HsPBGS inhibition by hexamer-stabilizing drugs
Each of the compounds that was confirmed to increase the mole fraction of Hs
PBGS hexamer was subjected to a dose-response native PAGE analysis, and to kinetic inhibition studies as detailed in the experimental section. The Hs
PBGS hexamer-stabilizing compounds are chemically and pharmacologically diverse (), and are detailed below. Representative kinetic inhibition, dose response native PAGE gels, and quantified native PAGE results are shown in for two of the compounds, nitrofurazone, which inhibited Hs
PBGS activity to 0%, and tolefenamic acid, which inhibited Hs
PBGS activity to a plateau of 41 %. All inhibition and dose-response native PAGE data are summarized in , and presented in detail in Supplemental Figures S1 and S2
. Because the native PAGE experiments were performed at a high protein concentration (0.3 mg mL−1
), while the kinetic experiments were performed at a low protein concentration (10μg mL−1
), apparent K0.5
values estimated from the gels appear higher than the IC50
values calculated from the inhibition kinetics. This is consistent with the law of mass action, the equilibrium illustrated in , and previous studies that utilized low-protein concentration native PAGE, protein concentration-dependent chromatography, and thermodynamic analyses of the oligomeric interconversion to establish this relationship.[5c, 9]
Parameters extracted from kinetic inhibition and hexamer-stabilization experiments.
Nitrofurazone is a bactericidal agent used in ointments marketed under the trade name Furacin. It is not frequently used since alternative antibacterial ointments are available that are more effective and less toxic.
Nitrofurazone completely inhibited Hs
PBGS activity in assays with an IC50
of 2.1 ± 0.2 μM (), and yielded approximately 50% conversion to the hexamer in the native PAGE assay at the highest concentration tested ().
Copper sulfate is used topically in the treatment of phosphorous burns.
In the past it was used an emetic agent, but is now considered too toxic for ingestion.
Copper sulfate (insoluble in DMSO) was dissolved in H2
O, and the copper sulfate data is presented relative to a H2
O control. Copper sulfate inhibits Hs
PBGS with comparable potency to Nitrofurazone and exhibits an IC50
of 2.2 ± 0.1 μM (), and yields conversion to greater than 50% hexamer in the native PAGE assay at the highest concentration tested (Supplemental Fig. S1
). Based on comparable results with copper chloride (not shown) we conclude that copper is the chemical responsible for the inhibition and hexamer stabilization.
Rose Bengal is a fluorescein derivative used in diagnostic eye drops to stain and identify damaged corneal tissue.
Rose Bengal labeled with I125
had been used as a radioactive tracer marketed under the trade name Robengatope, though this product is no longer in use. A formulation of Rose Bengal (PV-10) is in clinical trials as a treatment for melanoma and breast cancer.
Rose Bengal completely inhibited Hs
PBGS with an IC50
of 6.9 ± 0.2 μM (); however, the native PAGE assay results did not support Hs
PBGS hexamer stabilization as the likely mode of inhibition. While the band representing the octamer decreased as a function of increasing Rose Bengal concentration, the band representing the hexamer did not increase. Rather, the appearance of smears and bands running at the dye front suggests that this compound is simply destabilizing the higher-order oligomers and perhaps denaturing the protein (Supplemental Fig. S1
). Of those compounds that we characterized, Rose Bengal is the only compound for which protein migrated farther than the position of the hexamer band in the gels.
2,4-dinitroanisole is a compound used in lice repellants, insecticides, and pesticides. It is metabolized to 2,4-dinitrophenol, a compound that uncouples oxidative phosphorylation and was used to promote weight loss in the 1930’s.
Its use as a weight-loss agent was terminated due to the side effect of dangerously high body temperature. 2,4-dinitroanisole inhibited Hs
PBGS activity with an IC50
of 15 ± 1 μM (), and yielded about 50% hexamer in the native PAGE assay at the highest concentration tested (Supplemental Fig. S1
3,5-dibromosalicylaldehyde was labeled as an antiseptic in the JHCCL library. This compound also completely inhibited Hs
PBGS with an IC50
of 22 ± 1 μM (). However, it is one of the most effective compounds for stabilizing the hexamer, yielding well over 50% of the protein as hexamer in the native PAGE assay (Supplemental Fig. S1
Five structurally-related non-steroidal anti-inflammatory drugs from the fenamate class were identified by the screen, and were found to induce hexamer stabilization and inhibit Hs
PBGS activity very similarly to each other. Tolfenamic acid is marketed under the trade name Clotam and is in use in Europe. Flufenamic acid is marketed under the trade name Arlef, and the KEGG database suggests current use in Europe and Japan. Diclofenac is widely used in the U.S. and abroad, and can be obtained over the counter in many countries. Different prescription formulations are available in the U.S. under trade names including Arthrotec, Cataflam, Pennsaid, Solaraz, Voltaren, and Zipsor. Meclofenamic acid is currently in use in the U.S. and abroad, and is available in the U.S. as meclofenamate sodium. Niflumic acid is marketed in a topical formulation under the trade name Niflugel. Data is shown for a representative compound, tolfenamic acid (). All of the fenamates reduce Hs
PBGS activity to a plateau of 40–50% activity with similar IC50
values: tolfenamic acid – 2.1 ± 0.3 μM, flufenamic acid – 2.5 ± 0.4 μM, diclofenac – 9 ± 2 μM, meclofenamic acid – 9 ± 2 μM, and niflumic acid – 13 ± 2 μM (). These compounds also exhibit similar results in the native PAGE assay, with less than 50% conversion to hexamer observed at the highest concentration tested (Supplemental Fig. S1
The final two compounds that conclusively stabilize the Hs
PBGS hexamer and inhibit Hs
PBGS activity are less potent than those described above ( and Supplemental Fig. S1
). Oxantel is an antihelminthic agent marketed under the trade name Telopar and is utilized to treat intestinal worms. This compound inhibited Hs
PBGS to a plateau to 46 ± 3% with an IC50
18 ± 3 μM, but was relatively potent in the native PAGE assay, yielding over 50% of the protein as hexamer at the highest concentration tested. Bendroflumethiazide is a diuretic and antihypertensive marketed under the trade names Naturetin and Corzide. Bendroflumethiazide, the least potent inhibitor identified in this screen, inhibits Hs
PBGS activity with an IC50
of 130μM, and exhibits less than 40% Hs
PBGS conversion to hexamer at the highest concentration evaluated in the native PAGE assay.
We have established that there is complex interplay of factors that determines the position of the oligomeric equilibrium of Hs
For instance, basic pH favors the hexamer, the presence of substrate/product favors the octamer, and the law of mass action dictates that higher protein concentration favors octamer. The studies presented herein show hexamer stabilization in an alanine based acrylamide gel at pH 8.8 at very high ionic strength (880 mM alanine, 250 mM tris), in the absence of substrate, and at a modest protein concentration (0.3 mg mL−1
). In contrast, enzyme inhibition assays were carried out in 100 mM bis-tris propane, pH 8.0, in the presence of substrate, and at a lower protein concentration (0.01 mg mL−1
). Thus, it is not surprising that hexamer stabilization and enzyme inhibition, which are done under dissimilar conditions, are not quantitatively correlated. Nevertheless, our published studies on Hs
PBGS used an activity stained native PAGE technique and provided an unequivocal correlation between Hs
PBGS hexamer stabilization and enzyme inactivation.[6a]
Similarly, in the current study we find that clinical compounds that showed the most pronounced hexamer stabilization are not necessarily the most potent inhibitors. We also find that some clinical compounds inhibit Hs
PBGS activity completely while others inhibit to a non-zero plateau. In fact, no correlation is present between IC50
values and the end point of inhibition.
Clinical relevance of HsPBGS quaternary structure dynamics
Protein structure dynamics are increasingly appreciated as essential to protein function. Proteins that use the morpheein model of allosteric regulation are denoted as morpheeins () and their existence adds a quaternary structure dynamic component to the principle that protein structure changes can govern protein function. PBGS is the prototype morpheein and we previously established that small molecules can modulate PBGS function by preferentially binding to one component of the quaternary structure equilibrium.[1, 6]
Here we address the possibility that the morpheein model of allostery may contribute to drug side effects. In the case of PBGS, drug stabilization of the inactive hexameric assembly is predicted to potentiate the symptoms of lead poisoning and ALAD porphyria and may interfere with certain forms of photodynamic therapy. Thus, it is significant that a structure-blind screen of clinical compounds revealed twelve hexamer stabilizing Hs
PBGS inhibitors. If the inhibition of Hs
PBGS by these agents is found to translate into physiologically relevant outcomes, this would constitute an unprecedented mechanism for a drug side effect, and would suggest that these medications be contraindicated for those suffering from lead poisoning, ALAD porphyria, and other porphyrias that result in accumulation of 5-aminolevulinate. While several of the identified compounds are used topically, it is important to note that topical medications are absorbed into the body. For example, patients using topical preparations of the amebicidal Clioquinol were found to have the drug present at micromolar levels in their plasma 
Furthermore, those which are topical might be contraindicated for patients considering certain types of photodynamic therapy.
The connection between our observed in vitro results of drug induced Hs
PBGS hexamer stabilization and in vivo side effects remains speculative but intriguing, particularly with regard to normal allelic variations in the human population. The two predominant alleles encode Hs
PBGS exists primarily as the active octamer under physiologically relevant conditions.[5c]
However, there are eight disease associated alleles for which compound heterozygotes suffer from ALAD porphyria. Each of these alleles encodes a Hs
PBGS with an increased propensity to form the inactive hexamer.
Individuals carrying these alleles would be expected to be more sensitive to porphyrigenic side effects relative to the general population. In fact, the two drugs identified from our published computational screen, Clioquinol and Iodoquinol, are documented to have side effects similar to a porphyric attack.
When tested in vitro, both drugs are more potent as Hs
PBGS hexamer stabilizing agents for two porphyria-associated variants relative to wild-type Hs
This raises the possibility for application of results such as ours to the burgeoning science of personalized medicine.
Notable in our results is the identification of the widely prescribed anti-inflammatory agent diclofenac as a Hs
PBGS inhibitor. Diclofenac is specifically contraindicated in porphyric patients as per recommendations of the American Porphyria Foundation.
In some countries, this drug is available over the counter. The current work suggests one mechanism by which this drug may be porphyrigenic. The entire family of porphyric illnesses is episodic in nature and precipitating factors are poorly understood.
The drugs identified in this study, particularly the widely used diclofenac formulations, may contribute to the episodic nature of porphyric diseases.
Drug stabilization of alternate oligomers of other proteins
Intriguingly, flufenamic acid and diclofenac have previously been established to stabilize a specific oligomeric form of another human protein, transthyretin, and prevent its aggregation into amyloid plaques;
however, the mechanism for this effect likely differs from the hexamer stabilization that we observe for these compounds with Hs
PBGS. Transthyretin performs its biological function of binding and transporting thyroxine as a tetramer, and the binding of thyroxine to two sites at the quaternary structure interface stabilizes the tetrameric assembly. The tetramer can dissociate to monomers in which the subunits can refold to become amyloidogenic and then irreversibly assemble into fibrils. This amyloid forming process occurs more readily with transthyretin mutations associated with the human diseases familial amyloid polyneuropathy, senile systemic amyloidosis, and familial amyloid cardiomyopathy.
Flufenamic acid and diclofenac are structurally similar to thyroxine, and have been shown by X-ray crystallography to occupy the thyroxine binding site suggesting that they stabilize the tetramer via similar mechanisms as the native ligand. [21b]
This is different from the hexamer stabilization mechanism we propose for these drugs with Hs
PBGS. First, the drugs have no similarity to the enzyme substrate or product (see and ) and are unlikely to bind at the Hs
PBGS active site. Second, the alternate Hs
PBGS oligomers represent a reversible equilibrium of native states, rather than a process that requires refolding and an irreversible assembly into amyloid. Despite the presumably different mechanisms, the fact that the two chemically related drugs, flufenamic acid and diclofenac, can stabilize a specific oligomer of two different proteins suggests that this class of drugs may have an affinity for protein-protein interfaces.
The morpheein concept is new enough that PBGS is the only verified example, though suggestive data exist for other homo-oligomeric proteins.[1, 4, 6b, 23]
Here we show that a significant number of drugs can modulate Hs
PBGS activity by modulating the quaternary structure equilibrium and raise the possibility that some drug side effects may be due to this effect on Hs
PBGS. Since it is unlikely that PBGS is the only protein that regulates its activity through an equilibrium of alternate oligomers, we posit that numerous other proteins could also be subject to modulation via small molecule stabilization of an alternate oligomer and that this may be the mechanism through which some drugs induce side effects. It is documented that the quaternary structure equilibrium of Hs
PBGS is affected by naturally occurring sequence variation.
Other proteins that utilize the morpheein mechanism would be susceptible to similar sequence-related variability. As with PBGS, this could contribute to individual variation in drug side effects.