The isoprenoid biosynthetic pathway is an essential metabolic system that is responsible for the production of one of the largest and most diverse ranges of biomolecules ever identified. The termini of this pathway include: fat-soluble vitamins, cholesterol, reproductive hormones, and components of cellular signal transduction and electron transport pathways. With such a diverse set of biologically important metabolites, it has become one of the most targeted pathways for study in human pathology. Earlier pharmaceutical development has yielded clinically relevant classes of compounds that impact specific enzymatically catalyzed stages along the mevalonate pathway. Possibly the most commonly recognizable class would be the statins, which were developed to constrain the production of cholesterol and other sterols via the inhibition of an early stage enzyme HMG-CoA reductase, making them useful in the treatment of cardiovascular disease. Another important class of inhibitors would be nitrogenous bisphosphonates such as Pamidronate and Zoledronate, which have been shown to disrupt the more downstream enzyme farnesyl diphosphate synthase (FDPS). The bisphosphonate core of these compounds helps to impart a high affinity for bone mineral, making them useful in the treatment of bone diseases such as osteoporosis and multiple myeloma.
Noting the success of bisphosphonates in the treatment of certain bone diseases via action on isoprenoid biosynthesis, more recent research has yielded compounds that selectively inhibit the later stage enzyme geranylgeranyl diphosphate synthase (GGDPS). One promising compound is digeranyl bisphosphonate (DGBP) which has been shown to induce apoptosis in some cancer cell lines. Crystallographic data for GGDPS aided in the determination of important structural features that lead to the activity and selectivity of DGBP. The bisphosphonate head group likely coordinates with magnesium cations in the active site of GGDPS, and the long nonpolar side chains can occupy hydrophobic channels which normally allow the binding of the natural substrates. These data appear to suggest DGBP’s selective binding arises from its capacity to form a ‘V-shaped’ inhibitor with the geranyl groups at the alpha position extending into the hydrophobic regions within the enzyme and the bisphosphonate functionality establishing a strong electrostatic interaction with the magnesium ions.
Noting these features, the development of novel compounds that retain the seemingly important structural features of known inhibitors while modifying certain aspects intended to enhance the biological activity of the resulting compounds was undertaken. These novel compounds were envisioned through a synthetic approach that would incorporate one isoprenoid chain at the α-carbon of the bisphosphonate and a second as a phosphonic ester. The previously studied phosphonate salts also were modified to the corresponding pivaloyl oxymethyl (POM) protected esters. The resulting motif was intended to allow for the liberation of a highly anionic species within a cell, which could more closely resemble the charge of the naturally occurring pyrophosphate substrates while retaining more desirable ADMET properties in the prodrug form. After intracellular formation of the active salt, the isoprenoid chains in the new motif could adopt a structure with more gradual curvature through the central portion making the structure more closely resemble a ‘U-shaped’ inhibitor.
A short synthetic sequence for the production of these new bisphosphonate inhibitors was developed. Each of the triPOM species formed was tested for the ability to disrupt the action of GGDPS in multiple myeloma cells. The compounds showed rather potent activity in cellular bioassays, in the hands of our collaborators in the research group of Dr. Sarah A. Holstein, with EC50 values in the single digit micromolar range. These compounds confirmed that the use of POM ester functionality can act as a viable prodrug strategy for the intracellular delivery of these compounds to the myeloma cells. These studies also demonstrate that the incorporation of a methyl group at the α-carbon results in the enhanced ability of these bisphosphonates to inhibit GGDPS. In general, the low µM range of concentrations required for these compounds to express meaningful cytotoxic biological activity makes them unattractive candidates for further investigation. However, the recognition of improved activity through the use of POM prodrug functionality and alpha methylation led to the development of other compounds that display these features. This has resulted in the synthesis of the most active inhibitors of GGDPS yet reported.
Another focus of research in the Wiemer group is the generation of novel phosphoantigens. Phosphoantigens are small phosphorus containing compounds that are recognized by certain immune cells and stimulate proliferation. The development of drugs that can regulate immune function via immunostimulatory activity is of great interest. The specific target for these phosphoantigens (γδ T cells) has recently been shown to play an important role in native cancer immunosurveillance. Currently the most potent natural phosphoantigen known, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), is found in the non-mevalonate isoprenoid biosynthetic pathway of bacterial cells, which may help explain the development of its immunostimulatory effect. However, the inherent lack of metabolic stability of HMBPP, which translates to a short in vivo lifetime, provides a significant challenge to clinical utility. The mechanism for phosphoantigen stimulation of T cell production is not fully characterized, but recent work has demonstrated the importance of intracellular delivery of phosphoantigens that bind to an intracellular domain of the butyrophilin 3A1 protein necessary to induce proliferation.
Noting the potent activity of HMBPP, we have advanced the design and synthesis of structurally similar compounds with potentially enhanced metabolic stability and cellular permeability. Some more recent contributions to this project include the synthesis of tris-pivaloyloxymethyl prodrug phosphinophosphonates. These phosphinophosphonates were found to produce a strong immunostimulatory cellular response, while demonstrating greater metabolic stability by virtue of the –C-P-C-P linkage replacing the anhydride –O-P-O-P arrangement in HMBPP. The POM prodrug functionality appeared to provide improved cell permeability as reflected in a significant increase in T cell proliferation.
Efforts to develop phosphoantigens with activity that matches or surpasses that of HMBPP, while exhibiting more desirable ADMET properties, are ongoing. The most recently developed compounds vary by the introduction of aryl phosphonic esters. The success of phosphorous-containing prodrugs that incorporate phenolic acid esters such as Sofosbuvir has supported our interest in this area. Preliminary T cell data for the lead compounds show great promise with significant proliferation at nanomolar concentrations. Biological assays for the phosphoantigens developed herein were performed by Dr. Andrew Wiemer and coworkers, and most of the tested compounds were found to be exceptionally active for the stimulated proliferation of Vγ9Vδ2 T cells. Studies are currently being conducted to form novel phosphoantigen prodrugs that express potent activity and desirable pharmacokinetic properties and will no doubt lead to the development of interesting and potentially therapeutically relevant compounds.