High-Throughput Screening to Identify Plant Derived Human LDH-A Inhibitors

Aims Lactate dehydrogenase (LDH)-A is highly expressed in diverse human malignant tumors, parallel to aggressive metastatic disease, resistance to radiation/chemotherapy and clinically poor outcome. Although this enzyme constitutes a plausible target in treatment of advanced cancer, there are few known LDH-A inhibitors. Study Design In this work, we utilized a high-throughput enzyme micro-array format to screen and evaluate > 900 commonly used medicinal plant extracts (0.00001-.5 mg/ml) for capacity to inhibit activity of recombinant full length human LDHA; EC .1.1.1.27. Methodology The protein sequence of purified enzyme was confirmed using 1D gel electrophoresis- MALDI-TOF-MS/MS, enzyme activity was validated by oxidation of NADH (500μM) and kinetic inhibition established in the presence of a known inhibitor (Oxalic Acid). Results Of the natural extracts tested, the lowest IC50s [<0.001 mg/ml] were obtained by: Chinese Gallnut (Melaphis chinensis gallnut), Bladderwrack (Fucus vesiculosus), Kelp (Laminaria Japonica) and Babul (Acacia Arabica). Forty-six additional herbs contained significant LDH-A inhibitory properties with IC50s [<0.07 mg/ml], some of which have common names of Arjun, Pipsissewa, Cinnamon, Pink Rose Buds/Petals, Wintergreen, Cat’s Claw, Witch Hazel Root and Rhodiola Root. Conclusion These findings reflect relative potency by rank of commonly used herbs and plants that contain human LDH-A inhibitory properties. Future research will be required to isolate chemical constituents within these plants responsible for LDH-A inhibition and investigate potential therapeutic application.


Herbal Extraction
Plant and herbal extracts were macerated, diced, chopped and homogenized in 100% ethanol at 50mg/ml. Samples were placed on a rocker shaker for 24 hours and stored in air tight containers at −20°C in the dark. All serial dilutions were made using a diluent consisting of HBSS with 10mM HEPES adjusted to a pH 7.4.

MALDI MS/MS Protein Identification
Recombinant full length Human LDHA (amino acids 1-332) with N terminal His tag; 352 amino acids with tag, MW 38.8 kDa: Enzyme Commission (EC) Number 1.1.1.27 (BRENDA | IUBMB) (Abcam, Cambridge, MA) was utilized. The protein was validated by proteomic analysis using Matrix Assisted Laser Desorption Ionization (MALDI) Mass Spec (MS/MS) and analyzed by Mascot ID. Briefly, pure enzyme was solubilized, denatured and subjected to 1 D SDS page gel electrophoresis using a 5-20% Tris-HCL gradient gel with a running buffer 25 mM Tris, 192 mM glycine, 0.1% SDS at 200 V for 35 minutes. High intensity bands for LDH-A at 38 KD were visualized with G-Biosciences' LabSafe GEL Blue ™ stain, then excised, followed by in gel digestion of peptides with trypsin, followed by reduction/alkylation with DTT and iodoacetamide, respectively. Samples were analyzed using MALDI MS/MS (Applied Biosystems) and protein sequence identified by Mascot analysis.

LDH-A Activity
A continuous LDH-A assay was used to conduct high-throughput screening (HTS). Briefly, a buffer consisted of HBSS + calcium and magnesium pH adjusted to 7.0. LDH-A enzyme (final concentration .02 Units/ml) was added to treatments of tier one, with concentrations of .5 mg/ml. After addition of β-Nicotinamide Adenine Dinucleotide, Reduced Form Solution (β-NADH) (final working concentration of 500μM) a pre-reading @ 340nm was established and the reaction was started with a solution of substrate pyruvate (final concentration = 3mM).

High Throughput Design
A rapid screening model was used based on works previously described [24]. An enzyme micro-array format was adapted to where a 96 well plate contained a known concentration of enzyme, and treatments of equal concentration dissolved in buffered HBSS and β-NADH. After addition of the substrate (pyruvate) a curve for time dependent NADH oxidation was monitored continuously over 75 minutes @340nm. A first tier investigation was established at a final working concentration of 0.5 mg/ml for each herbal extract. All compounds that inhibited LDH-A with in the first tier screen below 50% of control, were then placed in a second tier (final concentration = 0.25 mg/ml), third tier (final concentration =0.1 mg/ml) and fourth tier (final concentrations with extended range at 0.006, 0.03 and 0.16 mg/ml). Extracts were ranked for potency, and the most potent were further evaluated over a minimum of 6 concentrations from 1mg/ml to less than 0.00001 mg/ml to establish an IC 50 . The enzyme micro-array format was rapid, reproducible and repeatedly corroborated by a four-tier evaluation process.

Data Analysis
Statistical analysis was performed using Graph Pad Prism (version 3.0; Graph Pad Software Inc. San Diego, CA, USA) with significance of difference between the groups assessed using a one-way ANOVA, followed by Tukey post hoc means comparison test, a two way ANOVA or Student's t test. IC 50 s were determined by regression analysis using Origin Software (OriginLab, Northampton, MA).

RESULTS AND DISCUSSION
Method validation was established by monitoring continuous NADH oxidation initiated by addition of the substrate pyruvic acid (3mM) in the presence of varying enzyme concentration/time (Fig. 1a) A screening validation process was established using 0.02 U/ml LDH-A over 75 minutes (Fig. 1b) ± a known inhibitor; oxalic acid ( Fig. 1c). The data shows a slow but steady rate of reaction, resulting in time dependent O.D. decay over 65-70 minutes with high signal/noise ratio.
Although the enzyme used in this screening, was described as a recombinant full length Human LDHA (amino acids 1-332) with N terminal His tag; 352 amino acids with tag, MW 38.8 kDa: Enzyme Commission (EC) Number 1.1.1.27 (BRENDA | IUBMB) (Abcam, Cambridge, MA), we confirmed the identity of the enzyme using Matrix Assisted Laser Desorption Ionisation (MALDI) Mass Spec (MS/MS) and analysis by Mascot ID (Fig. 2). Fig. 2 (Top panel) shows the 1 D SDS page gel electrophoresis of the purified enzyme at three concentrations (right), along with a molecular marker standard (left). The gel band was excised, digested and analyzed by MSMS for peptide sequence and protein identify (Bottom Panel). The data showed a positive hit for human LDH-A with a 95% confidence interval for peptide/sequence mass.
A high throughput enzyme micro-array model was used in this work. Over 900 extracts of equal concentration (0.5 mg/ml) were dissolved in buffered HBSS and incubated with the enzyme, for 5 minutes prior to start of the reaction. After addition of the substrate, a curve for time dependent product formation was monitored continuously over 75 minutes. Fig. 3 represents the 75 minute reading taken from the original screening with values for each compound sorted according to inhibitory potency. Of the initially tested extracts, only 115 inhibited LDH-A within the first tier below 50% of control, denoted by red dotted line --- (Fig. 3). These plant extracts were then subject to a second tier screenings (final concentration = 0.25 mg/ml), third tier screenings (final concentration =0.1 mg/ml) and fourth tier screenings (final concentrations with extended range at 0.006 to 0.16 mg/ml) to which regression analysis was used to calculate IC 50 s.
Of the 115 retested, 46 extracts showed an IC 50 <0.077 mg/ml Table 1 as listed by potency rank. Full inhibitory dose response curves are shown for the top four inhibitors (Fig. 4). Table 1 Human LDH-A inhibitors by potency. Extract IC 50 s are listed by both mg/ml and μg/ml for inhibiting NADH oxidation on .02U/ml of LDHA.

DISCUSSION
In this HTS study, we investigated the ethanol extract of 905 natural products to identify those with human LDH-A inhibitor properties. Our results show Melaphis chinensis gallnut, also known as Rhus chinensis (RC) nut, to be the most potent and within a therapeutic range. Rhus chinensis belongs to family Anacardiaceae and genus Rhus. This family, consists of 250 species found in China [25] and many other locations around the world. Numerous traditional Chinese herbalists recommend RC for ailments such as chronic cough due to lung deficiency, chronic diarrhea and for clearing toxins. Unfortunately, these claims are not based on scientific grounds, yet the interest in this herb continues to increase for its numerous scientifically based findings. There is an abundance of research on the biological and pharmacological benefits of RC. For example, it was shown to be very effective in harmful intestinal and periodontal bacterial growth inhibition by a mechanism mediated in parts by its constituent's gallic acid and gallotaninns [26][27][28]. As an antiviral, RC ethyl acetate extract has inhibitory effects against hepatitis carcinoma virus [29,30] and severe acute respiratory syndrome corona virus [30]. Penta-1,2,3,4,6-O-galloyl-β-D-glucose (PGG) isolated from RC shows promising hepatoprotective properties [31] and the anticancer effects of RC are believed to involve inhibition of dcd25A phosphotase activity [32] or gallic acid as one of the bioactive components in RC [26,33,34] which directly induces apoptosis in prostate cancer cells [35]. PGG was also shown by Huh et al, to be a constituent in RC with ability to inhibit angiogenesis and stimulate apoptosis [36]. In addition, PGG reduced cancer cell viability [37] as well as suppressed prostate cancer, bone metastasis [38] and caused cell cycle arrest at the G1 phase [39].

CONCLUSION
The findings in this study, while broad show that a number of natural products have the ability to inhibit LDH-A, which may adversely affect cancer cell survival. We present evidence for LDH-A inhibitory properties of a number of commonly used herbs and spices with previously reported anti-cancer properties including bladderwrack [40], kelp [41], cinnamon [42], cats claw bark [43], arjun [44], polygonum multiflorum [45] and witch hazel [46]. Future research will be required to evaluate if LDH-A inhibition is a contributing factor to tumoricidal or anti-proliferative properties of these herbs on diverse human cancer cells.  Fig. 1a. Human LDH-A Activity-time and enzyme concentration dependent NADH oxidation in the presence of 3mM pyruvate. The data represent μM NADH reduced from 0-75 minutes (incubation at RT) and are presented as the Mean ± S.E.M, n=4. Significance of difference for product formation between Time 0 vs Time 15 -75 minutes were determined using a two-way ANOVA.* p< 0.05 Fig. 1b. Human LDH Activity at .02 Units/ml with time dependent NADH oxidation in the presence of 3mM pyurvate. The data represent μM NADH and are presented as the Mean ± S.E.M, n=4. Significance of difference for product formation between Time 0 vs Time 75 minutes was determined using a one-way ANOVA followed by a Tukey post hoc test. * p<0 .05 Fig. 1c. Human LDH Activity -Inhibitor Control. The data represent % Enzyme Activity @ 75 Minutes and are presented as the Mean ± S.E.M, n=4. Significance of difference for enzyme activity between the control and oxalic acid (0.9-11.3 mM) was determined using a one-way ANOVA followed by a Tukey post hoc test. * p<0 .05

Fig. 3.
A high-throughput enzyme experimental micro-array design. 905 extracts were evaluated for capacity to inhibit Human LDH-A. A first tier screening was conducted at a final working concentration of 0.5 mg/ml for each herbal extract. Enzyme activity was continuously monitored over a 75 min period. Extracts demonstrating an IC 50 <0.5 mg/ml (red dotted line -----) were screened through subsequent tier evaluations Most Potent Herbal Extract Inhibitors of Human LDH-A activity. The data represent LDH-A activity as % control in the presence or absence of extracts and are presented as Mean ± S.E.M., n=4. IC 50 concentrations were established from a sigmoidal fit dose-response equation and significance of difference between the controls vs. treatment was determined using a one-way ANOVA followed by a Tukey post hoc test. * p<0.05 Table 1 Natural source aqueous extracts with human LDH-A inhibitory potency by rank