Functionalized Congeners of Bioactive Compounds

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Work in our laboratory spanning more than three decades has demonstrated that certain drugs may be attached to well-defined carrier molecules and still retain the ability to bind to the receptor site and induce biological activity. This synthetic strategy for the attachment of drugs to carriers is termed the functionalized congener approach. The carrier molecule may be many times larger than the parent drug; indeed, there is practically no maximum size limitation for a fully potent analogue. Unlike the prodrug approach or the immobilization of drugs for slow release, the functionalized congener approach is designed to produce analogues for which no metabolic cleavage step is necessary for activation. Moreover, the attachment of the drug to a carrier such as a peptide may result in enhanced affinity at an extracellular receptor or other protein target site and an improvement in the pharmacological profile of the parent drug through energetically favorable interaction with distal sites on a receptor, as modeled for our high affinity fluorescent A2A and P2Y14 receptor antagonists. Recently determined experimental structures of the adenosine receptors (ARs) and P2Y receptors (P2YRs) for nucleotides follow upon a two-decade long progression of knowledge of the AR binding site(s) and other structural features, based on: empirical probing of structure activity relationships (SARs) of ligands, molecular modeling, and mutagenesis analysis. Early modifications of both AR agonists and alkylxanthine antagonists located sites in these two ligand scaffolds that were amenable to extension through a chemically functionalized chain without losing the ability to bind to the ARs. This logically suggested that the putative binding sites had regions that were accessible to the external environment and therefore less demanding sterically, i.e. the functionalized chains protruded beyond the conformationally and sterically restricted binding region of the core pharmacophore. However, the binding of such bitopic ligands can be complex, as we recently demonstrated cooperativity in fluorescent A2A and A3 antagonists. Points on the adenosine scaffold that displayed flexibility of substitution and could be extended chemically, without a limiting length, included the N6 position for the A1AR and the C2 position for the A2AAR. Both of these positions on adenosine derivatives are predicted by receptor docking to point toward the extracellular loops of the receptor. The C2-extended derivatives are most useful in the design of fluorescent agonists of the A2A receptor. A2A adenosine receptor agonists prevent bone loss in mice but have cardiovascular side effects. We synthesized novel alendronate-CGS21680 conjugates that specifically localize to bone, targeting the agonist to the site of tissue injury and thereby diminishing the frequency of administration and curtailing systemic side effects. MRS7216 contained the optimal tether length for receptor affinity. This compound reduced wear particle-induced bone degeneration in wild type, but not A2A-knockout mice. At the P2Y14R, which is a potential target for treatment of inflammation, the glucose moiety of native agonist UDP-glucose is suitable for chain extension (through an amide linkage to glucuronate). We used both of these points of substitution to design high affinity fluorescent ligands, which have advantages over radioligands as tools in drug discovery. We have used these fluorescent ligands for compound screening by flow cytometry. The fluorescent P2Y14 antagonist, MRS4174, is highly potent and has become our standard screening tool in our drug discovery efforts on that receptor. We are currently working on additional fluorescent P2Y14 antagonists, as research tools, and other divalent and biotopic ligands containing P2Y14 antagonists. We recently examined a bivalent A3 agonist/P2Y14 antagonist in pulmonary inflammation. A terminal amino group of the high affinity AR antagonist XAC (a xanthine amine congener) served as a site for generalized coupling to much larger moieties that extended into the extracellular medium, without losing AR affinity. The X-ray structures of XAC (Dore et al., 2011) bound to a thermostabilized hA2AAR show that the terminal amino chain of XAC proved to be very flexible and can be anchored to the receptor in various conformations. Thus, the objective in the design of XAC as a functionalized congener, in which the distal amino group escapes the steric constraints of the pharmacophore binding site, was finally explained structurally. We have explored other multivalent carriers to study GPCR action. For example, dendrimers are tree-like polymers that have multiple functional sites on the periphery for attachment of ligands. We have explored polyamidoamine (PAMAM) dendrimers, quantum dots and gold nanoparticles (AuNPs) as nanocarriers for functionalized congeners, i.e. strategically derivatized ligands for tethering, for ARs and P2YRs. However, we note with caution that terminal-amino PAMAM dendrimers undergo chemical rearrangements that make them less than optimal as drug carriers. allow the tuning of pharmacokinetic and pharmacodynamic properties by active or passive targeting of drugs for cancer and other diseases. It is apparent that the receptors have higher degrees of organization, i.e. they form functionally complex aggregates, and there is a need for pharmacological tools that address this multiplicity. We synthesized fluorescent A2AR agonists and antagonists and evaluated, by means of a flow cytometry homogeneous no-wash assay and a real-time fluorescence resonance energy transfer (FRET)-based approach. Our results provided evidence for the existence of a differential dopamine D2R-mediated negative allosteric modulation on A2AAR agonist binding that was oligomer-formation dependent, and with apomorphine being the best antiparkinsonian drug attenuating A2AAR agonist binding. In bitopic fluorescent conjugates of AR ligands, the interaction of the fluorophore with the receptor is proposed to modulate the potency or selectivity of the ligand or the binding kinetics. According to our modeling, the fluorescent conjugates bridge the orthosteric site on the receptor and an outer vestibule, where the fluorophore acts as distal secondary anchor to enhance the affinity of the conjugate. These bitopic probes can potentially be used for receptor detection and characterization in native tissues. However, their binding to the receptor may not be as straightforward as radioligands, due to cooperativity between the two binding regions for these long-chain compounds. Bivalent ligands are being developed to either activate or antagonize various combinations of GPCRs. We have also introduced functional congeners of uracil nucleotide-based inhibitors of CD73, which have extended chains that the 4 position of the nucleobase that enhance the affinity by acting as distal anchors. We have modeled the interactions of a range of enzyme-bound derivatives based on the X-ray structures of two of complexes with two of our inhibitors. Recently, we have applied this approach to a clinically important cancer target, Polo-like kinase1 (Plk1), with the introduction of allosteric inhibitors that target the Polo-box domain. An X-ray structure of our active inhibitor allopole-A is in agreement with the empirical result that functionalize chains can be appended to the molecule at an insensitive (not protein-constrained) site.