George W. A. Milne

Conformational changes of small molecules binding to proteins

Flexible molecules change their conformation upon binding to a protein. This was shown by the analysis of small molecules whose structures have been determined by X-ray crystallography of both the pure compound and the compound bound to a protein. Thirty-three compounds present both in the Cambridge Structural Database and the Brookhaven Protein Data Bank were analyzed, and both were compared with the global energy minimum conformation calculated by the molecular mechanics program CHARMm. It was found that the conformation bound to the protein differs from that in the crystal structure and also from that of the global energy minimum, and the degree of deformation depends upon the number of freely rotatable bonds in the molecule. Analysis of the conformational energies of the flexible molecules showed that, for most of those compounds, both the crystal and the protein-bound conformations are energetically well above the global minimum, and, in many cases, not even in any local energy minimum. Semi-empirical calculations performed for a select number of structures, using both the AM1 and PM3 hamiltonians, confirmed these results. These findings are discussed as to their impact upon contemporary methods of drug design.

Residues in the second cysteine-rich region of protein kinase C delta relevant to phorbol ester binding as revealed by site-directed mutagenesis.

Phorbol esters bind with high affinity to protein kinase C (PKC) isozymes as well as to two novel receptors, n-chimaerin and Unc-13. The cysteine-rich regions present in these proteins were identified as the binding sites for the phorbol ester tumor promoters and the lipophilic second messenger sn-diacylglycerol. A 50-amino-acid peptide comprising the second cysteine-rich region of PKC , expressed in Escherichia coli as a glutathione S-transferase (GST)-fusion protein, bound phorbol 12,13-dibutyrate (PDBu) with high affinity (K =0.8 nM). Using the cDNA of that cysteine-rich region as a template, a series of 37 point mutations was generated by site-directed mutagenesis, and the mutated proteins were analyzed quantitatively for binding of [H]PDBu and, as appropriate, for binding of the ultrapotent analog [H]bryostatin 1. Mutants displayed one of three patterns of behavior: phorbol ester binding was completely abolished, binding affinity was reduced, or binding was not significantly modified. As expected, five of the six cysteines as well as the two histidines involved in Zn coordination are critical for the interaction of the protein with the phorbol esters. In addition, mutations in several positions, including phenylalanine 3, tyrosine 8, proline 11, leucines 20, 21, and 24, tryptophan 21, glutamine 27, and valine 38 drastically reduced the interaction with the ligands. The effect of these mutations can be rationalized from the three-dimensional (NMR) structure of the cysteine-rich region. In particular, the C-terminal portion of the protein does not appear to be essential, and the loop comprising amino acids 20 to 28 is implicated in the binding activity.

National Cancer Institute Drug Information System 3D database

A searcheable database of three-dimensional structures has been developed from the chemistry database of the NCI Drug Information System (DIS), a file of about 450,000 primarily organic compounds which have been tested by NCI for anticancer activity. The DIS database is very similar in size and content to the proprietary databases used in the pharmaceutical industry; its development began in the 1950s; and this history led to a number of problems in the generation of 3D structures.

Alpha-decarboxylation, an important pathway for degradation of phytanic acid in animals

Abstract Patients suffering from Refsums disease, an inherited disorder of the nervous system ( Refsum, 1946 ), accumulate large stores of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) in their blood and tissues ( Klenk and Kahlke, 1963 ). There appears to be little or no endogenous synthesis of phytanic acid either in normal experimental animals ( Avigan, Steinberg and Cammermeyer, 1966 ; Mize et al, 1966 ) or in patients with Refsums disease ( Steinberg et al, 1965 , 1966a ) and the metabolic error is presumed to lie in a relative inability to degrade phytanic acid. It has been suggested on the basis of indirect evidence that an error in omega-oxidation might be involved ( Eldjarn, 1965 ), but there is as yet no direct information on the normal pathway for degradation of phytanic acid. The results reported below show that the normal rat rapidly converts phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) to its α-decarboxylation product, pristanic acid (2,6,10,14-tetramethylpentadecanoic acid).

Enantioselective synthesis of nonphosphorus-containing phosphotyrosyl mimetics and their use in the preparation of tyrosine phosphatase inhibitory peptides

Abstract Three new L-amino acid analogues 12, 18 and 25 have been prepared in protected form suitable for incorporation into peptides by solid-phase synthesis using Fmoc protocols. These agents represent nonphosphorus-containing phosphotyrosyl (pTyr) mimetics, which utilize carboxylic groups to provide functionality normally afforded by the pTyr phosphate group. To demonstrate the utility of these analogues, the protein-tyrosine phosphatase-directed peptides Ac-D-A-D-E-X-L-amide (28 – 30) were prepared, where X = pTyr mimetic. In the case where X = 3-carboxy-4-(O-carboxymethyl)-L-tyrosine (8) a Ki value of 3.6 μM was obtained against PTP1, which equals the Km of the parent pTyr containing peptide. Besides tyrosine phosphatases, these analogues may be useful in a number of contexts, including SH2 domain and phosphotyrosine binding domain systems.

The occurrence of pristane and phytane in man and animals

Abstract 1. 1. The concentration of the branched-chain hydrocarbons, pristane and phytane, was determined in a number of human and animal tissues by quantitative thin-layer and gas chromatography. 2. 2. In human serum and liver, pristane was found at concentrations of 2–9 μg/g, and of 52 μg/g in a single sample of human skin. The amounts of phytane were significantly lower and represented 1–35% of those in pristane in the various tissue samples analyzed. 3. 3. Rat liver and a number of bovine tissue samples contained similar concentrations of pristane and phytane to that found in human organs. 4. 4. Pristane was found in livers from a number of shark species at a concentration of 2–5 μg/g and, in contrast to the mammalian tissues, phytane was present at similar concentrations to those of pristane. 5. 5. The possible sources of these and other hydrocarbons found in animals are briefly discussed.

Identification of HIV-1 integrase inhibitors based on a four-point pharmacophore.

The rapid emergence of human immunodeficiency virus (HIV) strains resistant to available drugs implies that effective treatment modalities will require the use of a combination of drugs targeting different sites of the HIV life cycle. Because the virus cannot replicate without integration into a host chromosome, HIV-1 integrase (IN) is an attractive therapeutic target. Thus, an effective IN inhibitor should provide additional benefit in combination chemotherapy. A four-point pharmacophore has been identified based on the structures of quinalizarin and purpurin, which were found to be potent IN inhibitors using both a preintegration complex assay and a purified enzyme assay in vitro. Searching with this four-point pharmacophore in the ‘open” part of the National Cancer Institute three-dimensional structure database produced 234 compounds containing the pharmacophore. Sixty of these compounds were tested for their inhibitory activity against IN using the purified enzyme; 19 were found to be active against IN with IC50 values of less than 100 µM, among which 10 had IC50 values of less than 10 µM. These inhibitors can further serve as leads, and studies are in progress to design novel inhibitors based on the results presented in this study.

Recognition and Interaction of Small Rings With the Ricin A-Chain Binding Site

Ricin A‐chain is an N‐glucosidase that attacks ribosomal RNA at a highly conserved adenine residue. Our recent crystallographic studies show that not only adenine and formycin, but also pterin‐based rings can bind in the active site of ricin. For a better understanding of the means by which ricin recognizes adenine rings, the geometries and interaction energies were calculated for a number of complexes between ricin and tautomeric modifications of formycin, adenine, pterin, and guanine. These were studied by molecular mechanics, semi‐empirical quantum mechanics, and ab initio quantum mechanical methods. The calculations indicate that the formycin ring binds better than adenine and pterin better than formycin, a result that is consistent with the crystallographic data. A tautomer of pterin that is not in the low energy form in either the gas phase or in aqueous solution has the best interaction with the enzyme. The net interaction energy, defined as the interaction energy calculated in vacuo between the receptor and an inhibitor minus the solvation energy of the inhibitor, provides a good prediction of the ability of the inhibitor to bind to the receptor. The results from experimental and molecular modeling work suggest that the ricin binding site is not flexible and may only recognize a limited range of adenine‐like rings. Proteins 31:33–41, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Resiniferatoxin-amide and analogues as ligands for protein kinase C and vanilloid receptors and determination of their biological activities as vanilloids

Abstract: The naturally occurring diterpene resiniferatoxin (RTX) is an ultrapotent analogue of capsaicin. Acting on polymodal afferent neurons, RTX induces a generally similar pattern of responses as does capsaicin. However, the two compounds, as well as other vanilloid derivatives, display different relative potencies for different responses. In the present study, we examined the vanilloid‐like activities of two new derivatives, the amide analogue of RTX and phorbol 12,13‐dibenzoate 20‐homovanillylamide. Structurally, RTX‐amide resembles capsaicin more closely than does RTX, and after cleavage of the amide bond the resulting amine would be predicted to not bind to protein kinase C in contrast to resiniferonol 9,13,14‐orthophenylacetate, the parent diterpene of RTX. In contrast to our expectations the binding potency of the RTX‐amide for the vanilloid receptor present in rat spinal cord was 450‐fold lower than that of RTX (Ki values for the RTX‐amide and RTX were 10.4 ± 0.7 nM and 23.1 ± 3.2 pM, respectively). In the case of phorbol 12,13‐dibenzoate 20‐homovanillylamide, there was a further loss of affinity for the vanilloid receptor compared with RTX; nonetheless, the Ki (8.56 ± 0.61 µM) was comparable with that of capsaicin (5.31 ± 0.37 µM). Computer fitting of the binding data yielded Hill coefficient values of 2.25 ± 0.03, 2.33 ± 0.03, and 1.84 ± 0.05 for RTX, RTX‐amide, and phorbol 12,13‐dibenzoate 20‐homovanillylamide, respectively, indicating that both new compounds induced apparent positive cooperativity among vanilloid binding sites. We found that the RTX‐amide was also 20‐ and 300‐fold less potent than RTX in inducing chemogenic pain and hypothermia, respectively. The affinities of the compounds for protein kinase C‐α were evaluated by competition of [3H]phorbol 12,13‐dibutyrate binding. Replacement of the C‐20 hydroxyl group in phorbol 12,13‐dibenzoate by an amine led to a 750‐fold drop in binding affinity, and the conversion of the phorbol 12,13‐dibenzoate 20‐amine to the amide resulted in a further 60‐fold drop in binding activity (Ki values for phorbol 12,13‐dibenzoate, phorbol 12,13‐dibenzoate 20‐amine, and phorbol 12,13‐dibenzoate 20‐homovanillylamide were 0.96 ± 0.01, 720 ± 80, and 43,500 ± 4,060 nM, respectively). The Ki of the RTX‐amide for protein kinase C‐α was >100 µM, whereas RTX and the parent alcohol resiniferonol 9,13,14‐orthophenylacetate yielded Ki values of 1.49 ± 0.19 µM and 36.5 ± 1.3 nM, respectively.

QSAR of conformationally flexible molecules: Comparative Molecular Field Analysis of protein-tyrosine kinase inhibitors

SummaryComparative Molecular Field Analysis (CoMFA) has been applied to a study of quantitative structureactivity relationships (QSAR) of conformationally flexible molecules. The relationship between three-dimensional structure and activity of 20 styrene derivatives which inhibit protein-tyrosine kinase was determined. A technique was developed that allows accurate prediction of the inhibitory activity of these molecules and identification in each case of the active conformation. The problem of multiple energetically acceptable conformations was approached in an iterative procedure. Use was made of the varying degrees of symmetry among the molecules. First, CoMFA QSAR models were developed using only those compounds that possess a symmetrical substituent pattern on the phenyl ring. These CoMFA models were then used to select the active conformers of the less symmetrical compounds in the set. Allowing multiple conformers for each compound in the dataset yielded higher crossvalidated r2 values and better predictivity of the QSAR models. Different probe atoms (C+, O−, neutral C) were explored, the O− probe atom exhibiting the highest selectivity in the conformer selection process.