Design,synthesis and biological evaluation of a bivalent μ opiate and adenosine A1 receptor antagonist

The cross talk between different membrane receptors is the source of increasing research. We designed and synthesized a new hetero-bivalent ligand that has antagonist properties on both A₁ adenosine and μ opiate receptors with a K_i of 0.8 ± 0.05 and 0.7 ± 0.03 μM, respectively. This hybrid molecule increases cAMP production in cells that over express the μ receptor as well as those over expressing the A₁ adenosine receptor and reverses the antalgic effects of μ and A₁ adenosine receptor agonists in animals.     

Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas

Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)–Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)–biotin MRI contrast agent) to trap tumor-associated free radicals. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p < 0.001) in MR signal intensity or a significant decrease (p < 0.001) in T₁ relaxation, measured as %T₁ change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p < 0.0001) in T₁ relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin–Gd-DTPA–biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p < 0.001) and 3-nitrotyrosine (3-NT) (p < 0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model.     

Sulfur incorporation generally improves Ricin inhibition in pterin-appended glycine-phenylalanine dipeptide mimics

Several 7-aminoamido-pterins were synthesized to evaluate the electronic and biochemical subtleties observed in the ‘linker space’ when N-{N-(pterin-7-yl)carbonylglycyl}-l-phenylalanine 1 was bound to the active site of RTA. The gylcine–phenylalanine dipeptide analogs included both amides and thioamides. Decarboxy gly-phe analog 2 showed a 6.4-fold decrease in potency (IC₅₀ = 128 μM), yet the analogous thioamide 7 recovered the lost activity and performed similarly to the parent inhibitor (IC₅₀ = 29 μM). Thiourea 12 exhibited an IC₅₀ nearly six times lower than the oxo analog 13. All inhibitors showed the pterin head-group firmly bound in their X-ray structures yet the pendants were not fully resolved suggesting that all pendants are not firmly bound in the RTA linker space. Calculated log P values do not correlate to the increase in bioactivity suggesting other factors dominate.     

Topography of human cytochrome b5/cytochrome b5 reductase interacting domain and redox alterations upon complex formation

Cytochrome b₅ is the main electron acceptor of cytochrome b₅ reductase. The interacting domain between both human proteins has been unidentified up to date and very little is known about its redox properties modulation upon complex formation. In this article, we characterized the protein/protein interacting interface by solution NMR and molecular docking. In addition, upon complex formation, we measured an increase of cytochrome b₅ reductase flavin autofluorescence that was dependent upon the presence of cytochrome b_5. Data analysis of these results allowed us to calculate a dissociation constant value between proteins of 0.5 ± 0.1 μM and a 1:1 stoichiometry for the complex formation. In addition, a 30 mV negative shift of cytochrome b₅ reductase redox potential in presence of cytochrome b₅ was also measured. These experiments suggest that the FAD group of cytochrome b₅ reductase increase its solvent exposition upon complex formation promoting an efficient electron transfer between the proteins.     

Synthesis,characterization and biological evaluation of novel chalcone sulfonamide hybrids as potent intestinal alkaline phosphatase inhibitors

Alkaline phosphatase (AP) and ecto-5′-nucleotidase (e5′NT) belong to same family that hydrolyze the extracellular nucleotides and ensure the bioavailability of nucleotides and nucleosides at purinergic receptors. During pathophysiological conditions, the over expression of AP and e5′NT lead to an increased production of adenosine that enhance tumor proliferation, invasiveness, neoangiogenesis and disrupts the body antitumor response. As both enzymes are abundantly expressed in above mentioned conditions, therefore it is of great interest to synthesize and develop potent inhibitors of these enzymes that augment the antitumor therapy. Herein we reported the synthesis and biological activity of a new series of chalcone-sulfonamide hybrids (4a-j). These derivatives were then evaluated for their inhibitory potential against two members of ecto-nucleotidase family, e5′NT (human and rat) and APs isozyme (intestinal and tissue nonspecific). Only six derivatives were found to inhibit both human and rat e5′NT enzymes. Compounds 4e and 4d showed maximum inhibition of human and rat e5′NT with an IC₅₀ ± SEM = 0.26 ± 0.01 and 0.33 ± 0.004 μM, respectively. Moreover, on APs, these derivatives were identified as the selective inhibitors of calf intestinal AP (c-IAP). The derivative 4a exhibited maximum inhibition of c-IAP with an IC₅₀ ± SEM = 0.12 ± 0.02 μM. In conclusion, these chalcone-sulfonamide hybrids exhibited dual inhibition of both family of isozymes but was more selective towards c-IAP enzyme.     

In vivo detection of free radicals in mouse septic encephalopathy using molecular MRI and immuno-spin trapping

Free radicals are known to play a major role in sepsis. Combined immuno-spin trapping and molecular magnetic resonance imaging (MRI) was used to detect in vivo and in situ levels of free radicals in murine septic encephalopathy after cecal ligation and puncture (CLP). DMPO (5,5-dimethyl pyrroline N-oxide) was injected over 6 h after CLP, before administration of an anti-DMPO probe (anti-DMPO antibody bound to albumin–gadolinium–diethylene triamine pentaacetic acid–biotin MRI targeting contrast agent). In vitro assessment of the anti-DMPO probe in oxidatively stressed mouse astrocytes significantly decreased T₁ relaxation (p < 0.0001) compared to controls. MRI detected the presence of anti-DMPO adducts via a substantial decrease in %T₁ change within the hippocampus, striatum, occipital, and medial cortex brain regions (p < 0.01 for all) in septic animals compared to shams, which was sustained for over 60 min (p < 0.05 for all). Fluorescently labeled streptavidin was used to target the anti-DMPO probe biotin, which was elevated in septic brain, liver, and lungs compared to sham. Ex vivo DMPO adducts (qualitative) and oxidative products, including 4-hydroxynonenal and 3-nitrotyrosine (quantitative, p < 0.05 for both), were elevated in septic brains compared to shams. This is the first study that has reported on the detection of in vivo and in situ levels of free radicals in murine septic encephalopathy.     

Kinesin family member 14 is a candidate prognostic marker for outcome of glioma patients

Background & aim: Human kinesin superfamily proteins (KIFs) are a conserved class of microtubule-dependent molecular motor proteins with adenosine triphosphatase activity and motion characteristics. As a member of KIFs, KIF14 plays an important role in the regulation of cell cycle and mitotic progression. Deregulation of KIF14 has been found in several human malignancies and also has been demonstrated to be involved in tumor progression and related to patient survival. The aim of this study was to investigate the clinicopathological significance of KIF14 expression in glioma. Methods: Real-time quantitative RT-PCR assay was performed to detect KIF14 mRNA expression, and Western blot and immunohistochemistry analyses were performed to detect KIF14 protein expression in human gliomas and non-neoplastic brain tissues, respectively. Then, the association of KIF14 immunostaining with clinicopathological factors and prognosis of glioma patients was also statistically analyzed. Results: KIF14 mRNA and protein expression were respectively increased 5.5- and 4.2-fold on average in glioma tissues relative to non-neoplastic brain tissues (both P < 0.001). Additionally, both KIF14 mRNA and protein expression increased with ascending pathological grade. Then, the high KIF14 immunostaining in glioma tissues was significantly associated with advanced pathological grade (P = 0.008), low Karnofsky performance score (KPS) (P = 0.02), high mitotic index (P = 0.005) and Ki-67 index (P = 0.008). Furthermore, both univariate and multivariate Cox regression analyses determined that KIF14 overexpression effectively predicted decreased overall survival in patients with gliomas. Conclusions: These findings offer the first convinced evidence that KIF14 expression in gliomas is tumor-specific and increased in more aggressive tumors. KIF14 might function as a candidate prognostic marker for human gliomas.     

Influence of phosphorus nutrition on growth and metabolism of Duo grass (Duo festulolium)

Use of suitable plants that can extract and concentrate excess P from contaminated soil serves as an attractive method of phytoremediation. Plants vary in their potential to assimilate different organic and inorganic P-substrates. In this study, the response of Duo grass (Duo festulolium) to variable rates of soil-applied potassium dihydrogen phosphate (KH₂PO₄) on biomass yield and P uptake were studied. Duo grown for 5 weeks in soil with 2.5, 5 and 7.5 g KH₂PO₄ kg^?1 soil showed a significantly higher biomass and shoot P content of 8.3, 11.4 and 12.3 g P kg^?1 dry weight respectively compared to plants that received no soil added P. Also, the ability of Duo to metabolize different forms of P-substrates was determined by growing them in sterile Hoagland's agar media with different organic and inorganic P-substrates, viz. KH₂PO₄, glucose-1-phosphate (G1P), inositiol hexaphosphate (IHP), adenosine triphosphate (ATP) and adenosine monophosphate (AMP) for 2 weeks. Plants on agar media with different P-substrates also showed enhanced biomass yield and shoot P relative to no P control and the P uptake was in the order of ATP > KH₂PO₄ > G1P > IHP = AMP > no P control. The activities of both phytase (E.C.3.1.3.26) and acid phosphatases (E.C.3.1.3.2) were higher in all the P received plants than the control. Duo grass is capable of extracting P from the soil and also from the agar media and thus it can serve as possible candidate for phytoextraction of high P-soil.     

Soluble expression and characterization of a mouse epididymis-specific protein lipocalin6

Mouse lipocalin6 (mLcn6) was recently identified to be specifically expressed in the epididymis and speculated to may play a role in sperm maturation. However, further studies were hindered due to the bottleneck to obtain enough recombinant mLcn6 proteins. In this article, GB1 tag was successfully applied to improve the soluble expression of mLcn6. Thermal unfolding experiments demonstrate that GB1 can enhance the structural stability of mLcn6. Fluorescence spectroscopy experiments show that mLcn6 prepared according to our procedure has high affinities to both retinoic acid (K_d = 810 nM) and retinol (K_d = 210 nM). In conclusion, soluble, stable and active mLcn6 was recombinantly prepared with the help of the GB1 tag, which will facilitate the structural and functional studies of mLcn6.     

Characterization of the N-oxygenase AurF from Streptomyces thioletus

AurF catalyzes the N-oxidation of p-aminobenzoic acid to p-nitrobenzoic acid in the biosynthesis of the antibiotic aureothin. Here we report the characterization of AurF under optimized conditions to explore its potential use in biocatalysis. The pH optimum of the enzyme was established to be 5.5 using phenazine methosulfate (PMS)/NADH as the enzyme mediator system, showing ∼10-fold higher activity than previous reports in literature. Kinetic characterization at optimized conditions give a K_m of 14.7 ± 1.1 μM, a k_cat of 47.5 ± 5.4 min⁻¹ and a k_cat/K_m of 3.2 ± 0.4 μM⁻¹ min⁻¹. PMS/NADH and the native electron transfer proteins showed significant formation of the p-hydroxylaminobenzoic acid intermediate, however H₂O₂ produced mostly p-nitrobenzoic acid. Alanine scanning identified the role of important active site residues. The substrate specificity of AurF was examined and rationalized based on the protein crystal structure. Kinetic studies indicate that the K_m is the main determinant of AurF activity toward alternative substrates.     

The D-galactose specific lectin of field bean (Dolichos lablab) seed binds sugars with extreme negative cooperativity and half-of-the-sites binding

The field bean (Dolichos lablab) lectin designated as PPO-haemagglutinin (DLL-II) is bifunctional, exhibiting both polyphenol oxidase and haemagglutinating activity. The lectin is unusual in that it binds galactose (Gal), lactose (Lac) and N-acetylgalactosamine (GalNAc) only in the presence of (NH₄)₂SO₄ and exhibits negative cooperativity and half-of-the-sites binding. Circular dichroism, isothermal titration calorimetry and fluorescence quenching were used to assess the sugar binding in the presence of (NH₄)₂SO₄. Comparison of the near-UV CD spectra with and without bound sugar revealed ligand induced conformational changes. The intrinsic fluorescence quenching data indicate that DLL-II exhibits weak binding to Gal in the presence of (NH₄)₂SO₄ with a stoichiometry of one bound ligand per dimer. ITC data fitted using a two sets of sites binding model presented a similar picture. The K_a’s for Gal, Lac and GalNAc in the presence of (NH₄)₂SO₄ were 0.16 ± 0.002, 0.21 ± 0.004 and 8.45 ± 0.78 (×10^?3) M^?1 respectively. The Hill plot for the binding of these sugars to DLL-II was curvilinear with a tangent slope <1.0 indicating negative cooperativity. DLL-II thus exhibits half-of-the-site binding, an extreme form of negative cooperativity in which the second ligand does not bind at all. This is the first report of a legume lectin, exhibiting half-of-the-sites binding.     

Anticancer activity of natural cytokinins: A structure–activity relationship study

Cytokinin ribosides (N⁶-substituted adenosine derivatives) have been shown to have anticancer activity both in vitro and in vivo. This study presents the first systematic analysis of the relationship between the chemical structure of cytokinins and their cytotoxic effects against a panel of human cancer cell lines with diverse histopathological origins. The results confirm the cytotoxic activity of N⁶-isopentenyladenosine, kinetin riboside, and N⁶-benzyladenosine and show that the spectrum of cell lines that are sensitive to these compounds and their tissues of origin are wider than previously reported. The first evidence that the hydroxylated aromatic cytokinins (ortho-, meta-, para-topolin riboside) and the isoprenoid cytokinin cis-zeatin riboside have cytotoxic activities is presented.Most cell lines in the panel showed greatest sensitivity to ortho-topolin riboside (IC₅₀ = 0.5–11.6 μM). Cytokinin nucleotides, some synthesized for the first time in this study, were usually active in a similar concentration range to the corresponding ribosides. However, cytokinin free bases, 2-methylthio derivatives and both O- and N-glucosides showed little or no toxicity. Overall the study shows that structural requirements for cytotoxic activity of cytokinins against human cancer cell lines differ from the requirements for their activity in plant bioassays. The potent anticancer activity of ortho-topolin riboside (GI₅₀ = 0.07–84.60 μM, 1st quartile = 0.33 μM, median = 0.65 μM, 3rd quartile = 1.94 μM) was confirmed using NCI₆₀, a standard panel of 59 cell lines, originating from nine different tissues. Further, the activity pattern of oTR was distinctly different from those of standard anticancer drugs, suggesting that it has a unique mechanism of activity. In comparison with standard drugs, oTR showed exceptional cytotoxic activity against NCI₆₀ cell lines with a mutated p53 tumour suppressor gene. oTR also exhibited significant anticancer activity against several tumour models in in vivo hollow fibre assays.     

Novel l-adenosine analogs as cardioprotective agents

Two l-nucleosides, l-3′-amino-3′-deoxy-N⁶-dimethyladenosine (l-3′-ADMdA) 1, previously synthesized in our laboratory, and the novel l-3′-amino-3′-deoxy-N⁶-methyladenosine-5′-N-methyluronamide (l-3′-AM-MECA) 2 were evaluated in an ischemia/reperfusion model on Langendorff perfused mouse heart. l-3′-ADMdA 1 was found to enhance functional recovery from ischemia (32.2 ± 3.7 cm H₂O/s % rate pressure product, compared to 21.3 ± 1.4 for the control and 30.7 ± 3.4 for adenosine) and increase the time to onset of ischemic contracture (14.5 ± 0.9 min, compared to 10.5 ± 1.0 min for the control and 13.6 ± 0.6 min for adenosine) comparable to adenosine. Consistent with the functional recovery data, decreased infarction area was seen in the case of 1 (19.1 ± 8.4, compared to 40.5 ± 7.2% for the control and 11.5 ± 2.1% for adenosine). In contrast, l-3′-AM-MECA 2 did not show significant functional recovery, increased onset of contracture, nor decreased infarction area compared to control. Unlike adenosine, neither 1 nor 2 induced cardiac standstill in mouse heart.     

Structural changes that occur upon photolysis of the Fe(II)a3–CO complex in the cytochrome ba3-oxidase of Thermus thermophilus: A combined X-ray crystallographic and infrared spectral study demonstrates CO binding to CuB

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a₃ moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba₃-oxidase from Thermus thermophilus, determined at ~ 2.8–3.2 Å resolution, reveal a Fe–C distance of ~ 2.0 Å, a Cu–O distance of 2.4 Å and a Fe–C–O angle of ~ 126°. Upon photodissociation at 100 K, X-ray structures indicate loss of Fe_a3–CO and appearance of Cu_B–CO having a Cu–C distance of ~ 1.9 Å and an O–Fe distance of ~ 2.3 Å. Absolute FTIR spectra recorded from single crystals of reduced ba₃–CO that had not been exposed to X-ray radiation, showed several peaks around 1975 cm^? 1; after photolysis at 100 K, the absolute FTIR spectra also showed a significant peak at 2050 cm^? 1. Analysis of the ‘light’ minus ‘dark’ difference spectra showed four very sharp CO stretching bands at 1970 cm^? 1, 1977 cm^? 1, 1981 cm^? 1, and 1985 cm^? 1, previously assigned to the Fe_a3–CO complex, and a significantly broader CO stretching band centered at ~ 2050 cm^? 1, previously assigned to the CO stretching frequency of Cu_B bound CO. As expected for light propagating along the tetragonal axis of the P4₃2₁2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba₃ crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO₂ at 2337 cm^? 1 and one from traces of CO at 2133 cm^? 1; while bands associated with CO bound to either Fe_a3 or to Cu_B in “light” minus “dark” FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2 Å and FTIR spectra support the long-held position that photolysis of Fe_a3–CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu_B atom; Fe_a3 and Cu_B ligation, at the resolutions reported here, are otherwise unaltered. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Synthesis and pharmacological characterization of novel xanthine carboxylate amides as A2A adenosine receptor ligands exhibiting bronchospasmolytic activity

The carboxylate amides of 8-phenyl-1,3-dimethylxanthine described herein represent a new series of selective ligands of the adenosine A_2A receptors exhibiting bronchospasmolytic activity. The effects of location of 8-phenyl substitutions on the adenosine receptor (AR) binding affinities of the newly synthesized xanthines have also been studied. The compounds displayed moderate to potent binding affinities toward various adenosine receptor subtypes when evaluated through radioligand binding studies. However, most of the compounds showed the maximum affinity for the A_2A subtype, some with high selectivity versus all other subtypes. Xanthine carboxylate amide 13b with a diethylaminoethylamino moiety at the para-position of the 8-phenylxanthine scaffold was identified as the most potent A_2A adenosine receptor ligand with K_i = 0.06 μM. Similarly potent and highly A_2A-selective are the isovanillin derivatives 16a and 16d. In addition, the newly synthesized xanthine derivatives showed good in vivo bronchospasmolytic activity when tested in guinea pigs.     

Comparison of two type IV hyperthermophilic adenylyl cyclases characterizations from the archaeon Pyrococcus furiosus

In this paper, two genes that encoded two soluble type IV adenylyl cyclases (AC) from the hyperthermophilic archaeon Pyrococcus furiosus (PFAC I and PFAC II) were cloned and expressed in Escherichia coli (E. coli) BL21 (DE3). Amino acid sequence analysis of the two enzymes showed 29% homology. PFAC I and PFAC II were both Mn²⁺ activated enzyme. They were purified by His-trap chromatography and had a specific activity of 3.1 × 10³ U/mg at pH 10.0, 95 °C (PFAC I) and 2.0 × 10³ U/mg at pH 11.0, 95 °C (PFAC II), respectively. The K_m and k_cat of PFAC I was 1.38 mM and 1.11 s⁻¹. The K_m and k_cat of PFAC II was 1.44 mM and 0.80 s⁻¹. The thermostability of PFAC I and PFAC II were higher than the soluble type IV adenylyl cyclases from Yersinia pestis (YpAC-IV). All of the properties suggested that these two adenylyl cyclases may be useful for the industrial producing of cyclic adenosine 3′,5′-monophosphate (cAMP).     

Characterization of two peptides isolated from the venom of social wasp Chartergellus communis (Hymenoptera: Vespidae): Influence of multiple alanine residues and C-terminal amidation on biological effects

Chatergellus communis is a wasp species endemic to the neotropical region and its venom constituents have never been described. In this study, two peptides from C. communis venom, denominated Communis and Communis-AAAA, were chemically and biologically characterized. In respect to the chemical characterization, the following amino acid sequences and molecular masses were identified:Communis: Ile-Asn-Trp-Lys-Ala-Ile-Leu-Gly-Lys-Ile-Gly-Lys-COOH (1340.9 Da)Communis-AAAA: Ile-Asn-Trp-Lys-Ala-Ile-Leu-Gly-Lys-Ile-Gly-Lys-Ala-Ala-Ala-Ala-Val-Xle-NH₂ (1836.3 Da).Furthermore, their biological effects were compared, accounting for the differences in structural characteristics between the two peptides. To this end, three biological assays were performed in order to evaluate the hyperalgesic, edematogenic and hemolytic effects of these molecules. Communis-AAAA, unlike Communis, showed a potent hemolytic activity with EC₅₀ = 142.6 μM. Moreover, the highest dose of Communis-AAAA (2nmol/animal) induced hyperalgesia in mice. On the other hand, Communis (10nmol/animal) was able to induce edema but did not present hemolytic or hyperalgesic activity. Although both peptides have similarities in linear structures, we demonstrated the distinct biological effects of Communis and Communis-AAAA. This is the first study with Chartegellus communis venom, and both Communis and Communis-AAAA are unpublished peptides.     

NAD-based inhibitors with anticancer potential

Three classes of novel inhibitors of inosine monophosphate dehydrogenase have been prepared and their anti-proliferative properties were evaluated against several cancer cell lines.(1) Mycophenolic adenine dinucleotide analogues (8–13) containing a substituent at the C2 of adenine ring were found to be potent inhibitors of IMPDH (K_i’s in range of 0.6–82 nM) and sub-μM inhibitors of leukemic K562 cell proliferation. (2) Mycophenolic adenosine (d and l) esters (20 and 21) showed a potent inhibition of IMPDH2 (K_i = 102 and K_i = 231 nM, respectively) and inhibition of K562 cell growth (IC₅₀ = 0.5 and IC₅₀ = 1.6 μM). These compounds serve both as inhibitors of the enzyme and as a depot form of mycophenolic acid. The corresponding amide analogue 22, also a potent inhibitor of IMPDH (K_i = 84 nM), did not inhibit cancer cell proliferation. (3) Mycophenolic-(l)- and (d)-valine adenine di-amide derivatives 25 (K_i = 9 nM) and 28 (K_i = 3 nM) were found to be very potent enzymatically, but did not inhibit proliferation of cancer cells.     

Stereochemical modification of geminal dialkyl substituents on pantothenamides alters antimicrobial activity

Pantothenamides are N-substituted pantothenate derivatives which are known to exert antimicrobial activity through interference with coenzyme A (CoA) biosynthesis or downstream CoA-utilizing proteins. A previous report has shown that replacement of the ProR methyl group of the benchmark N-pentylpantothenamide with an allyl group (R-anti configuration) yielded one of the most potent antibacterial pantothenamides reported so far (MIC of 3.2 μM for both sensitive and resistant Staphylococcus aureus). We describe herein a synthetic route for accessing the corresponding R-syn diastereomer using a key diastereoselective reduction with Baker’s yeast, and report on the scope of this reaction for modified systems. Interestingly, whilst the R-anti diastereomer is the only one to show antibacterial activity, the R-syn isomer proved to be significantly more potent against the malaria parasite (IC₅₀ of 2.4 ± 0.2 μM). Our research underlines the striking influence that stereochemistry has on the biological activity of pantothenamides, and may find utility in the study of various CoA-utilizing systems.     

Towards understanding the unbound state of drug compounds: Implications for the intramolecular reorganization energy upon binding

There has been an explosion of structural information for pharmaceutical compounds bound to biological targets, but the conformations and dynamics of compounds free in solution are poorly characterized, if at all. Yet, knowledge of the unbound state is essential to understand the fundamentals of molecular recognition, including the much debated conformational intramolecular reorganization energy of a compound upon binding (ΔE_Reorg). Also, dependable observation of the unbound compounds is important for ligand-based drug discovery, e.g. with pharmacophore modelling. Here, these questions are addressed with long (⩾0.5 μs) state-of-the-art molecular dynamics (MD) simulations of 26 compounds (including 7 approved drugs) unbound in explicit solvent. These compounds were selected to be chemically diverse, with a range of flexibility, and good quality bioactive X-ray structures. The MD-simulated free compounds are compared to their bioactive structure and conformers generated with ad hoc sampling in vacuo or with implicit generalized Born (GB) aqueous solvation models. The GB conformational models clearly depart from those obtained in explicit solvent, and suffer from conformational collapse almost as severe as in vacuo. Thus, the global energy minima in vacuo or with GB are not suitable representations of the unbound state, which can instead be extensively sampled by MD simulations. Many, but not all, MD-simulated compounds displayed some structural similarity to their bioactive structure, supporting the notion of conformational pre-organization for binding. The ligand–protein complexes were also simulated in explicit solvent, to estimate ΔE_Reorg as an enthalpic difference ΔH_Reorg between the intramolecular energies in the bound and unbound states. This fresh approach yielded ΔH_Reorg values ⩽ 6 kcal/mol for 18 out of 26 compounds. For three particularly polar compounds 15 ⩽ ΔH_Reorg ⩽ 20 kcal/mol, supporting the notion that ΔH_Reorg can be substantial. Those large ΔH_Reorg values correspond to a redistribution of electrostatic interactions upon binding. Overall, the study illustrates how MD simulations offer a promising avenue to characterize the unbound state of medicinal compounds.