Development of machine learning models to predict inhibition of 3‐dehydroquinate dehydratase

In this study, we describe the development of new machine learning models to predict inhibition of the enzyme 3‐dehydroquinate dehydratase (DHQD). This enzyme is the third step of the shikimate pathway and is responsible for the synthesis of chorismate, which is a natural precursor of aromatic amino acids. The enzymes of shikimate pathway are absent in humans, which make them protein targets for the design of antimicrobial drugs. We focus our study on the crystallographic structures of DHQD in complex with competitive inhibitors, for which experimental inhibition constant data is available. Application of supervised machine learning techniques was able to elaborate a robust DHQD‐targeted model to predict binding affinity. Combination of high‐resolution crystallographic structures and binding information indicates that the prevalence of intermolecular electrostatic interactions between DHQD and competitive inhibitors is of pivotal importance for the binding affinity against this enzyme. The present findings can be used to speed up virtual screening studies focused on the DHQD structure.     

Topography of the neurotensin (NT)(8−9) binding site of human NT receptor‐1 probed with NT(8–13) analogs

Abstract: A series of neurotensin (NT)(8–13) analogs featuring substitution of the Arg⁸ and/or Arg⁹ residues with non‐natural cationic amino acids was synthesized and evaluated for binding to the human NT receptor‐1 (hNTR‐1). The modifications were designed to probe specific steric and electrostatic requirements in the N‐terminal cationic region of NT(8–13) for receptor binding as a general evaluation of the feasibility of incorporating minor structural changes into a peptide at a crucial polar receptor binding site. Many of the non‐natural amino acids are more or less isosteric to Arg but more lipophilic as a result of addition of alkyl groups or through removal or replacement of NH character with methylene or methyl substituents, whereas others vary the distance between the cation and the α–amino acid carbon. Substitution of Arg⁸ with N^G‐alkylated Arg derivatives or homolysine (Hlys) maintained the subnanomolar affinity of NT(8–13) to the hNTR‐1. Position 8 incorporation of Hlys produced the most favorable primary amine side‐chain substitution to date. Moderate losses in affinity observed with position 9 substitutions were attributed to adverse steric effects. Doubly substituted [Hlys⁸, DAB⁹]NT(8–13), in which DAB is 2,4‐diaminobutyric acid, was also prepared and tested as the shorter side‐chain of DAB is known to be favored in position 9 of NT(8–13). This analog maintained 60% of NT(8–13) binding affinity making it the most favored des‐guanidinium‐containing analog known. These results demonstrate that adequate receptor binding affinity can be maintained over a structural range of Arg analogs, thus providing a range of peptides expected to exhibit altered pharmacokinetic properties. From the standpoint of the hNTR‐1 cationic binding sites, these results help to map out the structural stringency inherent in the formation of a tight binding complex with NT(8–13) and related analogs.     

Isoform-selective inhibition of phosphoinositide 3-kinase: identification of a new region of nonconserved amino acids critical for p110α inhibition

The combination of molecular modeling and X-ray crystallography has failed to yield a consensus model of the mechanism for selective binding of inhibitors to the phosphoinositide 3-kinase (PI3K) p110 α-isoform. Here we have used kinetic analysis to determine that the p110α-selective inhibitor 2-methyl-5-nitro-2-[(6-bromoimidazo[1,2-α]pyridin-3-yl)methylene]-1-methylhydrazide-benzenesulfonic acid (PIK-75) is a competitive inhibitor with respect to a substrate, phosphatidylinositol (PI) in contrast to most other PI3K inhibitors, which bind at or near the ATP site. Using sequence analysis and the existing crystal structures of inhibitor complexes with the p110γ and -δ isoforms, we have identified a new region of nonconserved amino acids (region 2) that was postulated to be involved in PIK-75 p110α selectivity. Analysis of region 2, using in vitro mutation of identified nonconserved amino acids to alanine, showed that Ser773 was a critical amino acid involved in PIK-75 binding, with an 8-fold-increase in the IC(50) compared with wild-type. Kinetic analysis showed that, with respect to PI, the PIK-75 K(i) for the isoform mutant S773D increased 64-fold compared with wild-type enzyme. In addition, a nonconserved amino acid, His855, from the previously identified region 1 of nonconserved amino acids, was found to be involved in PIK-75 binding. These results show that these two regions of nonconserved amino acids that are close to the substrate binding site could be targeted to produce p110α isoform-selective inhibitors.     

Mutational analysis of a blood coagulation factor VIII‐binding peptide

Abstract: A peptide screened from a combinatorial peptide library with the sequence EYKSWEYC performed best as a ligand for affinity chromatography of human blood coagulation factor VIII (FVIII). With this peptide immobilized on monolithic CIM columns via epoxy groups we were able to capture FVIII from diluted plasma. Rational substitution of amino acids by spot synthesis revealed that lysine and cysteine can be exchanged for almost all other proteinogenic amino acids without loss of affinity to FVIII. This offers the possibility of site‐specific attachment via either one of these residues or the N‐ or C‐terminus. The aliphatic positions O₅ (tryptophan) and O₇ (tyrosine), together with the charged position O₆ (glutamic acid), seem to form the core of the binding unit. In the positions with aliphatic amino acids, substitution by tyrosine or phenylalanine, and in the positions with charged amino acids, substitution by aspartic acid or lysine, preserved the affinity to FVIII. The functionality of the selected peptides was confirmed by affinity chromatography. Selective binding and elution could be achieved.     

Analogs of Substance P modified at the C‐terminus which are both agonist and antagonist of the NK‐1 receptor depending on the second messenger pathway

Abstract: The initial goal of this study was to analyze, using photolabeling, the interactions between Substance P and its tachykinin NK‐1 receptor. Therefore, the photoreactive amino acid para‐benzoyl‐phenylalanine (pBzl)Phe was incorporated into the Substance P sequence from position 4 to 11 leading to Bapa⁰[(pBzl)Phe^x]SP analogs. Biotinyl sulfone‐5‐aminopentanoic acid (Bapa) was introduced in order to purify the covalent complex. These photoreactive SP analogs were first assayed for their affinity for the two binding sites associated with the NK‐1 receptor, as well as for their potency in activating the phospholipase C and adenylate cyclase pathways. All analogs photoreactive from position 4 to 11 have moderate to high affinity for the two NK‐1 receptor‐binding sites, except for the analog modified at position 7. This affinity could be correlated to their potency to activate the phospholipase C and adenylate cyclase pathways, except for the analog photoreactive at position 11. Bapa⁰[(pBzl)Phe¹¹]SP was found to be an agonist in the phospholipase C pathway and an antagonist in the adenylate cyclase pathway, other analogs modified at position 11 were therefore analyzed. Among these, Bapa⁰[Pro⁹, (pBzl)Hcy(O₂)¹¹]SP is a partial agonist, whereas Bapa⁰[Hcy(ethylaminodansyl)¹¹]SP is a full agonist in the phospholipase C pathway, the two analogs being antagonist in the adenylate cyclase pathway. These results show that analogs of SP can be simultaneously agonist at one binding site and antagonist at the other binding site associated with the NK‐1 receptor.     

Synthesis of enantiopure non‐natural α‐amino acids using tert‐butyl (2S)‐2‐[bis‐(tert‐butoxycarbonyl)amino]‐5‐oxopentanoate as key‐intermediate:the first synthesis of(S)‐2‐amino‐oleic acid

Abstract: A general method for the synthesis of enantiopure non‐natural α‐amino acids is described. The key intermediate tert‐butyl (2S)‐2‐[bis(tert‐butoxycarbonyl)amino]‐5‐oxopentanoate was obtained from l ‐glutamic acid after suitable protection and selective reduction of the γ‐methyl ester group by DIBALH. Wittig reaction of this chiral aldehyde with various ylides led to a variety of δ,ε‐unsaturated α‐amino acids. This methodology was applied to the synthesis of (S)‐2‐amino‐oleic acid.     

Effects of urethane and ketamine on substance P- and excitatory amino acid-induced behavior in mice.

Urethane and ketamine were tested for their ability to alter the caudally directed binding and scratching response elicited by the intrathecal (i.t.) injection of excitatory amino acids (EAAs) or substance P (SP). EAAs, such as N-methyl-D-aspartate (NMDA), kainate acid and quisqualic acid, but not SP, were inhibited by subanesthetic doses of urethane. In contrast, SP was more sensitive than NMDA to the inhibitory effect of (+)-ketamine. (-)-Ketamine produced much less inhibition of the SP-induced behaviors than the (+)isomer. These results have important implications regarding the use of urethane and ketamine as anesthetics for studies in which these excitatory compounds are potential mediators.     

A kinetic analysis of substance P trafficking

The potential for administering substance P (SP) nocitoxins for the treatment of chronic pain has been identified. To characterize treatment protocols for the spinal cord or elsewhere, binding/internalization of these compounds at the cellular targets must be understood quantitatively. Thus, a kinetic model of SP binding and intracellular trafficking has been developed from data. The eight differential equation model describes surface binding between SP and neurokinin 1 receptor, clathrin-mediated endocytosis followed by spatial translation to a perinuclear endosome where SP is sorted from its receptor, SP degradation in late endosomes/early lysosomes, and return of sorted receptor to plasma membrane via recycling endosomes. With suitably optimized parameters, the model accounts for the kinetics of total, membrane-associated, and internalized SP in cells continuously exposed to SP, as well as the fractions of internalized SP remaining intact at 30 and 60 min. Simultaneously, the model accounts for the kinetics of internalization and receptor recycling after SP preloading of membrane and subsequent exposure to SP-free media. Rate constants (min(-1)) are: 0.034 +/- 0.004 (receptor off-rate), 0.15 +/- 0.03 (internalization), 0.048 +/- 0.003 (exit from sorting endosome), 0.062 +/- 0.008 (exit of labeled SP amino acids from prelysosome), and 0.029 +/- 0.004 (receptor return from recycling endosome to plasma membrane). The SP kinetics resemble those of transferrin and its receptor at the internalization step, but are several-fold slower in the sorting and recycling steps.     

Modifications of the cyclic mu receptor selective tetrapeptide Tyr‐c[d‐Cys‐Phe‐d‐Pen]NH2 (Et): effects on opioid receptor binding and activation

Abstract: The previously described cyclic mu opioid receptor‐selective tetrapeptide Tyr‐c[d ‐Cys‐Phe‐d ‐Pen]NH₂ (Et) (JOM‐6) was modified at residues 1 and 3 by substitution with various natural and synthetic amino acids, and/or by alteration of the cyclic system. Effects on mu and delta opioid receptor binding affinities, and on potencies and efficacies as measured by the [³⁵S]‐GTPγS assay, were evaluated. Affinities at mu and delta receptors were not influenced dramatically by substitution of Tyr¹ with conformationally restricted phenolic amino acids. In the [³⁵S]‐GTPγS assay, all of the peptides tested exhibited a maximal response comparable with that of fentanyl at the mu opioid receptor, and all showed high potency, in the range0.4–9 nm . However, potency changes did not always correlate with affinity, suggesting that the conformation required for binding and the conformation required for activation of the opioid receptors are different. At the delta opioid receptor, none of the peptides were able to produce a response equivalent to that of the full delta agonist BW 373,U86 and only one had an EC₅₀ value of less than 100 nm . Lastly, we have identified a peptide, d ‐Hat‐c[d ‐Cys‐Phe‐d ‐Pen]NH₂ (Et), with high potency and > 1000‐fold functional selectivity for the mu over delta opioid receptor as measured by the [³⁵S]‐GTPγS assay.     

Alanine Scan of an Immunosuppressive Peptide (CP): Analysis of Structure–Function Relationships

Core peptide is a hydrophobic peptide, the sequence of which is derived from the T‐cell antigen receptor alpha‐chain transmembrane region. Previous studies have shown that core peptide can inhibit T‐cell‐mediated immune responses both in vitro and in vivo. Here, we report the role each constituent amino acid plays within core peptide using an alanine scan and the amino acid effect on function using a biological antigen presentation assay. The biophysical behaviour of these analogues in model membranes was analysed using surface plasmon resonance studies and then binding correlated with T‐cell function. Removal of any single hydrophobic amino acid between the two charged amino acids in core peptide (R, K) resulted in lower binding. Changing the overall net charge of core peptide, by removing either of the positively charged residues (R or K), had varying effects on peptide binding and IL‐2 production. There was a direct correlation (ρ = 0.718) between peptide binding to model membranes and peptide ability to inhibit IL‐2. Except for IL‐2 inhibition, production of other T‐cell cytokines such as GM‐CSF, IFN‐γ, IL‐1α, IL‐4, IL‐5, IL‐6, IL‐10, IL‐17 and T‐cell antigen receptor alpha‐chain was not detected using a fluorescent bead immunoassay. This study provides important structure–function relationships essential for further drug design.     

Identification of curcumin targets in neuroinflammatory pathways: molecular docking scores with GSK-3β, p38 MAPK, COX, ICE and TACE enzymes

In the present study, the multiple targets have been identified in the mediation of anti-inflammatory response of curcumin. The anti-inflammatory pathway of curcumin was identified through docking with of curcumin with various inflammation inducing enzymes like glycogen synthase kinase (GSK-3β), p38 mitogen activated protein kinase (MAPK), COX, interleukin-1β converting enzyme (ICE) and tumor necrosis factor-α converting enzyme (TACE). Theoretical docking study was used for the prediction of the conformation orientation and position (pose) of the bioactive compound into the binding pocket and estimation of effective target-ligand interactions (scoring) was utilized for conformational sampling. The final docked conformations were selected according to their scores. The binding target GSK-3β (-6.44) was found to be more selective for curcumin binding when compared with MAPK (-4.08), COX (-7.35), ICE (-4.02), TACE (-6.38) and their respective native ligand. The binding takes place through hydrogen bonding interactions of curcumin with the amino acids in the substrate enzyme. The key amino acids involved were Vall35, Gln185 and Lys85 in GSK-3β. The binding efficiency of curcumin was compared with a standard molecule GF109203 which showed a docking score of - 4.97. These findings enabled us to identify the keto form of curcumin as a best choice of lead compound to target GSK-3β.     

Design rationale,synthesis, and characterization ofnon‐natural analogs of the cationic amino acids arginine and lysine

Abstract: A series of non‐natural isosteric analogs of the cationic, ion‐pairing, natural amino acids arginine and lysine have been synthesized, characterized with regard to relevant physical parameters, and protected for routine inclusion in Merrifield solid‐phase synthesis. The design of these molecules is based on the concept of steric inhibition of solvation, in that judicious placement of alkyl groups can destabilize aqueous ion solvation and favor ion‐pairing [see Beeson & Dix (1993) J. Am. Chem. Soc. 115, 10275]. When the residues are substituted for the natural amino acids in biologically active peptides, enhanced ion‐pairing of the peptides to their receptors to increase the peptides’ biological activities can result. The increased lipophilicity of the non‐natural residues can also improve pharmacokinetic parameters and agonist/antagonist behaviors of peptides. While the synthesis of the L‐series is described, theD‐isomers were also prepared using identical chemistry     

DiSSiMiL: Diverse Small Size Mini‐Libraries applied to simple and rapid epitope mapping of a monoclonal antibody

Abstract: Methods for screening protein–protein interactions are useful in protein science and for the generation of drug leads. We set out to develop a simplified assay to rapidly test protein–protein interactions, with a library of 400 pentapeptides comprising the 20 natural amino acids at two variable positions followed by three glycines (NH₂‐X₁X₂GGG). The library was used to identify the epitope of monoclonal antibody (mAb) 10D11 directed against the HOXD4 protein. Three pentapeptide ‘hits’ were selected (VYGGG, PWGGG and WKGGG) from direct binding assays screening for pentapeptide?mAb interactions; and from assays using pentapeptides in solution to competitively block HOXD4?mAb interactions. Alignment of the three ‘hit’ pentapeptides to the HOXD4 sequence predicts the mAb 10D11 epitope as NH₂‐VYPWMK. Synthesis of NH₂‐VYPWMK hexapeptide confirmed this prediction; and an alanine scan of HOXD4 ablated binding by mAb 10D11 when amino acids in the putative epitope were mutated. We propose that these simplified but diverse libraries can be used for rapid epitope mapping of some mAbs, and for generating lead small peptide analogs that interfere with receptor–ligand or other protein–protein interactions, or with enzymatic activity.     

Chemical synthesis of insulin-like growth factor II

Human insulin-like growth factor II (IGF-II) with 67 amino acids and three disulfide bridges has been synthesized by the solid-phase method. The synthetic hormone is shown to be homogeneous in high performance liquid chromatography (HPLC), high performance partition chromatography (HPPC), and chromatofocusing. It is indistinguishable from natural hormone in HPLC, peptide map of thermolysin digests, amino acid composition and radioreceptor binding assay. Thus, synthetic and natural IGF-II are identical.     

Synthesis and analysis of potent,more lipophilic derivatives of the bradykinin B2 receptor antagonist peptide Hoe 140

Abstract: Bradykinin (BK) is an endogenous peptide that has been implicated in several pathological conditions, hence antagonists of its activity have therapeutic potential. The decapeptide Hoe 140 is currently one of the best BK antagonists, but interest remains in finding even more potent compounds. A library of Hoe 140 derivatives was synthesized that incorporated non‐natural analogs of the cationic, naturally occurring amino acids arginine (Arg) and lysine (Lys). The modified amino acids were designed to form enhanced ionic interactions due to an increase in local hydrophobicity, which promotes desolvation of the cation in water. The potencies of the resulting peptides were determined by competitive binding assays in human A431 cells expressing the BK B2 receptor. Two of the peptides synthesized were equipotent to Hoe 140 (IC_50s 2.99 and 3.36 nm ) and the most potent was demonstrated as a functional antagonist in vitro by blocking BK‐mediated phosphorylation of mitogen‐activated protein (MAP) kinases. The new derivatives are more hydrophobic than Hoe 140 and thus may exhibit changes in pharmacokinetic properties when evaluated in vivo.     

Sialogogic effects on rat submandibular gland of analogs of the C-terminal hexapeptide of substance P.

The sialogogic response of submandibular glands to analogs of the C-terminal hexapeptide of substance P with various amino acids at the N-terminus was investigated in urethane-anesthetized rats. The rank order of potencies was as follows: SP greater than (pGlu6)SP6-11 much greater than (Dab6)SP6-11 greater than (Orn6)SP6-11 greater than (Gln6)SP6-11 greater than (Lys6)SP6-11 much greater than (Ala6)SP6-11. These results suggest that the sialogogic activity of the analogs of the C-terminal hexapeptide is influenced by the steric effects of the N-terminal amino acid, and the nature of its side chain is of particular importance.     

Conformational mapping of the N‐terminal segment of surfactant protein B in lipid using 13C‐enhanced Fourier transform infrared spectroscopy

Abstract: Synthetic peptides based on the N‐terminal domain of human surfactant protein B (SP‐B_1?25; 25 amino acid residues; NH₂‐FPIPLPYCWLCRALIKRIQAMIPKG) retain important lung activities of the full‐length, 79‐residue protein. Here, we used physical techniques to examine the secondary conformation of SP‐B_1?25 in aqueous, lipid and structure‐promoting environments. Circular dichroism and conventional, ¹²C‐Fourier transform infrared (FTIR) spectroscopy each indicated a predominateα‐helical conformation for SP‐B_1?25 in phosphate‐buffered saline, liposomes of 1‐palmitoyl‐2‐oleoyl phosphatidylglycerol and the structure‐promoting solvent hexafluoroisopropanol; FTIR spectra also showed significant β‐ and random conformations for peptide in these three environments. In further experiments designed to map secondary structure to specific residues, isotope‐enhanced FTIR spectroscopy was performed with 1‐palmitoyl‐2‐oleoyl phosphatidylglycerol liposomes and a suite of SP‐B_1?25 peptides labeled with ¹³C‐carbonyl groups at either single or multiple sites. Combining these ¹³C‐enhanced FTIR results with energy minimizations and molecular simulations indicated the following model for SP‐B_1?25 in 1‐palmitoyl‐2‐oleoyl phosphatidylglycerol: β‐sheet (residues 1–6), α‐helix (residues 8–22) and random (residues 23–25) conformations. Analogous structural motifs are observed in the corresponding homologous N‐terminal regions of several proteins that also share the ‘saposin‐like’ (i.e. 5‐helix bundle) folding pattern of full‐length, human SP‐B. In future studies, ¹³C‐enhanced FTIR spectroscopy and energy minimizations may be of general use in defining backbone conformations at amino acid resolution, particularly for peptides or proteins in membrane
environments.     

Fatty Acid Binding Receptors and Their Physiological Role in Type 2 Diabetes

G‐protein‐coupled receptors (GPCRs) respond to various physiological ligands such as photons, ions, and small molecules that include amines, fatty acids, and amino acids to peptides, proteins and steroids. Therefore, this family of proteins represents an attractive target for biopharmaceutical research [1]. The physiological role of fatty acids and other lipid molecules as important signal mediators is well studied in various metabolic pathways [2]. Acute administration of free fatty acids (FFAs) stimulates insulin release. Conversely, chronic exposure to high levels of free fatty acids leads to impairment of β cell function and lipotoxicity. However, the receptors through which these fatty acids and lipids act were unknown, until the identification of fatty acid binding receptors: GPR40, GPR41, GPR43, and GPR119. Based on their tissue‐expression profile, and pharmacologic analysis, the fatty acid binding receptors along with lipid binding receptor GPR119 are linked to diabetes and obesity. They play a critical role in the metabolic regulation of insulin release and glucose homeostasis. In this review, the mechanism of receptor activation, pharmacology, and the physiological functions of the fatty acid binding receptors will be discussed.     

Design and discovery of a novel dipeptidyl-peptidase IV (CD26)-based prodrug approach

Here we describe a novel type of enzyme-based prodrug approach in which a dipeptide moiety is linked to a nonpeptidic therapeutic drug through an amide bond which is specifically cleaved by the dipeptidyl-peptidase IV (DPP IV/CD26) enzyme activity present in plasma and on the surface of certain cells. DPP IV has high substrate selectivity for peptides with a proline (or an alanine) at the penultimate amino acid position at the N-terminus but tolerates a wide range of natural amino acids at the amino terminal end. A variety of dipeptidyl amide prodrugs of anti-HIV TSAO molecules were synthesized and evaluated for their ability to act as substrates for the enzyme. Our data revealed that DPP IV/CD26 can efficiently recognize such prodrugs as substrates, releasing the parent compound. Moreover, it is possible to modify the half-life and the lipophilicity of the prodrugs by changing the nature of the dipeptide. All conjugates have shown marked in vitro antiviral activities irrespective the the nature of the terminal and/or the penultimate amino acid moiety.     

Distribution and pharmacological characterization of primate NK-1 and NK-3 tachykinin receptors in the central nervous system of the rhesus monkey

Much attention has focused on tachykinin receptors as therapeutic targets for neuropsychiatric disorders, although their expressional distributions in the primate central nervous system (CNS) remain unclear. We cloned the genes encoding the NK-1 and NK-3 tachykinin receptors (referred to as rmNK-1 and rmNK-3) from the rhesus monkey (Macaca mulatta) brain and examined their pharmacological profiles and regional distributions in the CNS. The deduced rmNK-1 amino-acid sequence differed by only two amino acids from the human NK-1 (hNK-1). The deduced rmNK-3 amino-acid sequence was two amino acids shorter than human NK-3 (hNK-3), with a seven-amino-acid difference in sequence. Ligand binding studies revealed that the affinity of rmNK-1 to substance P (SP) was comparable to that of hNK-1 in cell lines that expressed individual receptors stably. Nonpeptide antagonists had similar effects on the binding of rmNK-1 and hNK-1. Affinity of rmNK-3 for NKB was stronger than for SP and the IC50 value was comparable with that of hNK-3. Ca2+ imaging showed that activations of both rmNK-1 and rmNK-3 by specific ligands, SP and senktide, induced increased intracellular Ca2+ in cell lines that stably expressed individual primate tachykinin receptors. The amounts of rmNK-1 and rmNK-3 mRNAs were quantitatively determined in the monkey CNS. The expression of rmNK-1 was observed in all of the cortical and subcortical regions, including the hippocampus and the amygdala. The putamen contained the most NK-1 mRNA in the brain, with less rmNK-3 mRNA found in the cortex compared to rmNK-1 mRNA. In the monkey hippocampus and amygdala, rmNK-1 mRNA was present at markedly higher concentrations than rmNK-3 mRNA. The present results provide an insight into the distinct physiological nature and significance of the NK-1 and NK-3 tachykinin systems in the primate CNS. These findings are indispensable for establishing model systems in the search for a subtype-specific tachykinin receptor agonist and antagonist for the treatment of neuropsychiatric disorders.