Novel biologically active nonpeptidic inhibitors of myristoylCoA:protein N-myristoyltransferase.

A new class of biologically active nonpeptidic inhibitors of Candida albicans NMT has been synthesized starting from the octapeptide ALYASKLS-NH2 (2). The synthetic strategy entailed the preparation of novel protected Ser-Lys mimics 9 and 12 from (S)- or (R)-3-iodotyrosine and then grafting key enzyme recognition elements in a stepwise manner. Like 2, compounds 16, 17, and 18 are competitive Candida NMT inhibitors that bind to the peptide recognition site of the enzyme. Moreover, 16-18 have an affinity comparable to that of 2 even though they are devoid of peptide bonds. In contrast to 2, these nonpeptidic inhibitors exhibit antifungal activity.

Design and synthesis of novel imidazole-substituted dipeptide amides as potent and selective inhibitors of Candida albicans myristoylCoA:protein N-myristoyltransferase and identification of related tripeptide inhibitors with mechanism-based antifungal activity.

A new class of antifungal agents has been discovered which exert their activity by blockade of myristoylCoA: protein N-myristoyltransferase (NMT; EC 2.1.3.97). Genetic experiments have established that NMT is needed to maintain the viability of Candida albicans and Cryptococcus neoformans,the two principal causes of systemic fungal infections in immunocompromised humans. Beginning with a weak octapeptide inhibitor ALYASKLS-NH2 (2, Ki = 15.3 +/- 6.4 microM), a series of imidazole-substituted Ser-Lys dipeptide amides have been designed and synthesized as potent and selective inhibitors of Candida albicans NMT. The strategy that led to these inhibitors evolved from the identification of those functional groups in the high-affinity octapeptide substrate GLYASKLS-NH2 1a necessary for tight binding, truncation of the C-terminus, replacement of the four amino acids at the N-terminus by a spacer group, and substitution of the glycine amino group with an N-linked 2-methylimidazole moiety. Initial structure-activity studies led to the identification of 31 as a potent and selective peptidomimetic inhibitor with an IC50 of 56 nM and 250-fold selectivity versus human NMT. 2-Methylimidazole as the N-terminal amine replacement in combination with a 4-substituted phenacetyl moiety imparts remarkable potency and selectivity to this novel class of inhibitors. The (S,S) stereochemistry of serine and lysine residues is critical for the inhibitory activity, since the (R,R) enantiomer 40 is 10(3)-fold less active than the (S,S) isomer 31. The inhibitory profile exhibited by this new class of NMT ligands is a function of the pKa of the imidazole substituent as illustrated by the benzimidazole analog 35 which is about 10-fold less potent than 31. The measured pKa (7.1 +/- 0.5) of 2-methylimidazole in 31 is comparable with the estimated pKa (approximately 8.0) of the glycyl residue in the high-affinity substrate 1a. Groups bulkier than methyl, such as ethyl, isopropyl, or iodo, at the imidazole 2-position have a detrimental effect on potency. Further refinement of 31 by grafting an alpha-methyl group at the benzylic position adjacent to the serine residue led to 61 with an IC50 of 40 nM. Subsequent chiral chromatography of 61 culminated in the discovery of the most potent Candida NMT inhibitor 61a reported to date with an IC50 of 20 nM and 400-fold selectivity versus the human enzyme. Both 31 and 61a are competitive inhibitors of Candida NMT with respect to the octapeptide substrate GNAASARR-NH2 with Ki(app) = 30 and 27 nM, respectively. The potency and selectivity displayed by these inhibitors are dependent upon the size and orientation of the alpha-substituent. An alpha-methyl group with the R configuration corresponding to the (S)-methyl-4-alanine in 2 confers maximum potency and selectivity. Structural modification of 31 and 61 by appending an (S)-carboxyl group beta to the cyclohexyl moiety provided the less potent tripeptide inhibitors 73a and 73b with an IC50 of 1.45 +/- 0.08 and 0.38 +/- 0.03 microM, respectively. However, these tripeptides (73a and 73b) exhibited a pronounced selectivity of 560- and 2200-fold versus the human NMT. More importantly 73a displayed fungistatic activity against C albicans with an EC50 of 51 +/- 17 microM in cell culture. Compound 73b also exhibited a similar antifungal activity. An Arf protein gel mobility shift assay for monitoring intracellular myristoylation revealed that a single dose of 200 microM of 73a or 73b produced < 50% reduction in Arf N-myristoylation, after 24 and 48 h, consistent with their fungistatic rather than fungicidal activity. In contrast, the enantiomer 73d which had an IC50 > 1000 microM against C. albicans NMT did not exhibit antifungal activity and produced no detectable reduction in Arf N-myristoylation in cultures of C. albicans. These studies confirm that the observed antifungal activity of 73a and 73b is due to the attenuation of NMT activity and that NMT represents an attractive tar

Conformationally constrained [p-(omega-aminoalkyl)phenacetyl]-L-seryl-L-lysyl dipeptide amides as potent peptidomimetic inhibitors of Candida albicans and human myristoyl-CoA:protein N-myristoyl transferase.

MyristoylCoA:protein N-myristoyltransferase (NMT) covalently attaches the 14-carbon saturated fatty acid myristate, via an amide bond, to the N-terminal glycine residues of a variety of cellular proteins. Genetic studies have shown that NMT is essential for the viability of the principal fungal pathogens which cause systemic infection in immunosuppressed humans and hence is a target for development of fungicidal drugs. We have generated a class of potent peptidomimetic inhibitors of the NMT from one such fungal pathogen, Candida albicans. The N-terminal tetrapeptide from a substrate analog inhibitor, ALYASKL-NH2, was replaced with an omega-aminoalkanoyl moiety having an optimal 11-carbon chain for inhibition (11-aminoundecanoyl-SKL-NH2, 3a, IC50 = 1.2 +/- 0.14 microM). A series of replacements for the C-terminal Leu established that residues containing a lipophilic side chain were most effective, with cyclohexylalanine having the greatest potency (3g, IC50 = 0.36 +/- 0.06 microM). Removal of the carboxamide moiety led to a metabolically stable dipeptide inhibitor containing an N-(cyclohexylethyl)lysinamide (17e, IC50 = 0.11 +/- 0.03 microM). Partial rigidification of the flexible aminoundecanoyl chain produced the dipeptide p-(omega-aminohexyl)phenacetyl-L-seryl-L-lysyl-N-(cyclohexyleth yl)amide (26b, IC50 = 0.11 +/- 0.04 microM). Subsequent incorporation of an alpha-methyl substituent into 26b provided the dipeptide analog [2-[p-(omega-aminohexyl)phenyl]propionyl]-L-seryl-L-lysyl-N-(cyclohex ylethyl)amide, a very potent inhibitor (48, IC50 = 0.043 +/- 0.006 microM), which retained the three essential elements required for recognition by the acyl transferase's peptide binding site.

Scanning alanine mutagenesis and de-peptidization of a Candida albicans myristoyl-CoA:protein N-myristoyltransferase octapeptide substrate reveals three elements critical for molecular recognition.

Candida albicans produces a single myristoyl-CoA:protein N-myristoyltransferase (Nmt) that is essential for its viability. An ADP-ribosylation factor (Arf) is included among the few cellular protein substrates of this enzyme. An octapeptide (GLYASKLS-NH2) derived from a N-terminal Arf sequence was used as the starting point to identify elements critical for recognition by the acyltransferases's peptide-binding site. In vitro kinetic studies, employing purified Nmt and a panel of peptides with single Ala substitutions at each position of GLYASKLS-NH2, established that its Gly1, Ser5, and Lys6 residues play predominant roles in binding. ALYASKLS-NH2 was found to be an inhibitor competitive for peptide (Ki = 15.3 +/- 6.4 microM) and noncompetitive for myristoyl-CoA (Ki = 31.2 +/- 0.7 microM). A survey of 26 derivatives of this inhibitor, representing (i) a complete alanine scan, (ii) progressive C-terminal truncations, and (iii) manipulation of the physical-chemical properties of its residues 1, 5, and 6, confirmed the important stereochemical requirements for the N-terminal amine, the beta-hydroxyl of Ser5, and the epsilon-amino group of Lys6. Remarkably, replacement of the the N-terminal tetrapeptide of ALYASKLS-NH2 with an 11-aminoundecanoyl group produced a competitive inhibitor, 11-aminoundecanoyl-SKLS-NH2, that was 38-fold more potent (Ki = 0.40 +/- 0.03 microM) than the starting octapeptide. Removing the primary amine (undecanoyl-SKLS-NH2), or replacing it with a methyl group (dodecanoyl-SKLS-NH2), resulted in 26- and 34-fold increases in IC50, confirming the important contribution of the amine to recognition. Removal of LeuSer from the C terminus (11-aminoundecanoyl-SK-NH2) yielded a competitive dipeptide inhibitor with a Ki (11.7 +/- 0.4 microM) equivalent to that of the starting octapeptide, ALYASKLS-NH2. Substitution of Ser with homoserine, cis-4-hydroxyproline, or tyrosine reduces potency by 3-70-fold, emphasizing the requirement for proper presentation of the hydroxyl group in the dipeptide inhibitor. Substituting D- for L-Lys decreases its inhibitory activity >100-fold, while deletion of the epsilon-amino group (Nle) or masking its charge (epsilon-N-acetyl-lysine) produces 4-7-fold attenuations. L-His, but not its D-isomer, can fully substitute for L-Lys, producing a competitive dipeptide inhibitor with similar potency (Ki = 11.9 +/- 1.0 microM). 11-Aminoundecanoyl-SK-NH2 and 11-aminoundecanoyl-SH-NH2 establish that a simple alkyl backbone can maintain an appropriate distance between three elements critical for recognition by the fungal enzyme's peptide-binding site: a simple omega-terminal amino group, a beta-hydroxyl, and an epsilon-amino group or an imidazole. These compounds contain one peptide bond and two chiral centers, suggesting that it may be feasible to incorporate these elements of recognition, or functionally equivalent mimics, into a fully de-peptidized Nmt inhibitor.

Selective peptidic and peptidomimetic inhibitors of Candida albicans myristoylCoA: protein N-myristoyltransferase: a new approach to antifungal therapy.

MyristoylCoA: protein N-myristoyltransferase (NMT) catalyzes the cotranslational covalent attachment of a rare cellular fatty acid, myristate, to the N-terminal Gly residue of a variety of eukaryotic proteins. The myristoyl moiety is often essential for expression of the biological functions for these proteins. Attachment of C14:0 alone provides barely enough hydrophobicity to allow stable association with membranes. The partitioning of N-myrisotylproteins is therefore often modulated by "switches" that function through additional covalent or noncovalent modifications. Candida albicans, the principal cause of systemic fungal infection in immunocompromised humans, contains a single NMT gene that is essential for its viability. The functional properties of the acylCoA binding site of human and C. albicans NMT are very similar. However, there are distinct differences in their peptide binding sites. An ADP ribosylation factor (Arf) is included among the few cellular protein substrates of the fungal enzyme. Alanine scanning mutagenesis of an octapeptide derived from an N-terminal Arf sequence (GLYASKLS-NH2) disclosed that Gly1, Ser5, and Lys6 play predominant roles in binding. ALYASKLS-NH2 is an inhibitor competitive for peptide [Ki(app) = 15.3 +/- 6.4 microM] and noncompetitive for myristoylCoA. Remarkably, replacement of the N-terminal tetrapeptide with an 11-aminoundecanoyl group results in a competitive inhibitor (11-aminoundecanoyl-SKLS-NH2) that is approximately 40-fold more potent [Ki(app) = 0.40 +/- 0.03 microM] than the starting octapeptide. Removal of Leu-Ser from the C-terminus generates a competitive dipeptide inhibitor (11-aminoundecanoyl-SK-NH2) with a Ki(app) of 11.7 +/- 0.4 microM, equivalent to that of the starting octapeptide. A derivative dipeptide inhibitor containing a C-terminal N-cyclohexylethyl lysinamide moiety has the advantage of being more potent (IC50 = 0.11 +/- 0.03 microM) and resistant to digestion by cellular carboxypeptidases. Rigidifying the flexible aminoundecanoyl chain results in very potent general NMT inhibitors (IC50 = 40-50 nM). Substituting a 2-methylimidazole for the N-terminal amine and adding a benzylic alpha-methyl group with R stereochemistry to the rigidifying element produces even more potent inhibitors (IC50 = 20-50 nM) that are up to 500-fold selective for the fungal compared to human enzyme. A related less potent member of this series of compounds is fungistatic. Its growth inhibitory effects are associated with a reduction in cellular protein N-myristoylation, judged using cellular Arf as a reporter. These studies establish that NMT is a new antifungal target.

Genetic studies reveal that myristoylCoA:protein N-myristoyltransferase is an essential enzyme in Candida albicans.

MyristoylCoA:protein N-myristoyltransferase (Nmt) catalyses the co-translational, covalent attachment of myristate (C14:0) to the amino-terminal glycine residue of a number of eukaryotic proteins involved in cellular growth and signal transduction. The NMT1 gene is essential for vegetative growth of Saccharomyces cerevisiae. Studies were carried out to determine if Nmt is also essential for vegetative growth of the pathogenic fungus Candida albicans. A strain of C. albicans was constructed in which one copy of NMT was partially deleted and disrupted. A Gly-447-->Asp mutation was introduced into the second NMT allele. This mutation produced marked reductions in catalytic efficiency at 24 and 37 degrees C, as judged by in vitro kinetic studies of the wild-type and mutant enzymes which had been expressed in, and purified from, Escherichia coli. The growth characteristics of isogenic NMT/NMT, NMT/delta nmt, and nmt delta/nmtG447D C. albicans strains were assessed under a variety of conditions. Only the nmt delta/nmtG447D strain required myristate for growth. This was true at both 24 and 37 degrees C. Palmitate could not substitute for myristate. Incubation of nmt delta/nmtG447D cells at 37 degrees C in the absence of myristate resulted in cell death as observed by the inability to form colonies on media supplemented with 500 microM myristate. Studies in an immunosuppressed-mouse model of C. albicans infection revealed that the NMT/delta nmt strain produced 100% lethality within 7 d after intravenous administration while the isogenic nmt delta/nmtG447G strain produced no deaths even after 21 d. These observations establish that Nmt is essential for vegetative growth of C. albicans and suggest that inhibitors of this acyltransferase may be therapeutically useful fungicidal agents.

Antibodies specific for branched ribonucleic acids.

A chemically synthesized branched tetranucleotide, G3'p5'A [2'p5'G]3'p5'C corresponding to the consensus sequence at the branch point in introns undergoing RNA splicing, was used as a hapten to elicit antibranch antibodies. Binding assays with 32P-labeled hapten and unlabeled structurally related haptens indicated that the antibodies are highly specific for the branch structure and have some specificity for the A2'p5'G sequence at the branch point, but have essentially none for a variety of other 2'p5' or 3'p5' dinucleotides or for the linear trinucleotide G3'p5'A3'p5'C. Purification of these antibodies by binding to A2'p5'G covalently linked to Sepharose followed by covalent attachment of the purified antibodies to protein A-Sepharose has provided an adsorbent that immunospecifically retains branched oligonucleotides as well as branched introns released from RNAs during in vitro splicing.

Antibody-nucleic acid complexes. Oligo(dG)n and -(dT)n specificities associated with anti-DNA antibodies from autoimmune MRL mice.

The specificity of anti-DNA antibodies in the sera of unimmunized autoimmune MRL mice was initially assessed via an enzyme-linked immunosorbent assay (ELISA). Antibody binding profiles to a panel of immobilized antigens (AMP-, GMP-, CMP-, UMP-, and TMP-BSA, ss- and dsDNA) demonstrated high levels of immunoglobulins reacting with GMP and ssDNA and intermediate levels with AMP, TMP, and dsDNA. Fractionation of serum anti-DNA antibodies into subsets on the basis of their binding to GMP- and TMP-agarose indicated that the resulting GMP- or TMP-reactive antibodies bound to their homologous nucleotides and ssDNA. Competition-inhibition studies with soluble mono-, oligo-, and polynucleotides revealed that GMP- and TMP-reactive antibodies were highly specific for oligo(dG)n and -(dT)n sequences, respectively. Whereas the relative affinity of TMP-reactive autoantibodies to oligo(dT)n increased with oligonucleotide length (n = 2, 4, 6, 8, 10, 15), GMP-reactive antibodies preferentially recognized oligo(dG)10 (Ka congruent to 1 x 10(7) M-1). While neither antibody recognized oligo(dA)8 and -(dC)8 competitors, mixed-base oligonucleotides were inhibitory at concentrations approximately 10-fold greater than similarly sized oligo(dG)n and -(dT)n sequences. Similar characterizations of both pooled and individual MRL sera indicated that anti-DNA antibodies represent 8-10% of the total serum IgG. More importantly, GMP-reactive autoantibodies predominated and accounted for 60-70% of the entire unbound anti-DNA antibody population.

TMP-reactive autoantibodies in human SLE sera demonstrate thymine-dependent oligonucleotide specificity.

Autoantibodies present in the sera of lupus patients and specific for single-stranded (ss) DNA were fractionated into subsets based upon their reactivity towards 5' nucleotide haptens. As evaluated by ELISA testing, antibodies retained by TMP-agarose bound to TMP-BSA and ssDNA but not to other nucleotide-BSA conjugates or to double-stranded (ds) DNA. Competition-inhibition studies further revealed that TMP-enriched oligo- and polynucleotides were the preferred antigens for these affinity purified antibodies. Similar assays with sequence- or size- defined oligonucleotides further implied that those oligonucleotides comprised entirely of TMP residues were most antigenic and that antigenicity increased with size (length). These results document the existence of a TMP-dependent oligonucleotide specificity among a diverse population of autoanti-ssDNA antibodies.

Distribution and specificity of nucleotide-reactive autoantibodies in human SLE.

An enzyme-linked immunosorbent assay (ELISA) was utilized to characterize nucleotide-reactive antibodies present in the sera of 67 human subjects: 27 active SLE, 20 inactive SLE, and 20 asymptomatic controls. This assay consisted of measuring the quantity of antibodies retained by a panel of immobilized 5'-nucleotide-BSA conjugates (AMP-, GMP-, CMP-, UMP-, and TMP-BSA) together with ssDNA and dsDNA antigens. Although the relative distribution of antibodies binding to nucleotide-BSA antigens (i.e., anti-GMP greater than anti-AMP greater than or equal to anti-TMP greater than anti-UMP greater than or equal to anti-CMP antibodies) was independent of clinical status, the sera of active SLE patients possessed three- and five-fold higher concentrations of these antibodies relative to those present in inactive SLE and control subjects, respectively. Affinity purification of the most dominant of these antibody populations with DNA- and GMP-agarose adsorbents suggested that the majority of anti-GMP antibodies were monospecific with respect to the guanine base moiety. For example, antibodies retained by GMP-agarose reacted with GMP-BSA and ssDNA but not with other nucleotide-BSA or dsDNA antigens. However, ELISA competition-inhibition studies with affinity-purified anti-GMP antibodies indicated that although the guanine base represents an important determinant, guanine-enriched oligo- and polynucleotides were preferred substrates (i.e., guanine-dependent, oligonucleotide specificity). This was exemplified by the finding that a 500- and 50-fold molar excess of dGMP and d(G)4 were required to achieve the same degree of inhibition as that observed with d(G)8. Finally, and as evaluated by indirect immunofluorescence with fixed HEp-2 cells, affinity-purified anti-GMP antibodies reacted with antigens restricted to nucleolar organelles.

Naturally occurring masked antibodies in murine sera recognize a component of the mitotic spindle apparatus.

Antibody activities previously masked in autoimmune MRL and normal Balb/c mice were expressed by briefly subjecting their sera to acidic (pH 2.0) or alkaline (pH 12.0) environments. An enzyme-linked immunosorbent assay (ELISA) revealed that these pH-expressable immunoglobulins reacted with specific nucleotide-BSA antigens (primarily 5'-AMP, -GMP, -TMP) but not with single (ss)- or double (ds)-stranded DNAs or with unconjugated BSA. ELISA analysis of pH-expressed antibodies purified via GMP-BSA/Sepharose indicated that they bound not only to the homologous hapten (GMP) but to AMP and TMP as well, i.e., anti-pAGT antibodies. Further, indirect immunofluorescent assays (IIF) with fixed HEp-2 cells demonstrated that purified anti-pAGT antibodies recognized an epitope within the mitotic spindle apparatus. These results document the existence of a previously undefined masked antibody population in murine sera with specificities directed toward certain nucleotides and a component of the mitotic spindle apparatus. Last, these anti-pAGT (or anti-mitotic spindle) antibodies are not restricted to murine systems inasmuch as they have been detected in every human serum (greater than 100 samples) examined thus far.

Detection of human autoantibodies specific for 5'-m7GMP and m7G(5')ppp(5')N.

An enzyme-linked immunosorbent assay was utilized for the detection of spontaneously occurring antibodies with apparent specificities for m7G, 5'-m7GMP, and m7G(5')ppp(5')C. From the sera of 50 patients containing anti-nuclear antibodies, 48 (96%) possessed antibodies which bound to one or more immobilized nucleoside-BSA antigens (A-, G-, C-, U-, and T-BSA). Additionally, 8 (16%) of these sera contained immunoglobulins that reacted with m7G-BSA antigen. In these latter sera, soluble competitors such as m7G, 5'm7GMP, and m7G(5')ppp(5')C (but not 5'-AMP, -GMP, -CMP, -UMP, and -TMP or m1G and m22G) effectively inhibited antibody-binding to immobilized m7G-BSA. These results indicate the existence of spontaneously occurring anti-m7G antibodies in autoimmune diseases which are distinct from anti-G antibody populations.

Preferred flavors and performance of weanling pigs.

To characterize the flavor preferences of weanling pigs, a T-maze test was designed and validated. After having sampled feed from one side, containing flavor, and the other side, containing no flavor, pigs were allowed to select feed from either side of the T-maze five times. The frequency of selecting the flavored feed and the percentage of total feed eaten in 20 s/run were used to establish degree of preference for flavors. A total of 129 flavors or flavor combinations was tested in 248 trials, with some flavors tested at several concentrations. At least four flavors were tested in each of eight major flavor groups; the frequency distribution of preference did not differ among groups. Five specific flavors, highly preferred by the pigs in the T-maze tests, were selected for sustained preference tests for two 5-d periods. In the tests, a preference was shown for three of the five flavors. Two flavors were then tested for effects on performance using three groups of 10 pigs for 5 wk after weaning. The feed intakes and body weight gains were increased during the first week in both groups fed flavored feed compared with those of the control group. In a growth trial with 1,219 pigs, one of the two flavors increased feed intake and improved weight gain during the first week after weaning. Thus, although pigs did not appear to prefer flavors in any one of the groups tested, they did prefer specific flavors, which, when added to feed, improved feed intake and weight gain during the critical first week after weaning.