Small Molecule Inhibitors Selection Guide

Slmolecule inhibitors are one of the most accessible and versatile tools for understanding protein function and its role in normal physiology and disease pathology. To be valuable in questioning biological processes or validating new targets, these tools must meet the criteria necessary to provide reliable and repeatable data. Since it is sometimes difficult to determine the most suitable inhibitor from the large number of molecules available, we collected a series of parameters that should be considered during the selection process.

When you consider using each new micromolecule in your research, you must pay attention to its 1) chemical properties, 2) potency, 3) selectivity and 4) environment of use. It is best to consult the scientific literature to learn about established tools in your field and the possible advantages and disadvantages of certain compounds (for example, cytotoxic or off-target effects). Detailed information about available probes can also be found by using a target-focused search in the Web-based chemical probe database. In addition, Aladdin provides relevant, detailed biological information in each product insert to help you select the right compound for your experiment. Often for more novel or relatively unidentified inhibitors, it is best to conduct initial tests yourself to see how the compound reacts in an experimental system. While not all compounds need to meet the recommended thresholds below in every case, considering these parameters will help you determine if the probe is appropriate for your purposes.

Chemistry
Structure	Structure should be clear, and the composition should be replicable. Avoid common toxic parts and extensive laboratory disturbance parts (PAINS). Also avoid chemically reactive groups unless necessary, such as covalent addition.
Stability	Stability (purity and chemical properties) should be maintained in relevant media, with attention to pH sensitivity. Activities should be reserved for cultural media. Molecules should not exhibit non-specific chemical reactivity (e.g., REDOX reactions or membrane instability).
Solubility	The solubility in aqueous medium should be adequate (e.g., 10 times IC50 or less than 0.05 μg/ml in % DMSO). Both solubility and lipophilicity need to be balanced. Highly charged, highly soluble compounds may exhibit low cellular or tissue permeability. Hydrophobic compounds may exhibit high permeability and potency with solubility problems. The use of hydrophobic molecules in the form of salt should improve water solubility.
Permeability	Permeability through passive or active transport is essential for cellular activity. Blood-brain barrier penetration is important for expected central nervous system (CNS) effects. Caco-2 cell permeability assay or parallel artificial membrane permeability assay (PAMPAs) is a commonly used assay to evaluate passive diffusion. MDCK-MDR1 permeability can be used to evaluate central nervous system permeability, including active efflux of P-glycoprotein (PGP).

Potency
IC50 and Ki	IC50 and Ki are the most commonly used methods to indicate the potency of inhibitors. In the context of enzyme inhibition, IC50 represents the concentration of the inhibitor required to reduce the rate of enzyme reaction by 50% under a given experimental condition. Ki represents the ratio of inhibitor-target complex decomposition (koff) to inhibitor-target complex formation (kon) for inhibitor-enzyme binding. The term Ki is a thermodynamic equilibrium constant and therefore a fixed value. IC50, on the other hand, is a representation of inhibition under a defined set of conditions and will vary according to factors such as substrate concentration present in the reaction environment. For competing inhibitors, IC50 can be related to Ki via the Cheng-Prusoff equation :IC50=Ki*(1+[S]0/Km). This is a useful web-based tool for converting IC50 values to Ki values using different inhibitor binding models. Note that for proteins other than enzymes (such as G-protein-coupled receptors or ion channels), the efficacy of antagonists can also be reported via Ki or IC50. In the case of GPCRs, IC50(or usually EC50) is an indication of inhibition of a cellular response (i.e. a reduction in cAMP concentration), whereas Ki refers only to binding to the receptor with a natural ligand.
In vitro	In vitro pharmacodynamic reference standards are usually IC50 or Ki values of 100 nM in biochemical assays and 1-10 μM in cellular assays. The potency of the enzyme assay should be related to the potency in the cell. Use the lowest possible concentration to avoid off-target effects. Inhibitors that are effective on cells only at concentrations of 10 μM may be non-specific targeting proteins.
In vivo	The baseline standards for in vivo potency should meet the levels associated with cellular potency in the target tissue. Attention must be paid to effects related to absorption, dissolution, metabolism or excretion of compounds. Metabolic stability is the key question if this compound will advance animal studies.
Dose-dependent activity	Dose-dependent activity should be evident when experiments are conducted using recommended concentrations in the low to high range. The near complete inhibition of the target at saturated concentration confirmed the efficacy of the compound.
Structure activity relationship	Structure activity relationship (SAR) analysis can interpret the binding of a range of compounds with different activities to an enzyme or receptor to understand the binding requirements, permissible amounts, and limitations of the target protein. It is important for the SAR family to cover 3-4 orders of magnitude of effectiveness. A "flat" synthetic aperture (SAR), where an entire range of compounds containing large structural differences have nearly equivalent weak potency, can indicate increased difficulty in delivering drugs to a target.

Selectivity
Profiling	The analysis will define selectivity for relevant objectives, which is often a more critical factor than effectiveness. Typically, in biochemical analysis, factors that are 10-100 times more potent than other family members define an inhibitor's selectivity for that target. Selective inhibitors designed for the target you're interested in can still bind to other proteins at higher concentrations. It is important to be aware of any additional activities associated with a particular chemical class.
Negative control experiments	Negative controlled experiments can be designed to demonstrate that the inhibitor does not effectively alter the function of any off-target protein at the concentration used to inhibit the desired target. Well-considered negative controls, such as exposing cells or proteins only to solvents or replacing closely related inactive structural analogs, such as R/S stereoisomers, help confirm the effect of inhibitors.
Positive control experiments	Positive control tests can be used to demonstrate that the inhibitor behaves as expected when the effect is not obvious. The control group did not receive the experimental treatment, but other treatments known to produce the desired effect.
Orthogonal probes	Orthogonal probes with similar activities but from different chemical types and/or modes of action may also be considered as additional controls. If the orthogonal probe produces a similar response, it is more likely that inhibition of the target is responsible for the observed phenotype. If not, then confounding off-target or toxic effects may be caused by the inhibitor.

Context
MOA	The mechanism of action is important to further define the properties of the inhibitor in order to determine how natural substrates modulate the efficacy of the inhibitor at physiological concentrations and to identify any vulnerabilities associated with that mechanism. Potential inhibiting events include:
	Irreversible Inhibition	Irreversible inhibition - Inhibitors inactivate enzymes irreversibly, usually by covalently attaching to them. Although irreversible inhibitors are usually covalent, non-covalent inhibitors can sometimes act as irreversible inhibitors for very long periods of time.
	Reversible Inhibition	●  Reversible inhibition can be broken down into the following types: ●  lcompetitive inhibition -- free enzyme inhibitors and the substrate competition, but the combination of each block each other, because the activity of the combination of events often occur in the target site (is a locus), is the place of the substrate is combined with. ●  noncompetitive inhibition - inhibitor combined with free enzyme and enzyme and substrate complex as well. Although non-competitive inhibition usually occurs at allosteric sites, non-competitive inhibition can also occur when binding to orthosteric sites, usually in cases where the active site is a double substrate site (the enzyme competes with one substrate but not the other). ●  noncompetitive inhibition - inhibitor can combine with enzyme - substrate compounds, the formation of reversible ternary complexes of inactive. Noncompetitive inhibition is a special case of mixed inhibition. ● mixed inhibition - inhibitor combined with the enzyme in the free enzyme and enzyme - compounds of different substrates. This type of inhibitor may exhibit a greater affinity for one state (non-competitive inhibition) or another (competitive inhibition). ●  allosteric inhibition - inhibitor binds to a target on the allosteric site rather than the active site, cause the conformational changes of target enzyme, it is necessary to suppress. These conformational changes can affect the formation of the usual enzyme-substrate active site complex, destabilize transition states, or reduce the ability to reduce catalytic activation energy. ●  partially inhibit enzyme - substrate inhibitor interact to produce compounds than enzyme - substrate compounds of turnover rate is low. Some activity remains because the catalytic center of the enzyme-submatter-inhibitor complex may retain some ability to arrange near the substrate and promote catalysis. ●  closely with inhibition of experience - the original enzyme inhibitors complex isomerization closer compounds to form the second. Tight binding inhibitors tend to show a non-competitive phenotype, even though they can bind to the target enzyme in a competitive, non-competitive, or non-competitive manner. Because this can manifest as slowly increasing enzyme inhibition, which is time dependent, traditional Michaelis-Menten dynamics would give an incorrect Ki value. More accurate Ki values can be obtained by analyzing kon and koff rate constants. ●  Time-dependent inhibition - The inhibitor slowly binds to the enzyme over a time scale of enzyme turnover, which has the effect of slowing the observed onset of inhibition. This may cause the catalyst rate constant (kcat) value to slow down.

Inhibitor-target kinetics	Inhibitor-target dynamics, including kon, residence time, and koff, add an extra dimension to the potency and selectivity of the compound. Compounds that dissociate slowly from the target may have longer activity at lower concentrations, resulting in a lower dose level or dose frequency. Residence time is often more relevant to in vivo activity than thermodynamic potency.
Target vulnerability	Target vulnerability is a function of the lowest level of target participation required to observe an effect. A highly vulnerable target requires a low level of target occupancy (i.e. a low level of inhibitor exposure) to achieve the desired effect. Cell-based irrigation experiments can provide insight into target vulnerability by helping to determine the phenotypic consequences of target contact once the inhibitor is removed from the system.
Physiological context	The physical environment of the target and the downstream consequences of target engagement must be considered. For example, disrupting an enzyme that catalyzes rate-limiting steps in the metabolic pathway may have greater consequences than other enzymes in the pathway. In an antibacterial or anticancer context, the last step in inhibiting biochemical pathways, downstream of high energy/high cost biochemical products, may prove to be particularly toxic to target cells.
Timeframe	Experiments should be designed with the time range required to anticipate phenotypic effects or trigger the cascade of signaling events in mind to capture this length of time accordingly.
Complementary experiments	Complementary experiments using available RNAi or mutants, when available, help build consensus on the role of a given target in a biological system.
Application fitness	The suitability of a compound for application to a target ultimately depends on the degree to which it relates to the biological context hypothesized under study, as the use of an inhibitor in one biological system does not necessarily infer from another. An inhibitor that is fit for purpose is appropriate for both the target and the broader scientific context.
Availability	The compound is widely available and can be obtained in quantities for subsequent use.

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