Critical Inhibitor Characteristics to Consider

Uncover the critical inhibitor characteristics to consider before working with enzyme inhibitors, such as cell permeability, the prozone effect, and Lipinski’s rule of 5. Also, discover practical examples of inhibitors used as pharmaceutical agents.

• Cell Permeability of Inhibitors
• Prozone Effect
• Enzyme Inhibitors as Pharmaceutical Agents
• Lipinski’s Rule of 5

Cell Permeability of Inhibitors

There is tremendous interest in developing synthetic molecules that can manipulate protein-protein interactions in living cells. This is the basis for many pharmaceutical development programs. However, the plasma membrane is selectively permeable and regulates the entry and exit of most molecules in the cell. The cell permeability of inhibitors, or how the inhibitor molecules cross the plasma membrane to reach their intracellular targets, depends on their size and lipophilic or hydrophobic characteristics. If a molecule is small enough, it can be transported across the membrane via passive diffusion, facilitated diffusion, or active transport. However, larger molecules may move via endocytosis.

Permeability of Peptide-Based Inhibitors

The permeability of peptide-based inhibitors depends on a variety of factors such as:

• Peptides with four or more amino acids may not be cell-permeable. However, attaching selective groups to these peptides can make them cell-permeable.
  • For example, the fluoro-methylketone (FMK)-based caspase inhibitors are cell-permeable because the carboxyl groups of aspartic and glutamic acid are esterified, making them more hydrophobic. These inhibitors covalently modify the thiol group of caspases, making them irreversible inhibitors. Also, at the amine end of the inhibitor, a Z group, biotin, or Ac group can be attached. These groups increase the hydrophobicity of the molecule, which makes them more cell-permeable. Compared to the inhibitors with an Ac or a biotin group, those inhibitors with a Z group are even more cell-permeable.
• Acyclic peptides are more permeable than corresponding cyclic peptides. This is due to the greater conformational freedom of the acyclic peptides.
• Basic peptides, such as HIV-1 Tat-(48-60) and Drosophila Antennapedia-(43-58) exhibit high membrane permeability and, when fused to peptide-based inhibitors, can perform as carriers for intracellular delivery.
• A short treatment of cells with digitonin can transiently permeabilize cells and allow peptide-based inhibitors to move into cells.
• Peptides can also be transported into cells by using protein transfection agents, such as ProteoJuice™ Transfection Reagent (Product No. 71281).

Permeability of Small Molecule Inhibitors

The following factors must be considered to determine the permeability of small molecule inhibitors:

• Some inhibitors are totally impermeable and cannot be used for cell-based studies. However, they are perfectly fine for use with lysates and homogenates.
• Some small molecule inhibitors are permeable due to their hydrophobic and lipophilic nature.
• In general, charged molecules are not cell-permeable.
• Most phosphorylated compounds are not cell-permeable. However, modified phosphorylated compounds, such as mono- and dibutyryl-cAMP, are cell-permeable.

 

Prozone Effect

The prozone effect, also known as the high dose-hook phenomenon, is classically used to describe an immunoassay in which adding excess antigen or excess antibody results in false-negative or false-low results. Although originally described in immunometric assays, the prozone effect can also be invoked in biochemical assays where the concentration of an inhibitor or a receptor antagonist far exceeds the concentration of the enzyme or receptor. The prozone phenomenon will cause the reaction to be either weak or negative (a false negative) in the first few dilutions, but upon further dilutions, the reaction proceeds as expected (Figure 1).

Figure 1. A graphical representation of the prozone effect.

In a typical immunoassay, antigens and antibodies bind to create a conjugate that can be detected and measured. However, when the prozone effect occurs, excess antigens or antibodies can bind all of the receptor sites, leaving nothing available to become a conjugate. Hence, the antibody-antigen conjugate cannot be detected, and a false negative result is produced, which can go undetected. In a clinical setting, this could lead to misdiagnosis, or in the case of inhibitors, it can reduce the efficacy of the drug or cause toxicity.

How To Avoid The Prozone Effect

The prozone effect can be avoided in the following ways:

• Use sequential dilutions of inhibitors
• Use concentrations that are 5- to 10- fold higher than the reported IC₅₀ values

Hence, when an inhibitor fails to perform as expected, it is best to reexamine the IC₅₀ values and use an appropriately diluted inhibitor.

Enzyme Inhibitors as Pharmaceutical Agents

Most drug therapies are based on inhibiting the activity of overactive enzyme(s). If an overactive enzyme can be inhibited, the progression of disease can be slowed, and symptoms can be alleviated. The utility of enzyme inhibitors as pharmaceutical agents is based on the concept of competitive enzyme inhibition, where inhibitors are structural analogs of normal biochemical substrates that compete with the natural substrate for the active site of the enzyme and block the formation of undesirable quantities of metabolic products.

Prodrugs

In addition to active compounds as pharmaceutical agents, prodrugs are preferred in some cases. Prodrugs are not effective until they are metabolized and converted to an active form. Some inhibitors can interfere with the in vivo conversion of prodrugs if administered at the same time, reducing the efficacy of the latter. For example, the anticancer prodrug, tamoxifen, requires the cytochrome P450 2D6 to become an active drug. However, antidepressants, such as paroxetine hydrochloride, can inhibit the activity of cytochrome P450 2D6, thereby severely reducing the efficacy of tamoxifen if taken in the same timeframe.

Structure-Based Drug Design

In the drug development process, an initial candidate compound often exhibits merely modest competitive inhibition. Medicinal chemists improve upon the initial candidate by slightly modifying its structure to make it more effective, specific, bioavailable, and less toxic. Inhibitors developed based on structure-based drug design often exhibit poor bioavailability, primarily due to poor solubility. Increasing the solubility helps reduce the toxicity of the compound.

The effectiveness of the final product depends on its potency, specificity, metabolic pathway, bioavailability, and pharmacokinetic properties of the inhibitor molecule. High specificity for a single reaction can avoid any unwanted side effects and potential toxicity. High specificity can also reduce the depletion of inhibitors by nonspecific pathways.

Common Pharmaceutical Examples

Common examples of inhibitors used as pharmaceuticals are:

• Analgesics: acetaminophen (COX inhibitor)
• Hypercholesterolemic agents: lovastatin (Product No. 438185; HMG-CoA reductase inhibitor)
• Gout control: allopurinol (Product No. A8003; xanthine oxidase inhibitor)
• Antibiotics: rifampicin (Product No. R3501; DNA-dependent RNA polymerase inhibitor)
• Erectile dysfunction: sildenafil citrate (Product No. SML3033; phosphodiesterase V inhibitor)
• Anti-cancer agents: doxorubicin (topoisomerase inhibitor), imatinib mesylate (Product No. SML1027; Bcr/Abl tyrosine kinase inhibitor), 5-fluorouracil (Product No. 343922; thymidylate synthase inhibitor)
• Blood pressure control: captopril (Product No. C4042; angiotensin converting enzyme (ACE) inhibitor)

More specifically, some examples of receptor antagonists as pharmaceuticals include: 

• Atropine: acts as a central and peripheral ACh muscarinic receptor blocker
• Phentolamine: α1 and α2 adrenergic receptor blocker
• Prazosin: α1-specific adrenergic blocker
• Atenolol: β1-specific adrenergic blocker
• Naloxone: opioid receptor antagonist; fast-acting competitive inhibitor of m, k, and s receptors
• Candesartan: angiotensin II type I receptor blocker
• Cyclothiazide: a noncompetitive antagonist of metabotropic glutamate receptor 1
• Memantine: uncompetitive antagonist of NMDA glutamate receptor

NOTE: All inhibitors and receptor agonists and antagonists provided by us are for research use only and are not for clinical, diagnostic, or veterinary applications.

Lipinski’s Rule of 5

Lipinski et. al. (1997) analyzed the physicochemical properties of more than 2,000 drugs and candidate drugs in clinical trials. They concluded that a compound is more likely to be membrane-permeable and easily absorbed by the body if it matches the following criteria. ¹

Lipinski’s Rule of 5:

• Its molecular weight is less than 500.
• The compound’s lipophilicity, expressed as a quantity known as logP (the logarithm of the partition coefficient between water and 1-octanol), is less than 5.
• The number of groups in the molecule that can donate hydrogen atoms to hydrogen bonds (usually the sum of hydroxyl and amine groups in a drug molecule) is less than 5.
• The number of groups that can accept hydrogen atoms to form hydrogen bonds (estimated by the sum of oxygen and nitrogen atoms) is less than 10.

Lipinski’s rule of 5 provides a general guideline to determine the likeliness of a chemical compound to be a successful oral drug. These rules, based on the 90-percentile values of the drugs’ property distributions, apply only to absorption by passive diffusion of compounds through cell membranes. Compounds that are actively transported through the cell membrane by transporter proteins are exceptions to the rule. Lipinski’s criteria are widely used by medicinal chemists to predict not only the absorption of compounds, but also overall drug-likeness.

Exceptions and Concerns

• Although this rule provides a powerful and simple tool to narrow down a pool of potential drugs, it could potentially exclude compounds that could become successful drugs.
  • For example, most tuberculosis (TB) drugs and antibacterials, in general, do not follow the rule.
• One concern is that the sharpness of the boundaries can cause one molecule to score a “0” and another extremely similar molecule to score a “4”.
• Another concern is that equal weight is given to each rule. There have been suggestions to modify the rule, particularly to soften sharp boundaries. In “Softening the Rule of Five—where to draw the line” Petit et. al. (2012) propose a new, in-depth approach to both soften the thresholds and assign each rule a specific weight, resulting in improved predictive power. ²

By fine-tuning Lipinski’s rule, drug discovery may be improved by avoiding premature and inappropriate discarding of potential drugs.

Learn more about specific inhibitor characteristics in our Receptor Agonists and Antagonists article.

For Research Use Only. Not For Use In Diagnostic Procedures.

References

1. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. 1997. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Reviews. 23(1-3):3-25. https://doi.org/10.1016/s0169-409x(96)00423-1

2. Petit J, Meurice N, Kaiser C, Maggiora G. 2012. Softening the Rule of Five—where to draw the line?. Bioorganic & Medicinal Chemistry. 20(18):5343-5351. https://doi.org/10.1016/j.bmc.2011.11.064