Small Molecule Complementarity As A Source of

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Current Pharmaceutical Design, 2007, 13, 000-000

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Small Molecule Complementarity As A Source of Novel Pharmaceutical Agents and Combination Therapies Robert S. Root-Bernstein* and Patrick F. Dillon Department of Physiology, Michigan State University, East Lansing, MI 48824 USA Abstract: Many examples of specific binding between small molecules are known that are associated with modified physiological and pharmacological activities. Conversely, the antagonism or synergism of small molecules is often correlated with specific binding between the molecules. It follows that small molecule binding can be used as a relatively quick, easy, and specific screen for functionally useful drug actions and interactions. These actions and interactions may manifest themselves as functional antagonisms; binding may correlate with enhancement or synergism; the formation of some complexes may yield clues about how drugs may be targeted to specific cell types in vivo and provide leads for the development of antidotes for drug overdoses or poisoning; the binding of one molecule to another may mimic receptor binding; and complexation may provide novel ways of protecting and delivering drugs. Relevant examples from each type of application are reviewed involving peptide-peptide interactions; peptide-aromatic compound interactions; aromatic-aromatic compound interactions; vitamin-aromatic compound interactions; and polycyclic compound interactions. We argue that screening for molecular complementarity of small molecules turns ligands such as neurotransmitters and their metabolites, hormones, and drugs themselves, into direct targets of drug development that can augment screening new compounds for activity against receptors and second messenger systems. We believe that the small molecule complementarity approach is novel, fruitful and under-utilized.

Key Words: Complexation; pi-pi bonding; charge-transfer complex; binding; antisense peptides; vitamins; catecholamines; indoleamines; drug interactions. INTRODUCTION Combinations of bioactive agents can have pharmaceutical and physiological effects that are different from their individual effects. In some cases, agents synergize or antagonize, but they may also enhance, alter, or generate novel responses. Binding may protect the combinations from oxidation, proteolysis, and other degradative processes. And binding may be useful for solubilization and other drug delivery purposes. Small molecule complexation may therefore be a source of beneficial and novel pharmaceutical effects. The purpose of this paper is to review some of the ways that small molecule drug interactions may be identified and used for therapeutic purposes. We suggest some novel applications as well. Our approach to small-molecule drug interactions is directed by the observation that, just as there is a link between structure and function in biological systems, so there is a link between the physicochemical properties of bioactive molecules and their physiological activity. In particular, molecules that bind to one another often (if not always) alter each others activity and, conversely, when two compounds are found to alter each others activity, they often (if not always) bind to one another [1]. We will demonstrate this alteredactivity-binding principle with reference to five classes of small molecule interactions: 1) peptides with peptides; 2) peptides with monoamines; 3) monoamines with small molecules such as vitamins; 4) monoamines with aromatic drugs; and 5) more complex aromatic molecular interactions. Strategies for identifying additional cases and classes of such interactions and applying them in novel ways for therapeutic purposes will conclude the paper. PEPTIDE-PEPTIDE INTERACTIONS Peptide-peptide binding can be useful to produce antagonism, functional enhancement, and for drug delivery purposes. There is clearly a correlation between the ability of peptides to antagonize each others activity and their ability to bind. The two key cases that have been worked out in detail involve bovine pineal antireproductive tripeptide (BPART) and luteinizing hormone*Address correspondence to this author at the Department of Physiology, Michigan State University, East Lansing, MI 48824 USA; Tel: 517-3556475; Emails: [email protected] or [email protected]

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releasing hormone (LHRH), on the one hand, and insulin and glucagon, on the other. BPART, as its name suggests, is an anti-ovulatory peptide isolated from cow brains that was found to antagonize LHRH activity and was investigated as a drug lead for the development of novel anti-reproductive drugs [2-6]. Subsequent studies demonstrated that BPART antagonism of LHRH was mediated by direct binding of the two peptides, resulting in lowered availability of free LHRH [7]. A similar case was then observed with regard to insulin and glucagon. Insulin lowers blood glucose; glucagon raises it. Based on the principle that physiological antagonism is mirrored in physicochemical complementarity, it was predicted that the two hormones would bind to each other, which they do with a Kd of about 13.5 uM [8] to 85 uM [9]. It appears that evolution has used the chemical complementarity of both the BPART-LHRH and insulinglucagon pairs in elaborating the metabolic control systems used by modern organisms [1, 10]. We suggest that other cases in which peptides have been observed to display mutually antagonistic activities will be characterized by a similar ability to bind, and therefore that the search for peptide complementarity may provide a rich source of peptide antagonists. The strategy of designing peptides to bind to one another has certainly yielded many physiological antagonists. Two general methods for designing peptide antagonists have been proposed, both based on the possibility that the complementary (non-coding) strand of DNA (cDNA) encodes peptides that are complementary to those encoded by the coding strand of DNA, just as cDNA encodes inhibitory RNA [11]. The first method is to read the cDNA strand in the normal reading frame (5-->3) [12-15] and the second method is to read the cDNA strand "backwards" (3-->5) (i.e., in parallel with the cDNA reading frame) [16]. Several limitations exist in the current literature on such "antisense peptides". Most of the more than 150 published examples of antisense peptides have originated from the "forward" reading of cDNA [reviewed in 17-19], but there are no unambiguous demonstrations using physicochemical methods that any of the antisense peptides designed by this method actually binds to its target peptide [reviewed in 20]. Moreover, the method is based on the controversial concept of “hydropathic anticomplementarity”, in which hydrophobic amino acids preferably © 2007 Bentham Science Publishers Ltd.

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bind to hydrophilic ones, against both common sense and much experimental data [20,21]. The only antisense peptides for which physicochemical techniques prove binding to target peptides are those designed by using the "backwards" reading frame of cDNA. Binding of these antisense peptides to their sense peptides has been demonstrated using NMR, u.v. spectroscopy, circular dichroism, and related methods [20, 22-23]. These structural binding studies show that the result of reading the cDNA sequence backwards is to produce a peptide that binds in a parallel beta ribbon to the sense peptide encoded by the coding strand of DNA. The beta ribbon aligns the side chains to form stereospecific amino acid pairs that are genetically encoded and physicochemically preferred. The requirement that the sense and antisense peptides be able to form a beta ribbon limits the possible applications of this technique, but the simplicity of the theory allows it to be easily applied within these limitations. “Backwards” antisense peptides are currently being used as drug leads for indications such as interference with A-beta protein aggregation in Alzheimers disease [24], regulation of nitric oxide synthase [25], and cytokine over-production [26]. It is intriguing to note that not all antisense peptides result in a physiological antagonism. In at least two cases -- an antisense peptide to angiotensin II [27] and antisense peptides to enkephalins [28] -- the antisense peptide enhanced peptide activity in vitro. The mechanism by which such enhancement occurs has not been elucidated, but might provide fertile ground for investigation. Peptide pairs may also be used for drug delivery purposes. Binding of one peptide to another should protect both against proteolytic enzymes, oxidation reactions, etc. [1,20]. Perhaps the best model so far characterized is the complex formed between fragments of neurophysin and either of the two hormones neurotensin or oxytocin [reviewed in 29]. Neurophysins act as protein carriers for neurotensin and oxytocin in vivo and peptide fragments of neurophysin have been found to incorporate most of the binding specificity and protective effects of the larger protein. It should be possible to engineer other such peptide-peptide interactions for the purposes of drug delivery whether using antisense peptide approaches or the types of interactions exemplified by insulin-glucagon and neurotensin-neurophysin binding. PEPTIDE-MONOAMINE COMPLEXES Peptides can sometimes bind smaller molecules as well as other peptides. Peptides hormones and neurotransmitters, such as adrenocorticotropic hormone, luteinizing hormone releasing hormone, substance P, the enkephalins and endorphins, etc., often contain sequences of amino acids that include pairs of adjacent aromatic side chains. When any pair of the side chains tryptophan, phenylalanine, tyrosine, or histamine, is separated by a single amino acid in a linear sequence, then they can form a "molecular sandwich" of exactly the appropriate dimensions for the intercalation of another aromatic molecule [30]. If a peptide forms an alpha helix, then a pair of aromatic residues that are separated by three others will also be brought into juxtaposition to form a molecular sandwich. Nearby side chains may also participate in the binding as well, especially if they can form hydrogen or ionic bonds to hydroxyls, amines, etc. of the aromatic molecule. These molecular sandwiches can bind aromatic monoamines and related compounds with high affinity and specificity with resultant alterations in physiological activity. Several well-characterized examples of peptide-monoamine molecular sandwiches exist. Dopamine binds to neurotensin with a kD of 7.5 x 10(-8), which represents sufficient affinity to explain many observations of antagonism of dopamine-induced locomotor activity by neurotensin in vivo [31]. Neurotensin also forms complexes with 4-ethylcatchol, 4-methlycatchol, 3-methoxytyramine, and norepinephrine although the specific physiological effects of such complexation have not been characterized [32].

Root-Bernstein and Dillon

Indoleamines bind to myelin basic protein (MBP), adrenocorticotropin-releasing hormone (ACTH), and LHRH. This case is particularly interesting in the context of drug discovery and design since complementarity theory played a large role in the elucidation of the binding and led to further, unexpected insights. It was known by 1980 that MBP contained a moderate affinity for the serotonin (5-OH tryptamine) binding site, but the structure and location of the site were not known [33, 34]. The concept of molecular sandwiching was used to predict that serotonin would bind by intercalating between phenylalanine and tryptophan residues and form hydrogen bonds to arginine and serine in the sequence Arg-Phe-Ser-Trp at positions 112-115 of MBP [35]. Subsequent nuclear magnetic resonance studies verified that this was, indeed, the binding site [36] and the peptide was shown to interfere with binding of serotonin to its receptor [37]. ACTH and LHRH, which have similar sequences (Phe-Arg-Trp and His-Trp-Ser-Tyr, respectively), were also found to bind serotonin [36]. Both of these peptide hormones are known to be regulated by serotonin in vivo [e.g., 38-40], so that binding correlates with functional interactions. Catecholamines, histamine, acetylcholine and related compounds had no measurable affinity for these peptides [36] and are not known to regulate the activity of these peptide hormones. The elucidation of the serotonin binding site on MBP led directly to the discovery that muramyl dipeptide is a serotonin mimic. Muramyl dipeptide had been identified as having two distinct activities. One was as the minimal active component of mycobacterial adjuvants [41, 42]. The other was as a human urine-derived “sleep factor”[43-45]. NMR experiments had shown that muramyl dipeptide binds to the same site on MBP (ACTH and LHRH) as does serotonin [46], leading to the hypothesis that muramyl dipeptide might exert its sleep-like activity by mimicking serotonin [47]. Muramyl dipeptide does, indeed, bind to serotonin receptors [37, 48-50]. This insight resulted in a wide range of serotonin-like activities being discovered for muramyl peptides in addition to their sleep-like properties, including smooth muscle activation, fever induction, immunological enhancement, analgesia, and platelet activation [51-56]. Thus, a small peptide “model” for serotonin binding was useful in discovering and predicting serotonin receptor behavior for a most unexpected peptidoglycan. As noted above, catecholamines do not bind to serotoninbinding peptides. Catecholamines such as epinephrine (EPI, or adrenalin), norepinephrine (NE, or noradrenalin), and dopamine (DA) bind to a distinct class of peptides including the enkaphalins and morphiceptin with mid-micromolar affinity [57]. Dopamine, but not EPI or NE, also binds to substance P and gastrin tetrapeptide [57]. Indolamines bind to none of these peptides [57]. Notably, EPI or NE are co-localized, co-released, and regulate enkephalin activity in vivo [reviewed in 57; see also 58-60], so that, once again, binding correlates with physiological interaction. This interaction has observable pharmacological effects. NE antagonizes enkephalins at physiological concentrations in vitro [61, 62], while opioids, including the antagonist naloxone, enhance adrenergic activity such as the inotropic effects of isoproterenol on cardiac muscle [63-65]. These data suggest two possibilities: 1) it might be possible to design small molecule antagonists to peptides, or peptide antagonists to small molecules, in which the antagonism is a direct result of binding; or 2) since the body apparently builds upon such chemical interactions in evolving complex receptor-mediated interactions, it may be possible to design molecularly complementary antagonists that act through complementary receptors rather than directly on the ligand of interest. AROMATIC-AROMATIC COMPLEXES Simple aromatic compounds can also bind to other aromatic compounds having complementary charge distributions and resi-

Small Molecule Complementarity

dues in appropriate positions, and of appropriate compositions such as hydroxyls, amines, etc., for forming hydrogen and ionic bonds. Thus, a logical implication of the fact that EPI, NE, and DA bind to endogenous opioids such as enkaphalins and morphiceptin is that they might also bind to exogenous opiates such as morphine, and this turned out to be the case. EPI and NE both bind to morphine with a Kd of between 1.35 x 10-5 [9] and 2 x 10-5 M [57], which is sufficient to produce a significant decrease in free serum EPI and NE. Thus, many investigators have reported drug interactions involving opiate and adrenergic agonists, with obvious implications for anesthesiology, blood pressure control and related processes [66-71]. This chemical interaction may explain why it is often recommended that adrenergic drugs and opiates not be mixed in treating patients [72]. On the other hand, as we have suggested elsewhere, every adverse drug interaction probably has a valuable therapeutic application to some medical problem [73]. Another example of aromatic-aromatic drug interactions involves fenfluramine, which lowers serum concentrations of serotonin, thereby suppressing appetite and other serotonin-associated functions [74, 75]. Based on this functional interaction, it was predicted that fenfluramine would bind directly to serotonin or a related compound. NMR studies showed that fenfluramine binds to the serotonin precursor 5OH-tryptophan by means of pi-pi overlap bonding and three hydrogen bonds [76]. This complexation is thought to interfere with 5OH-tryptophan conversion to serotonin. Similarly, dopamine complexes with typical antipsychotic drugs such as haloperidol and chlorpromazine, but not to the atypical antipsychotic drugs [77]. Binding to haloperidol is mediated by the piperidinyl moieties of such drugs [77]. Haloperidol, in turn, produces blockade of dopamine activity by interfering with its reuptake and turnover [78]. While most pharmacologists refer to haloperidol and chlorpromazine as dopamine D2 receptor antagonists, it is striking that some of the reuptake inhibition may also be mediated by competition of the drug for the ligand itself [79, 80]. Antagonism of hormone and neurotransmitter activity by direct binding to the ligand rather than by blockade of the receptor is an approach to drug design that has been exploited very little if at all to the present day. Even if such binding is simply correlated to receptor antagonist activity, such a correlation would be useful in drug design and screening. AROMATIC COMPOUND COMPLEXES INVOLVING VITAMINS Evidence from a variety of systems suggests that focusing on antagonizing or enhancing ligand activity rather than receptor activity might yield interesting effects. For example, it has been known for many decades that flavinoid compounds such as flavin and riboflavin will bind to betacarbolines and other indoles such as serotonin to form charge-transfer complexes [81-83]. The chemistry of these complexes has been very well-characterized [84-87] and has been implicated in the specificity of flavin binding to the indole tryptophan within flavoprotein enzymes such as flavin monooxygenase, N-methyl-tryptophan oxidase, and cytochrome P450 [88, 89]. Thus, the simple complexation reaction found in solution appears to have been adapted to complex protein binding sites by evolution, so that studying one yields clues about the other. Thus, dietary indoles have been found to alter flavoprotein enzyme activity, leading to unexpected drug-drug interactions [90, 91]. Intriguingly, just as the chemistry of these complexes in enzymes yields useful chemical reactions, flavin-indole complexes in solution can also undergo chemical reactions in the presence of light to yield novel antibacterial and antitumor agents [92, 93]. Even more intriguing is the fact that both indole-based serotonin agonists (triptans) and riboflavin are each currently being exploited as migraine medications, but there appear to be no studies examining the effects of a combination of the two agents simultaneously [94-98]. Given the fact that riboflavin binds to serotonin,

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we predict that riboflavin will bind to some or all of the triptans, and that a combination therapy based on a mixture of the two might be far more effective against migraines than either agent alone. An example of a vitamin-drug combination that has just such increased effect is that of ascorbic acid (AA, or vitamin C) combined with adrenergic compounds. AA has long been used as an antioxidant for EPI, NE and other catecholamines. The stability of the resulting solutions is, however, much greater than can be accounted for by the relative rates of reaction of each compound [99]. It was therefore hypothesized AA forms a complex with catecholamines [1], and subsequent physicochemical studies verified that prediction [100, 101]. The physiological effects of AA-catecholamine complexation was then investigated and, surprisingly, it was discovered that AA markedly enhances catecholamine-induced aortic contraction and trachealis relaxation in vitro and greatly increased duration of catecholamine activity [101, 102]. The same effects have been observed in vivo with adrenergic drugs [103-5]. The inotropic activity of dobutamine, for example, is increased approximately three-fold when infused into human patients with supra-physiological concentrations of ascorbate [106], while ascorbate potentiates the effects of isoproterenol and phenylephrine more than two-fold when administered together via the duodenum (but not intravenously) [107]. The mechanism by which this enhancement is induced is definitely not related to the antioxidant effects of ascorbate acting on catecholamines or adrenergic compounds [101, 102], and is likely due to ascorbate allosterically interacting with the adrenergic receptor [108, 109]. The possible uses of ascorbate enhancement of adrenergic drug activity and duration are nearly endless, running the gamut from improved stroke treatment [110], more effective heart failure treatments, better control of blood pressure during shock, and improved asthma formulations to novel approaches to hemostasis, glaucoma treatment, and cold medications [111]. Another interesting implication of the discovery that ascorbate binds to EPI and NE can be recognized by integrating this fact with the fact that morphine also binds to EPI and NE. If morphine exerts some of its physiological effects by binding directly to EPI and NE, then it would follow that ascorbate should act as an antagonist to morphine, and so be of use in treating withdrawal symptoms. A significant body of literature has accumulated suggesting that such ascorbate antagonism of morphine does occur [112-115], but these reports have generally been ignored because no mechanism of action has previously been suggested. The fact that both ascorbate and opiates bind directly to EPI and NE may provide the needed mechanism. A further implication of such a mechanism is that it might be possible to design less addictive opiate-type drugs by maximizing opiate receptor binding while minimizing binding to NE and EPI. COMPLEXES INVOLVING POLYCYCLIC AROMATIC MOLECULES Aromatic complexes involving polycyclic molecules have also been reported to have potential drug activity, particularly when one of the molecules is selected not only for its ability to bind to the other, but also to undergo a chemical reaction with it. Several mutagen-neutralizing compounds have been discovered according to these criteria. Colter, et al. [116, 117] demonstrated that pi-electron donor molecules that complex to 2,4, 7-trinitro-9-fluroenyl-ptoluenesulfonate are able to accelerate its rate of acetolysis. Wood, et al. [118] have reported that riboflavin 5-phosphate (flavin mononucleotide, FMN) binds to the ultimate carcinogenic metabolite of benzo[a]pyrene, (+)-7,8-diol 9,10-epoxide via pi-pi and hydrogen bonds before cleaving the epoxide ring to inactivate the carcinogen. Subsequently, Sayer, et al. [119] demonstrated an even more facile reaction between benzo[a]pyrene, (+)-7,8-diol 9,10-epoxide and ellagic acid, the intermediate complex again being formed by pi-pi and hydrogen bonds. Subsequent studies have demonstrated that

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ellagic acid has potent anti-cancer activity in in vitro and in vivo tests [120-123]. Ellagic acid has also been found to prevent mutation-associated activities of the carcinogen 2-aminofluorene, though there appear to be no studies of whether the two molecules bind to one another [124]. We predict that such binding will be found. Oddly, caffeine has also been found to bind to benzo[a]pyrene [125], opening up a wide range of possible drug development possibilities in the anticancer field. A final example of polycyclic aromatic-aromatic complexing that may provide clues to novel treatment strategies involves neurotoxins. Snyder and his colleagues demonstrated that MPP+ (methylphenylpyridine), the key metabolite of the Parkinsonian neurotoxin MPTP (methylphenyltetrahydropyridine) that destroys neuronal cell bodies, binds with an IC50 of 40 nM to melanin and neuromelanin. Chloroquine (190 nM), hyrdoxychloroquine (220 nM), paraquat (600 nM), quinicrine (750 nM), and haloperidol (810 nM), also bind to melanin with significant IC50s. MPTP itself binds to melanin with an IC50 of 200 nM [126]. Data such as these not only help to explain the reasons that certain neurotoxins such as MPTP and paraquat accumulate anatomically where they do, but also suggest that melanin-like compounds might be used as antidotes to poisoning, an experiment that appears not to have been performed thus far. CHARGE-TRANSFER COMPLEXES One of the best sources of drug development leads based on molecular complementarity may be analytical techniques involving binding assays of drugs to other small molecules. Many of these are based on the formation of charge-transfer complexes. For example, phenothiazines and structurally related drugs form colored charge transfer complexes with xanthine dyes such as fluorescein, eosin, erythrosin and rose bengal that can be used to quantitate the drug by differential spectrophotometry. The phenothiazines also have antibacterial activity that prevents the replication of bacterial plasmids. Charge transfer complexation of the phenothiazines to xanthines correlates with anti-plasmid activity when the binding energy to xanthine dyes is in the range of 0.23-2.31 kcal/mole – equal to or less than the strength of a hydrogen bond [127]. There is a reasonable probability, therefore, that any compound with this degree of binding to xanthine dyes will interfere with plasmid replication in bacteria, and such binding can therefore be used as a screen for identifying novel antibacterials. Notably, those tricyclic antidepressants such as clozapine, promethazine and imipramine that have anti-plasmid activity, also bind to some antibiotics, including penicillins, tetracycline, and gentamicin, resulting in a synergistic increase in antibacterial activity [128]. Thus, the search for novel antibacterial compounds that may augment antibiotic activity may be simplified by first searching for compounds that directly bind to the relevant antibiotics. Synergistic drug interactions associated with the formation of charge transfer complexes have also been found between the phenothiazine derivatives (promethazine, promazine, triflupromazine, methotrimeprazine, propiomazine, trifluoperazine and fluphenazine), and the anticancer agents 5-fluorouracil, methotrexate and sulindac. Formation of complexes increased anticancer activity, altering the solubility of the drugs so that they were more likely to enter target cells [129]. This observation suggests that smallmolecule complexes may be useful as drug delivery mechanisms. Other examples of charge-transfer and ion-pair complexes may yield similarly useful pharmaceutical applications when lifted out of their analytical contexts. Pharmaceutical piparazines such as ketoconazole, piribedil and prazosin hydrochlroide undergo charge -transfer complexation with 2,3-dichloro-5,6-dicyano-p-benzoquinone and also with bromophenol blue (sometimes used as a disinfectant), both of which are used to quantitate the drugs [130, 131]. Moclobemide and imipramine can be quantititated by their forma-

Root-Bernstein and Dillon

tion of charge-transfer complexes with chloranillic acid [132]. Many beta-blockers such as atenolol, metoprolol, stalol, and nadolol, form charge transfer complexes with 4-chloro-7-nitro2,1,3-benzoxadiazole [133]. We suggest that the pharmacological effects of these complexes, or similar ones designed to optimize biological activity, may yield useful synergisms and antagonisms of pharmaceutical value. CONCLUSION: GENERAL METHODS FOR USING COMPLEXATION IN DRUG DISCOVERY AND DEVELOPMENT To summarize, many examples of specific binding between small molecules are known that are associated with modified physiological and pharmacological activities. Conversely, the antagonism or synergism of small molecules in vivo and in vitro is often correlated with specific binding between the molecules. It follows that small molecule binding can be used as a relatively quick, easy, and specific screen for potentially useful drug actions and interactions. Conversely, wherever drug interactions are observed pharmacologically or physiologically, or small molecules exert antagonism, synergism, or functional modification of a pharmacological activity when present together, it may be hypothesized that the compounds also interact physicochemically. Thus, drug discovery and development based upon small-molecule complementarity can begin by searching for agents that bind to one another; by investigating the physicochemical basis of functional drug interactions; or proceed using modern molecular modeling techniques to search for, or to design, compounds that will bind specifically to any particular ligand of interest. The use to which information derived from complementarity studies is put depends upon several factors because the link between binding and function is complex. Complexation may result in interactions that manifest themselves as functional antagonisms (e.g., BPART antagonism of LHRH; fenfluramine inhibiting 5-OH tryptophan conversion to serotonin; morphine binding to EPI and NE lowering blood pressure; haloperidol binding to dopamine correlating with receptor antagonism). Binding may correlate with enhancement or synergism (e.g., ascorbate enhancement of adrenergic drug activity). The formation of some complexes may yield clues about how drugs may be targeted to specific cell types in vivo (e.g., MPTP binding to neuromelanins) and provide leads for the development of antidotes for drug overdoses or poisoning (e.g., ascorbate treatment for morphine overdose or melanin-like compounds as antidotes to MTPT poisoning). In some instances, the binding of one molecule to another may mimic receptor binding (e.g., flavins binding to indoles in solution and also to tryptophan within the enzymes, or peptide-serotonin binding that mimics serotonin receptor specificities) or other functions such as anti-tumor activity (e.g., FMN or ellagic acid and benzo-[a]-pyrene, or caffeine or phenothiazine derivatives with anticancer agents such as 5fluorouracil, methotrexate and sulindac). And finally, complexation may provide novel ways of protecting and delivering drugs (e.g., neurophysins acting as a carriers of oxytocin and neurotensin and other antisense peptides). If complementarity is being explored as a means to generate specific antagonists to ligands, then several guidelines need to be considered. The affinity of the binding must be on the order of kT in order for the binding to significantly alter the ratio of free-tobound ligand. In a related vein, the kD of the binding of the complement to the ligand must be on the order of the kD of the binding of the ligand to its receptor if the complement is to be used at pharmacological concentrations in the same range as the ligand. Weakly binding complements will be effective physiologically only if the concentration of the complement can be raised safely to very high concentrations relative to the ligand (high micromolar or millimolar), as is the case with ascorbate in relation to EPI and NE, for example. On the other hand, if complementarity is being used to model ligand interactions with receptors, as was done with sero-

Small Molecule Complementarity

tonin and muramyl dipeptide binding to myelin basic protein, the affinity of the model system need not approximate that of the real receptor system in order to be useful, as long as the specificities of the two systems are very similar. These examples demonstrate that nature has made use of molecular complementarity in a wide variety of ways to regulate physiological processes, all of which provide drug development clues for pharmacologists and medicinal chemists. What is truly unique about the molecular complementarity perspective on drug development is that rather than screening new compounds for activity against receptors and second messenger systems, ligands such as neurotransmitters and their metabolites, hormones, and drugs themselves, become the targets of drug development. We believe this approach is novel, fruitful and underutilized. REFERENCES [1] [2] [3]

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