Recombinant Human NAD-dependent malic enzyme, mitochondrial protein (ME2) (Active)

What is human NAD-dependent malic enzyme 2 (ME2) and what is its primary function?

Human NAD(P)+-dependent malic enzyme 2 (ME2) is an oxidative decarboxylase that catalyzes the conversion of L-malate to pyruvate and CO₂, using NAD+ or NADP+ as a cofactor. The enzyme plays a critical role in cellular metabolism, particularly in energy production and biosynthetic pathways . ME2 is predominantly located in the mitochondria and contributes to the malate-aspartate shuttle system, which is essential for maintaining redox balance within cells. The functional enzyme exists as an oligomeric complex, with specific structural domains that facilitate substrate binding and catalytic activity .

How is recombinant human ME2 typically produced for research purposes?

Recombinant human ME2 is commonly produced using an E. coli expression system. The process typically involves:

• Cloning ME2 cDNA into a suitable expression vector (e.g., pET29 or pET32) with a His-tag fusion

• Transforming competent E. coli cells (often BL21-CodonPlus strain) with the expression construct

• Inducing protein expression using lactose (1% w/v) or IPTG

• Growing cultures at reduced temperatures (16°C) to enhance protein solubility

• Harvesting cells by centrifugation and lysing through sonication

• Purifying the recombinant protein using Ni-NTA His-binding resin with sequential washing using increasing imidazole concentrations (10, 50, 100 mmol/L)

• Eluting the purified protein with 250 mmol/L imidazole solution

• Dialyzing at 4°C to remove imidazole

This procedure typically yields ME2 protein with a molecular mass of approximately 60 kDa, which can be confirmed by SDS-PAGE . The specific activity of purified ME2 has been reported as approximately 1652.25±11.69 Units·min⁻¹·mg⁻¹ of protein, representing a 142-fold purification from whole lysate with a 16% yield .

What are the standard assay conditions for measuring ME2 enzymatic activity?

The standard enzymatic assay for ME2 activity typically involves spectrophotometric measurement of NADH formation at 340 nm. The reaction mixture generally contains:

• Buffer system (often 50 mM Tris-HCl, pH 7.4)

• Divalent cation (usually 2-10 mM MgCl₂)

• NAD+ (4 mM)

• L-malate (10 mM)

• Enzyme sample in a final volume of 0.5 ml

The reaction is initiated by the addition of L-malate. Initial velocity studies are performed by varying the concentration of one substrate around its Kₘ value while maintaining the other substrate at saturating levels. Kinetic parameters are calculated using free concentrations of all substrates and data are fitted to the Michaelis-Menten equation or, in cases of sigmoidal kinetics, to the Hill equation by nonlinear regression .

When evaluating potential inhibitors or activators, ME2 activity is measured with non-saturating concentrations of malate (at the Kₘ value) in the presence of 0.5-2 mM of the test compound .

How does ME2 contribute to cancer cell metabolism and what evidence supports its role as a potential anticancer target?

ME2 plays a significant role in cancer cell metabolism through several mechanisms that support the proliferative and survival advantages of tumor cells:

• Enhanced proliferation: Studies have demonstrated that ME2 activity increases as cells progress toward neoplasia in rat tracheal epithelial lines and Morris hepatomas .

• Anti-apoptotic effects: Knockdown of endogenous ME2 has been shown to impair proliferation of K562 leukemia cells and induce apoptosis, as well as suppress tumor growth in vivo .

• Growth regulation: Silencing ME2 severely impairs the growth of HCT-116 and U2OS cancer cell lines, while overexpression enhances their growth .

• p53 interaction: ME2 is involved in the regulation of p53 during tumor metabolism, senescence, and growth, suggesting its involvement in key cancer-related pathways .

This evidence collectively suggests that ME2 is a promising anticancer target, particularly since inhibition of its activity could potentially disrupt the metabolic adaptations that support cancer cell growth and survival. The development of specific ME2 inhibitors represents an important approach in validating ME2 as a therapeutic target and potentially developing novel anticancer treatments .

What is known about the kinetic properties of ME2 and how do they differ from other malic enzyme isoforms?

ME2 displays complex kinetic properties that distinguish it from other malic enzyme isoforms:

ME2 exhibits complex regulatory properties, including activation by CoA. For ME2, activation studies typically involve varying the concentration of a substrate while keeping the other substrate at saturating levels and varying the concentration of CoA. The data for CoA activation of ME2 can be fitted to the equation:

v = v₀ + (Vₐ × A) / (A₅₀ + A)

Where v₀ is the rate in absence of activator, Vₐ is the maximum activated rate, A is the concentration of activator, and A₅₀ is the concentration of activator that gives 50% of Vₐ .

How do recently discovered ME2 inhibitors like NPD389 work, and what are their potential research applications?

NPD389, a derivative of NPD387, represents a novel class of ME2 inhibitors with a unique mechanism of action:

• Structural basis: NPD389 contains a 2,5-dihydroxy benzoquinone skeleton with 4-OMe-substituted phenyl groups at C3 and C6 positions, which enhances its inhibitory activity compared to the parent compound NPD387 .

• Inhibition mechanism: NPD389 acts as a fast-binding uncompetitive inhibitor with respect to the substrate NAD+ and a mixed-type inhibitor with respect to the substrate L-malate .

• Potency: NPD389 demonstrates significant potency with IC₅₀ values of 4.63±0.36 μmol/L or 5.59±0.38 μmol/L in the absence or presence of 0.01% Brij-35, respectively .

• Binding characteristics: Thermal shift assays confirm that NPD389 binds directly to ME2, causing conformational changes in the enzyme .

Research applications of NPD389 and similar ME2 inhibitors include:

• Tools for investigating ME2's role in cellular metabolism

• Probes for studying the effects of ME2 inhibition on cancer cell growth and survival

• Potential leads for developing anticancer therapeutics

• Reagents for validating ME2 as a therapeutic target in various disease contexts

Understanding the structure-activity relationships of these inhibitors provides valuable insights for the rational design of more potent and selective ME2 inhibitors for research and therapeutic applications .

What are the key considerations for developing a high-throughput screening assay for ME2 inhibitors?

Developing an effective high-throughput screening (HTS) assay for ME2 inhibitors requires careful attention to several critical factors:

• Assay quality control: The established HTS system for ME2 inhibitor discovery should demonstrate excellent reproducibility and reliability. An average Z' factor of 0.775 and a signal-to-noise ratio (S/N) of 9.80 indicate satisfactory HTS quality control .

• False-positive elimination: A common challenge in HTS is the accumulation of organic molecules into colloidal aggregates, which can inhibit enzymes nonspecifically. Including detergents such as 0.01% Brij-35 in secondary validation assays helps rule out promiscuous compounds that act by aggregating in solution or through undesirable precipitation in aqueous buffers .

• Hit validation strategy:

  • Primary screening at a fixed concentration (e.g., 40 μg/mL)

  • Secondary screening with dose-response curves

  • Counter-screening with detergent to exclude false positives

  • Verification of hits through orthogonal assays

In a case study of ME2 inhibitor screening, a library of 12,683 natural products yielded 47 initial hits (0.37% hit rate), but after excluding promiscuous compounds using 0.01% Brij-35 in dose-response determinations, only 15 ME2 inhibitors with diverse structures remained, resulting in a final hit rate of 0.12% .

What methods are available for characterizing the binding mode and kinetic parameters of ME2 inhibitors?

Several complementary methods are employed to thoroughly characterize ME2 inhibitor binding modes and kinetic parameters:

• Enzyme kinetics analysis:

  • Determination of inhibition type (competitive, non-competitive, uncompetitive, or mixed) by varying substrate concentrations in the presence of different inhibitor concentrations

  • Calculation of inhibition constants (Ki) through appropriate plotting methods (e.g., Lineweaver-Burk, Dixon, or direct nonlinear regression)

  • Time-dependence studies to distinguish between fast-binding and slow-binding inhibitors

• Thermal shift assay (TSA):

  • Measures changes in protein thermal stability upon inhibitor binding

  • Provides direct evidence of physical interaction between the inhibitor and the enzyme

  • Positive shifts in melting temperature (Tm) indicate stabilization of the enzyme structure by inhibitor binding

• Structural analysis:

  • X-ray crystallography to determine inhibitor binding sites

  • Molecular docking studies to predict binding modes

  • Structure-activity relationship (SAR) analyses to identify key pharmacophore features

For example, NPD389 was characterized as a fast-binding inhibitor of ME2 because its inhibition was independent of incubation time. Kinetic analysis revealed it to be a mixed-type inhibitor with respect to L-malate and an uncompetitive inhibitor with respect to NAD+, providing insights into its mechanism of action .

How can one express and purify recombinant ME2 variants to study structure-function relationships?

To express and purify ME2 variants for structure-function studies, researchers can follow this optimized protocol:

• Design of ME2 variants:

  • Point mutations can be introduced using site-directed mutagenesis

  • Chimeric constructs can be created by domain swapping with other malic enzyme isoforms

  • Truncated variants can be generated to study domain functions

• Co-expression system for hetero-oligomeric complexes:

  • Transform E. coli cells simultaneously with two compatible vectors (e.g., pET29-NAD-ME2 and pET32-NAD-ME1)

  • Select transformants on media containing both appropriate antibiotics (e.g., 100 μg/ml ampicillin and 30 μg/ml kanamycin)

  • Induce expression with lactose (1% w/v) at 16°C for 16 hours to enhance proper folding

• Purification strategy:

  • Harvest cells by centrifugation and resuspend in buffer containing protease inhibitors

  • Lyse cells by sonication and clarify lysate by centrifugation

  • For His-tagged variants, use Ni-NTA chromatography with imidazole gradient elution

  • For oligomeric analysis, subject purified protein to gel filtration chromatography on a Superdex 200 column at a flow rate of 0.5 ml/min

• Validation and characterization:

  • Confirm protein purity by SDS-PAGE

  • Verify oligomeric state by gel filtration chromatography

  • Assess enzymatic activity under standardized conditions

  • Compare kinetic parameters with wild-type enzyme

For hetero-oligomeric complexes, the molecular mass can be evaluated by gel filtration chromatography using a Superdex 200 column equilibrated with appropriate buffer (e.g., 25 mM Tris-HCl, pH 7.5, or 50 mM MES-NaOH, pH 6.5) .

What are common pitfalls in ME2 enzyme assays and how can they be addressed?

Several technical challenges can affect the reliability of ME2 enzyme assays. Here are common issues and their solutions:

• Nonspecific inhibition through compound aggregation:

  • Problem: Small molecules can form colloidal aggregates that nonspecifically inhibit enzymes.

  • Solution: Include detergents such as 0.01% Brij-35 in assay buffers to disrupt aggregates. This approach successfully eliminated 32 of 47 initial hits in a screening campaign for ME2 inhibitors .

• Enzyme stability issues:

  • Problem: ME2 may lose activity during storage or assay conditions.

  • Solution: Optimize buffer composition (pH, ionic strength), include stabilizing agents (glycerol, reducing agents), and store the enzyme at appropriate temperatures (-80°C for long-term, on ice during experiments).

• Interference from assay components:

  • Problem: Assay additives may absorb at 340 nm, interfering with NADH measurements.

  • Solution: Perform appropriate blank corrections and consider alternative detection methods for compounds with intrinsic absorbance at the wavelength used.

• Accurate determination of kinetic parameters:

  • Problem: Substrate inhibition or cooperative effects can complicate kinetic analysis.

  • Solution: Use appropriate kinetic models (Michaelis-Menten for hyperbolic kinetics, Hill equation for sigmoidal kinetics) and ensure sufficient data points across the substrate concentration range .

• Distinguishing true hits from artifacts in screening:

  • Problem: False positives can waste resources in follow-up studies.

  • Solution: Implement a robust validation cascade including dose-response curves, counter-screens, orthogonal assays, and thermal shift assays to confirm direct binding .

How should researchers interpret complex kinetic data when studying ME2 regulators?

Interpreting complex kinetic data for ME2 regulators requires systematic analysis and appropriate modeling:

• Distinguishing activation from inhibition mechanisms:

  • For activators like CoA, data should be fitted to the appropriate activation equation:
    v = v₀ + (Vₐ × A) / (A₅₀ + A)

  • This differentiates between effects on binding affinity versus catalytic efficiency

• Analyzing mixed-type inhibition patterns:

  • When an inhibitor affects both substrate binding and catalytic steps (as with NPD389), use global fitting to determine both competitive (Ki) and uncompetitive (Ki') inhibition constants

  • Examine the alpha factor (Ki'/Ki) to quantify the degree to which inhibitor binding affects substrate binding and vice versa

• Addressing multi-substrate enzyme kinetics:

  • For bisubstrate reactions like those catalyzed by ME2, systematically vary one substrate while keeping the other constant

  • Generate multiple datasets at different fixed concentrations of the second substrate

  • Use appropriate plotting methods (e.g., primary and secondary plots) to determine the kinetic mechanism

• Interpreting allosteric effects:

  • Sigmoidal kinetics indicate cooperative binding

  • Calculate Hill coefficients to quantify the degree of cooperativity

  • Consider the effect of regulators on the Hill coefficient as well as on Vmax and Km values

• Differentiating specific from nonspecific effects:

  • Compare IC50 values obtained in the presence and absence of detergent

  • Minimal change in potency with detergent (e.g., NPD389 showed IC50 values of 4.63±0.36 μmol/L vs. 5.59±0.38 μmol/L) suggests specific binding

What are effective strategies for validating potential ME2 inhibitors identified in primary screens?

A comprehensive validation strategy for potential ME2 inhibitors should include:

• Dose-response determination:

  • Test compounds at multiple concentrations (typically 8-12 point curves)

  • Calculate IC50 values with appropriate statistical analysis

  • Perform assays both with and without detergent (0.01% Brij-35) to identify promiscuous inhibitors

• Mechanism of action studies:

  • Determine inhibition type with respect to each substrate (NAD+ and L-malate)

  • NPD389, for example, was identified as an uncompetitive inhibitor with respect to NAD+ and a mixed-type inhibitor with respect to L-malate

  • Evaluate time-dependence of inhibition to classify as fast-binding or slow-binding

• Direct binding confirmation:

  • Employ thermal shift assays to verify physical interaction between inhibitor and enzyme

  • Positive shifts in melting temperature provide evidence of stabilizing interactions

  • For NPD389, thermal shift assays confirmed direct binding to ME2

• Structure-activity relationship (SAR) studies:

  • Test structurally related compounds to identify essential pharmacophore features

  • The 2,5-dihydroxy benzoquinone skeleton was identified as a key structural feature for ME2 inhibition

  • 4-OMe-substituted phenyl groups at C3 and C6 improved activity (NPD389), while 4-OH substituted phenyl groups decreased activity (NPD387)

• Selectivity assessment:

  • Test compounds against related enzymes (other malic enzyme isoforms)

  • Evaluate activity against unrelated enzymes to confirm specificity

  • Consider potential off-target effects based on structural features

By following this systematic validation approach, researchers identified NPD389 as a potent and specific ME2 inhibitor with an IC50 value of 4.63±0.36 μmol/L, providing a valuable tool for studying ME2 function and a potential lead for therapeutic development .

What are the potential implications of targeting ME2 in cancer therapy, and what research gaps need to be addressed?

Targeting ME2 in cancer therapy shows significant promise based on emerging evidence, but several research gaps must be addressed:

• Therapeutic potential:

  • ME2 knockdown impairs proliferation of K562 cells, induces apoptosis, and suppresses tumor growth in vivo

  • Silencing ME2 severely impairs growth of HCT-116 and U2OS cancer cell lines

  • ME2 is involved in p53 regulation during tumor metabolism, senescence, and growth

• Research gaps requiring investigation:

  • Cancer specificity: Define which cancer types are most dependent on ME2 activity

  • Resistance mechanisms: Identify potential compensatory pathways that might emerge upon ME2 inhibition

  • In vivo efficacy: Validate that ME2 inhibitors like NPD389 can achieve tumor suppression in animal models

  • Combination strategies: Determine optimal combination with existing therapies

  • Biomarkers: Develop predictive biomarkers to identify patients most likely to benefit

• Translational challenges:

  • Optimizing lead compounds for improved pharmacokinetic properties

  • Achieving sufficient selectivity against other malic enzyme isoforms

  • Balancing efficacy against potential metabolic side effects

  • Developing appropriate delivery strategies for maximal tumor targeting

The discovery of NPD389 as the first potent ME2 inhibitor represents an important milestone, providing a valuable tool to validate ME2 as an anticancer target and laying the foundation for developing novel therapeutic approaches .

How might structural biology approaches contribute to understanding ME2 function and developing more selective inhibitors?

Structural biology approaches offer powerful tools for advancing ME2 research:

• Structure determination methods:

  • X-ray crystallography of ME2 alone and in complex with substrates, cofactors, and inhibitors

  • Cryo-electron microscopy (cryo-EM) for visualization of oligomeric complexes

  • NMR spectroscopy for dynamic structural information and binding studies

• Structure-based inhibitor design:

  • Identification of binding pockets and interaction hotspots

  • Fragment-based approaches to develop high-affinity ligands

  • Structure-guided optimization of lead compounds like NPD389

  • Computational docking and molecular dynamics simulations to predict binding modes

• Understanding oligomerization and allostery:

  • Gel filtration chromatography reveals the quaternary structure of ME2

  • Characterization of homo- and hetero-oligomeric complexes

  • Mapping of allosteric sites for activators like CoA and inhibitors like ATP

  • Investigation of conformational changes during catalysis and regulation

• Domain function analysis:

  • Creation of chimeric constructs between different malic enzyme isoforms

  • Identification of domains responsible for substrate specificity and regulatory properties

  • Investigation of the structural basis for NAD+ vs. NADP+ preference

These approaches would significantly enhance our understanding of ME2 structure-function relationships and enable the rational design of more potent and selective inhibitors for both research and therapeutic applications.