Design and development of selective muscarinic agonists for the treatment of Alzheimer's disease: characterization of tetrahydropyrimidine derivatives and development of new approaches for improved affinity and selectivity for M1 receptors.

Cholinergic neurons degenerate in Alzheimer's disease, resulting in cognitive impairments and memory deficits, and drug development efforts have focused on selective M1 muscarinic agonists. 5-(3-Ethyl-1,2,4- oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine trifluoroacetic acid (CDD-0102) stimulates M1 muscarinic receptors in rat brain [Messer, W.S., Jr., Abuh, Y.F., Liu, Y., Periyasamy, S., Ngur, D.O., Edgar, M.A., El-Assadi, A.A., Sbeih, S., Dunbar, P.G., Roknich, S., Rho, T., Fang, Z., Ojo, B., Zhang, H., Huzl, J.J., III, Nagy, P.I., 1997a. J. Med. Chem. 40, 1230-1246.] and improves memory function in rats with lesions of the basal forebrain cholinergic system. Moreover, CDD-0102 exhibits oral bioavailability, few side effects and low toxicity, and thus represents a viable candidate for clinical studies. Despite the development of functionally selective agonists such as xanomeline and CDD-0102, there is room for improvements in ligand affinity and selectivity. The high degree of amino acid homology within transmembrane domains has hindered the development of truly selective agonists. Site-directed mutagenesis, biochemical and molecular modeling studies have identified key amino acid residues such as Thr192 and Asn382 in the binding of agonist to M1 receptors [Huang, X.P., Nagy, P.I., Williams, F.E., Peseckis, S.M., Messer, W.S., Jr., 1999. Br. J. Pharmacol. 126, 735-745.]. Recent work has implicated residues at the top of transmembrane domain VI in the binding of muscarinic agonists and activation of M1 receptors [Huang, X.P., Williams, F.E., Peseckis, S.M., Messer, W.S., Jr., 1998. J. Pharmacol. Exp. Ther. 286, 1129-1139.]. Thus, residues such as Ser388 represent molecular targets for the further development of agonists with improved M1 receptor affinity, selectivity and activity.

First fatty acylated dipeptides to affect muscarinic receptor ligand binding.

Fatty acylated dipeptides homologous to Gi alpha N-termini affect ligand binding to muscarinic acetylcholine receptors. Myristylglycine-serine containing dipeptides decrease antagonist binding at both M1 and M2 muscarinic receptors. Palmitate on the serine analogous to native palmitoylated cysteine affords dipeptide which selectively decreases the number of high affinity agonist binding sites at M2 but not M1 receptor.

Differential modulation of agonist potency and receptor coupling by mutations of Ser388Tyr and Thr389Pro at the junction of transmembrane domain VI and the third extracellular loop of human M(1) muscarinic acetylcholine receptors.

Transmembrane domain VI of muscarinic acetylcholine receptors plays an important role in ligand binding and receptor function. A human M(1) (HM(1)) mutant receptor, HM(1)(S388Y, T389P), displayed significantly enhanced agonist potency, binding affinity, and G protein coupling. The mutations are located at the top of transmembrane domain VI and about two helical turns above Tyr381 and Asn382, which are important for ligand binding and receptor function. To determine the functional role of individual mutations of Ser388Tyr and Thr389Pro, we created stable A9 L cell lines expressing HM(1)(S388Y) or HM(1)(T389P) receptors. In phosphatidylinositol hydrolysis assays, muscarinic agonists showed greater potency at the HM(1)(S388Y) and HM(1)(S388Y, T389P) mutants compared with the wild-type and HM(1)(T389P) receptors. Acetylcholine demonstrated 105-fold higher potency at HM(1)(S388Y) receptors than at HM(1)(T389P) receptors. Choline (30 microM, the concentration found in Dulbecco's modified Eagle's medium) exhibited 90% stimulation at HM(1)(S388Y) receptors but was inactive at HM(1)(T389P) receptors. In ligand binding experiments, mutation of Ser388Tyr resulted in significantly increased agonist binding affinity. In contrast, mutation of Thr389Pro did not change agonist binding affinity but rendered multiple agonist binding sites, and the high-affinity binding was sensitive to GTP analogs. These results demonstrate that the Ser388Tyr mutation is responsible for enhanced agonist potency and binding affinity, whereas the Thr389Pro mutation alters G protein interactions. The data suggest that Ser388 and Thr389 are potential targets for modulation of agonist binding and G protein coupling.

Roles of threonine 192 and asparagine 382 in agonist and antagonist interactions with M1 muscarinic receptors.

Conserved amino acids, such as Thr in transmembrane domains (TM) V and Asn in TM VI of muscarinic receptors, may be important in agonist binding and/or receptor activation. In order to determine the functional roles of Thr192 and Asn382 in human M1 receptors in ligand binding and receptor activation processes, we created and characterized mutant receptors with Thr192 or Asn382 substituted by Ala. HM1 wild-type (WT) and mutant receptors [HM1(Thr192Ala) and HM1(Asn382Ala)] were stably expressed in A9 L cells. The Kd values for 3H-(R)-QNB and Ki values for other classical muscarinic antagonists were similar at HM1(WT) and HM1(Thr192Ala) mutant receptors, yet higher at HM1(Asn382Ala) mutant receptors. Carbachol exhibited lower potency and efficacy in stimulating PI hydrolysis via HM1(Thr192Ala) mutant receptors, and intermediate agonist activity at the HM1(Asn382Ala) mutant receptors. The Asn382 residue in TM VI but not the Thr192 residue in TM V of the human M1 receptor appears to participate directly in antagonist binding. Both Thr192 and Asn382 residues are involved differentially in agonist binding and/or receptor activation processes, yet the Asn382 residue is less important than Thr192 in agonist activation of M1 receptors. Molecular modelling studies indicate that substitution of Thr192 or Asn382 results in the loss of hydrogen-bond interactions and changes in the agonist binding mode associated with an increase in hydrophobic interactions between ligand and receptor.

Pharmacological characterization of human m1 muscarinic acetylcholine receptors with double mutations at the junction of TM VI and the third extracellular domain.

A mutant human m5 receptor containing the mutations of Ser465 to Tyr and Thr466 to Pro showed constitutive activity. By replacing the equivalent Ser388 with Tyr and Thr389 with Pro, we created a mutant human m1 (Hm1) receptor with comparable double mutations. The mutant receptor, Hm1(Ser388Tyr, Thr389Pro), was stably expressed in A9 L cells and displayed enhanced responses to classical muscarinic agonists with significantly increased potencies. Choline, a normal component of growth media, showed an efficacy comparable to acetylcholine and carbachol at Hm1(Ser388Tyr, Thr389Pro) receptors. Methylcarbachol, a selective nicotinic agonist, exhibited partial agonist activity at human m1 wild-type receptors and full agonist activity at Hm1(Ser388Tyr, Thr389Pro) receptors. l-Hyoscyamine inhibited the activities of choline and methylcarbachol. Muscarinic antagonists displayed small reductions in binding affinities, although muscarinic agonists showed greatly increased binding affinities for Hm1(Ser388Tyr, Thr389Pro) receptors. All agonists, including choline and methylcarbachol, showed multiple affinity states at Hm1(Ser388Tyr, Thr389Pro) receptors in the absence of GppNHp. The high affinity binding sites for acetylcholine, arecoline and choline were shifted in the presence of GppNHp. These results suggest that Hm1(Ser388Tyr, Thr389Pro) is conformationally favorable for agonist binding and receptor activation.

Fatty acyl transfer by human N-myristyl transferase is dependent upon conserved cysteine and histidine residues.

N-Myristyl transferase (Nmt) catalyzes attachment of myristate onto the N terminus of suitable proteins. In order to identify amino acids important for catalytic functions, human Nmt and mutants representing all six conserved cysteine and histidine residues (Cys-169, Cys-214, His-131, His-171, His-218, and His-293) were expressed in Escherichia coli and analyzed for their ability to bind and transfer myristic acid. N-Terminal histidine-tagged fusion proteins displayed varying abilities to form an association with radiolabeled myristic acid indicative of an acyl-enzyme intermediate. When co-expressed with an acceptor substrate protein, pp60v-src, the mutants showed differential incorporation of radiolabeled myristic acid into v-Src protein. In vitro experiments monitoring transfer of myristyl CoA to a peptide homologous to the N terminus of pp60 v-src gave results similar to those obtained in vivo. Our studies showed that mutation at Cys-169, His-171, and especially His-293 interfered with formation of an acyl-enzyme intermediate, while human Nmts containing mutations at Cys-169, His-218, or His-293 showed greatly attenuated abilities to form acylated product. We propose a model for the Nmt reaction mechanism in which Cys-169 serves as the fatty acid attachment site for a covalent myristyl enzyme intermediate, while His-171 acts as a general acid/base and His-293 as a specific acid/base during acyl-enzyme intermediate formation. His-218 could then act as an acid or base needed to catalyze transfer of the acyl group from the acyl-enzyme intermediate to a polypeptide substrate. This working model will be useful for the design of regulators of Nmt function.

Dual myristylation and palmitylation of Src family member p59fyn affects subcellular localization.

The Src family consists of nine related tyrosine protein kinases with a common domain structure, including a myristylated N-terminal glycine residue. In this report, we identify cysteine residues within the N-terminal region of the Src family member Fyn which serve as sites for palmitylation. To facilitate detection of protein fatty acylation, p59fyn was overexpressed in COS cells and incubated with radioiodinated fatty acid analogs of myristate (IC13) or palmitate (IC16). Incorporation of both fatty acids into p59fyn was readily observed. Acylation with the palmitate analog was prevented when Gly-2 was mutated to alanine, implying that N-myristylation is required for palmitylation, and when either Cys-3 or Cys-6 was mutated to serine. Palmitylation was shown to alter the distribution of p59fyn between membrane-bound and soluble fractions. In contrast, no incorporation of the palmitate analog into pp60v-src, which lacks N-terminal cysteine residues, was observed. Mutation of Ser-3 of Src to cysteine, but not Ser-6, resulted in incorporation of the palmitate analog. These results serve to delineate sequence elements important for dual acylation of proteins, and further illustrate the utility of radioiodinated fatty acid analogs for studies of protein fatty acid acylation.

Regulation of enzymatic activity by active site fatty acylation. A new role for long chain fatty acid acylation of proteins.

Methylmalonate semialdehyde dehydrogenase (MMSDH) is a mitochondrial enzyme which can be acylated by myristoyl-CoA analogs (Deichaite, I., Berthiaume, L., Peseckis, S. M., Patton, W. F., and Resh, M. D. (1993) J. Biol. Chem. 268, 13788-13747). Here we describe the mechanisms which mediate regulation of the enzymatic activity of bovine MMSDH by long chain fatty acylation. The substrate specificity of the acylation reaction was measured in vitro using purified MMSDH and the coenzyme A derivative of an 125I-labeled long chain fatty acid (13-iodotridecanoate), an analog of myristoyl-CoA. Long chain fatty acyl CoAs (> 8 carbons) were able to inhibit radiolabeling of MMSDH. In order to study the physiological role of the acylation process in vivo, a system using highly purified mitochondria from COS-1 cells overexpressing MMSDH was exploited. MMSDH was shown to be processed properly, targeted to the mitochondrial fraction, and enzymatically active. The extent of fatty acylation of MMSDH as well as of other mitochondrial proteins was correlated with the mitochondrial energy level. Biochemical evidence as well as site-specific mutagenesis of cysteine 319 revealed that this highly conserved active site cysteine of MMSDH was the target of the fatty acylation. Another member of the aldehyde dehydrogenase family, yeast aldehyde dehydrogenase was also covalently modified by [125I]13-iodotridecanoyl-CoA and thereby inactivated. Furthermore, we demonstrate that glutamate dehydrogenase, an enzyme that has been previously shown to be strongly inhibited by palmitoyl-CoA, is fatty acylated by the 125I-labeled myristoyl-CoA analog. Our data suggest that attachment of long chain fatty acids to proteins is a new and potentially widespread type of enzyme regulation mechanism that we denote active site fatty acylation.

Novel use of an iodo-myristyl-CoA analog identifies a semialdehyde dehydrogenase in bovine liver.

We describe here the identification, purification, and characterization of a semialdehyde dehydrogenase with a novel fatty acid binding function. The coenzyme A derivative of an 125I-labeled long chain saturated fatty acid (13-iodo-tridecanoate) was used to tag proteins which bind myristoyl-CoA. A prominent 57 kDa band was identified, which was isolated from bovine liver by a high salt extraction followed by ammonium sulfate precipitation. Sequential chromatographic separation using phenyl-Sepharose, hydroxyapatite, DEAE-Sepharose, Mono Q, and Fast Flow S resins resulted in a purified protein that migrated as a single band of 57 kDa on denaturing gels. Sephacryl-200 gel filtration provided a native molecular mass estimation of 118 kDa suggesting that this protein exists as a dimer. Two-dimensional gel analysis resolved three isoform variants with pI values of 7.4, 7.7, and 7.9, respectively, and established that the pI = 7.9 form has the highest propensity for fatty acid binding. We proceeded to generate tryptic peptides from the purified protein and subjected several peptides to microchemical sequencing. Degenerate oligonucleotide probes were designed and polymerase chain reaction was used to generate a unique nucleotide sequence. Subsequent screening of a bovine liver cDNA library yielded a 1.7-kilobase clone which encodes a protein of 537 amino acids (58 kDa) with 95% identity to mammalian methylmalonate semialdehyde dehydrogenase (MMSDH). In vitro assays confirmed that the purified 57-kDa protein exhibited MMSDH activity, and that preincubation of the enzyme with fatty acyl-CoA inhibited its dehydrogenase activity. The myristyl-CoA analog therefore serves as an affinity label for MMSDH. We propose that fatty acyl CoAs may have the potential to function as enzyme regulators in vivo.

Iodinated fatty acids as probes for myristate processing and function. Incorporation into pp60v-src.

The myristyl group makes a critical contribution to the processing, trafficking, and function of myristylated proteins. A series of [omega-125I]iodo-fatty acids was synthesized in order to elucidate the myristyl group's contribution to the membrane association of pp60v-src, the transforming protein of Rous sarcoma virus. In vitro translation of v-src mRNA was employed to monitor incorporation of myristyl analogs into pp60v-src polypeptide. 12-Iodododecanoic, 13-iodotridecanoic, and 14-iodotetradecanoic acids were selectively incorporated in vitro into pp60v-src. One-dimensional peptide analysis confirmed that the analogs were attached to the N terminus of pp60v-src. Upon addition of membranes, the Src proteins modified by these analogs bound to membranes at levels comparable with or slightly less than the myristyl parent. Myristyl analogs were also shown to be incorporated into pp60v-src in vivo. Fractionation of [omega-125I]iodo-fatty acid labeled cells showed that 12-iododecanoic acid, 13-iodotridecanoic acid, and 14-iodotetradecanoic acid modified pp60v-src associated preferentially with the membrane fractions. These results demonstrate that fatty acyl groups one carbon longer or shorter than myristate can be accommodated within the membrane binding site for pp60v-src and illustrate the utility of in vitro systems for predicting analog behavior in vivo. We anticipate that iodinated fatty acids can be used as tools to aid in clarifying the role of the fatty acid in a variety of myristylated molecules.

Chemistry and positive inotropic effect of pelrinone and related derivatives. A novel class of 2-methylpyrimidones as inotropic agents.

A novel series of pyrimidine derivatives was synthesized and evaluated for positive inotropic activity. Inotropic and chronotropic effects were determined in vitro in cat papillary muscle and right atrium, respectively. Selected compounds were then evaluated in vivo in a dog heart failure model. Changes in ventricular dP/dt, heart rate, and blood pressure were monitored. Several of these agents produced relatively minor changes in heart rate. This class of agents demonstrated a varying degree of vasodilator effects concomitant with increases in ventricular contractility. The most potent analogues, 9, 48, and 49, were evaluated orally in conscious dogs with implanted Konisberg pressure transducers, and their effect on left ventricular dP/dt was compared with that of milrinone. Mechanistically, the agents of this novel class appear not to mediate their effect via beta-receptors or inhibition of Na+/K+-ATPase. A major component of their inotropic effect is mediated by the inhibition of cardiac phosphodiesterase (PDE)-Fr. III. This was clearly demonstrated by 9, 48, and 49. Compound 48 was found to be the most potent inhibitor of PDE-Fr. III from among the compounds tested in this assay.