Inhibitor screening of lactate dehydrogenase C4 from black-lipped pika in the Western Sichuan Plateau

Abstract

Studies indicated that lactate dehydrogenase C4 (LDH-C4) was a good target protein for development of contraceptive drugs. Virtual screening and in vitro enzyme assay using pika LDH-C4 as target protein revealed NSC61610, NSC215718, and NSC345647 with K_i of 7.8, 27, and 41 μM separately. This study might be helpful for development of pika contraceptive drugs.

Key words:

• virtual screening
• Ochotona curzoniae
• lactate dehydrogenase C4
• inhibitor

The black-lipped pika (Ochotona curzoniae) is a mammal species in the Ochotonidae family commonly known as pika. These animals prefer to live at elevations of 3,100–5,000 m, mostly in the Tibetan Plateau.¹⁾ In recent years, the frequent outbreak of rodents has been an important factor in plateau grassland degradation, where the black-lipped pika is one of the most serious rodent species.²⁾

Lactate dehydrogenase (hereinafter referred to as LDH; EC 1.1.1.27) is an oligomeric enzyme that functions at the end of the glycolysis pathway. It has the ability to specifically catalyze the redox reaction between pyruvic acid and lactic acid.³⁾ In mammals, there are three types of LDH isoenzymes: LDH-A, LDH-B, and LDH-C.⁴⁾ LDH-C is a tissue-specific isoenzyme. In mammals, LDH-C is expressed only in mature testis tissues and sperm cells. Only one form of the isoenzyme is expressed in these tissues: LDH-C4.⁵⁾ Antibodies against LDH-C4^6–7) and DNA vaccines based on LDH-C gene⁸⁾ effectively reduced the reproductive capacity of many mammals. Gene knockout experiments showed that the lack of LDH-C could seriously damage the reproductive capacity of male mice.⁹⁾ These studies indicate that LDH-C4 would be a good target protein for the development of contraceptive drugs. Thus, we have made attempt to screening inhibitors of pika LDH-C4 by virtual screening and in vitro enzyme assay.

For virtual screening, the 3D structure of pika LDH-C4 was built by method of homology modeling using software Swiss-PdbViewer^10–11) with human LDH (PDB Accession Number: 1i10) as template which showed 88% sequence similarity with pika LDH-C4 subunit deduced from pika LDH-C gene (GenBank Accession Number: GU076486). The model quality estimation of pika LDH-C4 got 0.738 QMEAN4 raw score and –0.36 z-score.^10–11) The scores indicated that the model of pika LDH-C4 could be used for molecular docking. The small molecules from National Cancer Institute (NCI) Diversity set II (http://dtp.nci.nih.gov/branches/dscb/div2_explanation.html) were used for screening. NADH (coenzyme of LDH) was added and set as the positive control. AutoDock version 4.2 with ADT-1.5.4¹²⁾ was used for the docking simulation and visual speculation. The docking grid encompassing one of the four LDH-C4 active sites (both pyruvate- and NADH-binding site) was size of 72 Å × 78 Å × 64 Å with points spacing of 0.375 Å. We selected the Lamarckian genetic algorithm for ligand conformational searching. For each compound, the docking parameters were trials of 100 dockings, 10,000,000 energy evaluations and 27,000 generations. Final docked conformations were clustered by using of a tolerance of 2.0 Å root-mean-square deviation. The virtual screening results were sorted on the basis of their predicted binding free energies (ΔG_AD4), which ranged from –1.84 to –12.44 kcal/mol. The ΔG_AD4 of NADH (the positive control) was –8.76 kcal/mol and the molecules whose ΔG_AD4≤ –8.76 kcal/mol were visually inspected (top 200 molecules). Finally, 38 compounds that occupied the pyruvate-binding site and were available from NCI were selected from the 200 compounds. The 38 Compounds were requested and received from NCI (http://dtp.nci.nih.gov/branches/dscb/repo_request.html). Chemical compounds were dissolved in DMSO to 10 mM final concentration and stored at room temperature.

To prepare sufficient pika LDH-C4 for in vitro inhibitor screening, we set out to express the native LDH-C4 protein (without tags) in Escherichia coli. The ORF of pika LDH-C was optimized for E. coli codon usage bias and synthesized (Supplemental data; see Biosci. Biotechnol. Biochem. http://dx.doi.org/10.1080/09168451.2014.890038). Then, the optimized ORF was amplified using a pair of PCR primers, cloned to pMD18-T vector, digested with NdeI and XhoI, and ligated into the corresponding sites of pET32a to obtain a recombinant plasmid pET32-pika-LDH-C. The PCR primers were expr-Ps (5ʹ-CAT ATG TCT ACT GTG AAA GAA CAG CT-3ʹ, the underlined sequence represented the NdeI site) and expr-Pa (5ʹ-CTC GAG GAA AAC CAG ATC TTT-3ʹ, the underlined sequence represented the XhoI site and the TTA shaded in gray was the stop codon). The NdeI site at the 5ʹ end insured that no tags were added to the N-terminus of the expressed protein, whereas the stop codon in the expr-Pa primer insured that no tags were added to the C-terminus of the expressed protein.

The E. coli BL21 (DE3) clone transformed with pET32-pika-LDH-C was induced to express pika LDH-C by 1.0 mM isopropyl-beta-D-thiogalactoside (IPTG) at 25 °C for 10 h. Bacterial cells were collected by centrifugation and disrupted by ultrasonication (36 × 5 s pulses with 5 s intervals). The lysates were centrifuged at 12,000 g for 15 min, and the obtained supernatant (crude extracts) was analyzed for lactate dehydrogenase activity using native Polyacrylamide gel electrophoresis (PAGE).¹³⁾ The crude extracts of E. coli transformed with pET32-pika-LDHC showed high LDH activity compared with the ones transformed with pET32 (Fig. 1(A)). This meant that the expression of pika LDH-C was successfully induced and that the crude extracts could be used for purifying of pika LDH-C4. After the affinity (Blue Sepharose) and ion-exchange (DEAE Sepharose) chromatography, the eluate showed a single band on SDS-PAGE¹⁴⁾ (Fig. 1(B)) with a purity of more than 99%, as assessed by capillary electrophoresis.¹⁵⁾ The purified pika LDH-C4 showed a high specific activity¹⁶⁾ of 478 U/mg and had a relative molecular weight of approximately 140 kDa analyzed by gel filtration (Superdex 200). Considering the subunit of the LDH-C4 was about 35 kDa by capillary electrophoresis, we concluded that the pika LDH-C4 expressed from E. coli was a tetramer, which was the same configuration found in spermatozoa. So, it was a good target protein for in vitro enzyme assay.

Fig. 1. Polyacrylamide gel electrophoresis.

Notes: (A) Staining for LDH activity after native PAGE. Lane 1: crude extracts of E. coli transformed with pET32a; lane 2: crude extracts of E. coli transformed with pET32-pika-LDH-C. (B) Coomassie brilliant blue staining after SDS-PAGE. Lane 1: crude extracts of E. coli transformed with pET32-pika-LDH-C; lane 2: affinity chromatography eluate; lane 3: ion-exchange chromatography eluate; lane M: protein molecular weight standards.

For in vitro assay, the reagent mixture contained 100 mM sodium phosphate buffer (pH 7.4), NADH, sodium pyruvate, and the enzyme. The enzyme was prepared by diluting it with the binding buffer, providing a ΔE₃₄₀ of 0.060–0.070 per min in a 1 cm light path (measuring the oxidation of NADH at 340 nm). To determine the Michaelis constants for pyruvate, pyruvate concentrations of 5, 20, 30, 50, 75, 100, and 150 μM were used with a constant concentration of 0.15 mM NADH. K_m (51.2 μM) and V_max (15.8 μM/min) values were calculated from a minimum of three velocity measurements by nonlinear regression method using the software Origin 8.0 (OriginLab). The selected compounds were added to the reaction buffer as mentioned above, and K and V were calculated using Origin 8.0. Only the compounds showed competitive inhibition against pyruvate (K increased and V remained stable) were selected. The Ki were calculated using equation K/K_m = [I] × K + 1¹⁷⁾ with at least 2 different inhibitor concentrations. As most of the compounds were water insoluble, these compounds were dissolved firstly in DMSO and added to the reaction buffer, so the effects of DMSO to the pika LDH-C4 were also analyzed, the results showed that up to 25% DMSO had no effects to the K_m and V_max, and 50% DMSO had no effects to the K_m, but decreased the V_max of pika LDH-C4. All assays were performed at 25 °C.

Nineteen compounds among the 38 compounds were soluble in 25% DMSO, and 8 compounds were soluble in 50% DMSO. The K and V of the 27 compounds were determined. Eight out of the 27 compounds did not show competitive inhibition and discarded. The K_i of the remaining 19 compounds were calculated. Among the 19 compounds, 3 compounds showed a K_i of lower than 50 μM (Table 1). Especially, NSC61610 showed a K_i of 7.8 μM, the binding pose of NSC61610 in the active site of pika LDH-C4 was shown in Fig. 2. NSC61610 shows high structural similarities and similar binding pose with NADH (Supplemental Fig. 1). The adenosine of NSC61610 bound in the hydrophobic crevice as the adenosine of NADH did (Fig. 2(B)), the residues form H-bonds with NSC61610 were Gly 96 and Asn137, which were different with the residues form H-bonds with NADH (Thr 94 and Tyr 82). The interactions indicate that NSC61610 can bind to LDH-C4 and forms a relatively stable complex as NADH do. NSC215718 shows some similarities with NADH, and NSC345647 did not show structural similarities with NADH and pyruvate. Both NSC215718 and NSC345647 occupied part of the pyruvate- and NADH-binding site of pika LDH-C4 (Supplemental Figs. 2 and 3). This study might be helpful for development of pika contraceptive drugs.

Table 1. Structures, autodock binding energies and inhibition constants of the 3 compounds.

NSC number	Structure	Ki (μM)	ΔGAD4
61610		7.8	−10.3
215718		27	−9.59
345647		41	−8.80

Fig. 2. Docking simulation of NSC61610 binding to the active site of pika LDH-C4.

Notes: (A) binding pose of NSC61610 in the active site of pika LDH-C4 subunit; (B) the enlarged active site of pika LDH-C4 bound with NSC61610; (C) interactions of NSC61610 with the active site of pika LDH-C4, hydrogen bonds were shown as little green spheres, non-bonded interactions (close contact atoms) were represented by wireframe spheres.

Supplemental material

The supplemental material for this paper is available at http://dx.doi.org/10.1080/09168451.2014.890038.

Funding

Funds were provided by the National Science Foundation of China (31071700); the Fundamental Research Funds for the Central Universities, Southwest University for Nationalities (12NZYBS07); the Foundation for Outstanding Scholars, Southwest University for Nationalities; the scientific research fund of Sichuan Provincial Education Department (12ZB022).

Notes

Abbreviations: DMSO, Dimethyl sulfoxide; LDH, Lactate Dehydrogenase; NADH, Nicotinamide adenine dinucleotide; IPTG, isopropyl-beta-D-thiogalactoside; ORF, open reading frame.