound 9, A Structural Analog of Compound 2

(A) and (B) were generated in the same manner with those of compound 2 in Figure 3. (C) is the superposition of compounds 2 and 9 at the catalytic site. See also Figures S2 and S4.