Ring size of somatostatin analogues (ODT-8) modulates receptor selectivity and binding affinity

The synthesis, biological testing, and NMR studies of several analogues of H-c[Cys (3)-Phe (6)-Phe (7)-DTrp (8)-Lys (9)-Thr (10)-Phe (11)-Cys (14)]-OH (ODT-8, a pan-somatostatin analogue, 1) have been performed to assess the effect of changing the stereochemistry and the number of atoms in the disulfide bridge on binding affinity. Cysteine at positions 3 and/or 14 (somatostatin numbering) were/was substituted with d-cysteine, norcysteine, D-norcysteine, homocysteine, and/or D-homocysteine. The 3D structure analysis of selected partially selective, bioactive analogues (3, 18, 19, and 21) was carried out in dimethylsulfoxide. Interestingly and not unexpectedly, the 3D structures of these analogues comprised the pharmacophore for which the analogues had the highest binding affinities (i.e., sst 4 in all cases).

Ring size in octreotide amide modulates differently agonist versus antagonist binding affinity and selectivity

H-DPhe (2)-c[Cys (3)-Phe (7)-DTrp (8)-Lys (9)-Thr (10)-Cys (14)]-Thr (15)-NH2 (1) (a somatostatin agonist, SRIF numbering) and H-Cpa (2)-c[DCys (3)-Tyr (7)-DTrp (8)-Lys (9)-Thr (10)-Cys (14)]-Nal (15)-NH2 (4) (a somatostatin antagonist) are based on the structure of octreotide that binds to three somatostatin receptor subtypes (sst 2/3/5) with significant binding affinity. Analogues of 1 and 4 were synthesized with norcysteine (Ncy), homocysteine (Hcy), or D-homocysteine (DHcy) at positions 3 and/or 14. Introducing Ncy at positions 3 and 14 constrained the backbone flexibility, resulting in loss of binding affinity at all sst s. The introduction of Hcy at positions 3 and 14 improved selectivity for sst 2 as a result of significant loss of binding affinity at the other sst s. Substitution by DHcy at position 3 in the antagonist scaffold (5), on the other hand, resulted in a significant loss of binding affinity at sst 2 and sst 3 as compared to the different affinities of the parent compound (4). The 3D NMR structures of the analogues in dimethylsulfoxide are consistent with the observed binding affinities.

New pansomatostatin ligands and their chelated versions: affinity profile, agonist activity, internalization, and tumor targeting

PURPOSE: Somatostatin receptor (sst) targeting is an established method to image and treat sst-positive tumors. Particularly, neuroendocrine tumors express the receptor subtype 2 in high density, but sst1, sst3, sst4, and sst5 are also expressed to some extent in different human tumors. Currently used targeting peptides mainly have sst2 affinity. We aimed at developing (radio)peptides that bind with high affinity to all receptor subtypes. EXPERIMENTAL DESIGN: Carbocyclic octapeptides were coupled with macrocyclic chelators for radiometal labeling. Affinity, internalization, and agonist potencies were determined on sst1- to sst5-expressing cell lines. Biodistribution was determined on nude mice bearing HEK-sst2 or AR4-2J and HEK-sst3 tumors. RESULTS: High affinity to all receptor subtypes was found. Y(III)-KE88 showed agonistic properties at all five sst receptor subtypes as it inhibits forskolin-stimulated cyclic AMP production. Surprisingly, very low or even absent sst2 receptor internalization was found compared with currently clinically established octapeptides, whereas the sst3 internalization was very efficient. Biodistribution studies of [(111)In]KE88 and [(67)Ga]KE88/[(68)Ga]KE88 reflected the in vitro data. In nude mice with s.c. implanted sst2 (HEK-sst2, AR4-2J)-expressing and sst3 (HEK-sst3)-expressing tumors, high and persistent uptake was found in sst3-expressing tumors, whereas the uptake in the sst2-expressing tumors was lower and showed fast washout. The kidney uptake was high but blockable by coinjection of lysine. CONCLUSION: This peptide family shows pansomatostatin potency. As radiopeptides, they are the first to show a full pansomatostatin profile. Despite some drawback, they should be useful for imaging sst2-expressing tumors with short-lived radiometals, such as (68)Ga, at early time points and for sst3-expressing tumors at later time points with longer-lived radiometals, such as (64)Cu or (86)Y.

Novel sst5-selective somatostatin dicarba-analogues: synthesis and conformation-affinity relationships

We describe synthesis, conformational studies, and binding to the five somatostatin receptors (sst 1-5) of a few analogues of the cyclic octapeptide octreotide (1), where the disulfide bridge was replaced by a dicarba group. These analogues were prepared by on-resin RCM of linear hepta-peptides containing two allylglycine residues; first- and second-generation Grubbs catalyst efficiencies were compared. The C=C bridge was hydrogenated via two different methods. Binding experiments showed that two analogues had good affinity and high selectivity for the sst5 receptor. Three-dimensional structures of the active analogues were determined by (1)H NMR spectroscopy. Conformation-affinity relationships confirmed the importance of D-Phe(2) orientation for sst2 affinity. Moreover, helical propensities well correlates with the peptide sst5 affinity. The presence of the bulky aromatic side chain of Tyr(Bzl)(10) favored the formation of a 3(10)-helix and enhanced the sst5 selectivity suppressing the sst2 affinity. Finally, a new pharmacophore model for the sst5 was developed.

closo-borane conjugated regulatory peptides retain high biological affinity: synthesis of closo-borane conjugated Tyr(3)-octreotate derivatives for BNCT

Despite the improvements in cancer therapy during the past years, high-grade gliomas and many other types of cancer are still extremely resistant to current forms of therapy. Boron neutron capture therapy (BNCT) provides a promising way to destroy cancer cells without damaging healthy tissue. However, BNCT in practice is still limited due to the lack of boron-containing compounds that selectively deliver boron to cancer cells. Since many neuroendocrine tumors show an overexpression of the somatostatin receptor, it was our aim to synthesize compounds that contain a large number of boron atoms and still show high affinity toward this transmembrane receptor. The synthetic peptide Tyr (3)-octreotate (TATE) was chosen as a high-affinity and internalizing tumor targeting vector (TTV). Novel boron cluster compounds, containing 10 or 20 boron atoms, were coupled to the N-terminus of TATE. The obtained affinity data demonstrate that the use of a spacer between TATE and the closo-borane moiety is the option to avoid a loss of biological affinity of closo-borane conjugated TATE. For the first time, it was shown that closo-borane conjugated regulatory peptides retain high biological affinity and selectivity toward their transmembrane tumor receptors. The results obtained and the improvement of spacer and boron building block chemistry may stimulate new directions for BNCT.

Effect of high-affinity anti-Pseudomonas aeruginosa lipopolysaccharide antibodies induced by immunization on the rate of Pseudomonas aeruginosa infection in patients with cystic fibrosis

Patients with cystic fibrosis (CF; N = 26) and with no prior history of infection with Pseudomonas aeruginosa were immunized with an octavalent O-polysaccharide-toxin A conjugate vaccine. During the next 4 years, 16 patients (61.5%) remained free of infection and 10 (38.5%) became infected. Total serum antilipopolysaccharide (LPS) antibody levels induced by immunization were comparable in infected and noninfected patients. In contrast, 12 of 16 noninfected versus 3 of 10 infected patients (p = 0.024) mounted and maintained a high-affinity anti-LPS antibody response. When compared retrospectively with the rate in a group of age- and gender-matched, nonimmunized, noncolonized patients with CF, the rate at which P. aeruginosa infections were acquired was significantly lower (p < or = 0.02) among all immunized versus nonimmunized patients during the first 2 years of observation. Subsequently, only those immunized patients who maintained a high-affinity anti-LPS antibody response had a significant reduction (p < or = 0.014) in the rate of infection during years 3 and 4. Smooth, typeable strains of P. aeruginosa predominated among immunized patients; rough, nontypeable strains were most frequently isolated from nonimmunized patients. Mucoid variants were isolated from one immunized patient versus six nonimmunized patients. These results indicate that the induction of a high-affinity P. aeruginosa anti-LPS antibody response can influence the rate of infection in patients with CF.

A 6'-Fluoro-Substituent in Bicyclo-DNA Increases Affinity to Complementary RNA Presumably by CF-HC Pseudohydrogen Bonds

The synthesis of a novel bicyclic thymidine analogue carrying a β-fluoro substituent at C6' (6'F-bcT) has been achieved. Key steps of the synthesis were an electrophilic fluorination/stereospecific hydrogenation sequence of a bicyclo sugar intermediate, followed by an N-iodo-succinimide-induced stereoselective nucleosidation. A corresponding phosphoramidite building block was then prepared and used for oligonucleotide synthesis. Tm measurements of oligonucleotides with single and double incorporations showed a remarkable stabilization of duplex formation particularly with RNA as complement without compromising pairing selectivity. Increases in Tm were in the range of +1-2 °C compared to thymidine and +1-3 °C compared to a standard bc-T residue. Structural investigations of the 6'F-bcT nucleoside by X-ray crystallography showed an in-line arrangement of the fluorine substituent with H6 of thymine, however, with a distance that is relatively long for a nonclassical CF-HC hydrogen bond. In contrast, structural investigations in solution by (1)H and (13)C NMR clearly showed scalar coupling of fluorine with H6 and C6 of the nucleobase, indicating the existence of at least weak electrostatic interactions. On the basis of these results, we put forward the hypothesis that these weak CF-HC6 electrostatic interactions increase duplex stability by orienting and partially freezing torsion angle χ of the 6'F-bcT nucleoside.

The SLC1 high-affinity glutamate and neutral amino acid transporter family

Glutamate transporters play important roles in the termination of excitatory neurotransmission and in providing cells throughout the body with glutamate for metabolic purposes. The high-affinity glutamate transporters EAAC1 (SLC1A1), GLT1 (SLC1A2), GLAST (SLC1A3), EAAT4 (SLC1A6), and EAAT5 (SLC1A7) mediate the cellular uptake of glutamate by the co-transport of three sodium ions (Na(+)) and one proton (H(+)), with the counter-transport of one potassium ion (K(+)). Thereby, they protect the CNS from glutamate-induced neurotoxicity. Loss of function of glutamate transporters has been implicated in the pathogenesis of several diseases, including amyotrophic lateral sclerosis and Alzheimer's disease. In addition, glutamate transporters play a role in glutamate excitotoxicity following an ischemic stroke, due to reversed glutamate transport. Besides glutamate transporters, the SLC1 family encompasses two transporters of neutral amino acids, ASCT1 (SLC1A4) and ASCT2 (SLC1A5). Both transporters facilitate electroneutral exchange of amino acids in neurons and/or cells of the peripheral tissues. Some years ago, a high resolution structure of an archaeal homologue of the SLC1 family was determined, followed by the elucidation of its structure in the presence of the substrate aspartate and the inhibitor d,l-threo-benzyloxy aspartate (d,l-TBOA). Historically, the first few known inhibitors of SLC1 transporters were based on constrained glutamate analogs which were active in the high micromolar range but often also showed off-target activity at glutamate receptors. Further development led to the discovery of l-threo-β-hydroxyaspartate derivatives, some of which effectively inhibited SLC1 transporters at nanomolar concentrations. More recently, small molecule inhibitors have been identified whose structures are not based on amino acids. Activators of SLC1 family members have also been discovered but there are only a few examples known.

Affinity retardation chromatography: characterization of the method and its application. The description of low affinity laminin self-interactions.

Affinity retardation chromatography (ARC), a method for the examination of low-affinity interactions, is mathematically described in order to characterize the method itself and to estimate binding coefficients of self-assembly domains of basement membrane protein laminin. Affinity retardation was determined by comparing the elutions on a "binding" and on a "nonreacting" column. It depends on the binding coefficient, the concentrations of both ligands, and the nonbinding elution position. Half maximal binding of the NH2-terminal domain of laminin B1-short arm to the A- and/or B2-short arms was estimated to occur at 10-17 microM for noncooperative and at < or = 3 microM for cooperative binding. A model of the laminin polymerization, postulating two levels of cooperative binding behavior, is described.

A low affinity penicillin-binding protein 2x is required for heteroresistance in Streptococcus pneumoniae

Heteroresistance to penicillin in Streptococcus pneumoniae is the ability of subpopulations to grow at a higher antibiotic concentration than expected from the minimal inhibitory concentration (MIC). This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary β-lactam resistance determinants, penicillin binding proteins PBP2b and PBP2x and secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from heteroresistant strain Spain(23F)2349 in non-heteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found that pbp2x, but not pbp2b or pbp1a alone, conferred heteroresistance to R6. However, a change of pbp2x expression is not observed and therefore expression does not correlate with an increased proportion of resistant subpopulations. Additional ciaR disruption mutants which have been described to mediate PBP-independent β-lactam resistance revealed no heteroresistant phenotype by PAP.We also showed, that the highly resistant subpopulations (HOM*) of transformants containing low affinity pbp2x undergo an increase in resistance upon selection on penicillin plates which partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins pstS, phoU, pstB and pstC in these highly resistant subpopulations.In conclusion, the presence of low affinity pbp2x enables certain pneumococcal colonies to survive in the presence of beta lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaption to antibiotic stress.

High affinity amino acid transporters specifically expressed in xylem parenchyma and developing seeds of Arabidopsis

Arabidopsis amino acid transporters (AAPs) show individual temporal and spatial expression patterns. A new amino acid transporter, AAP8 was isolated by reverse transcription-PCR. Growth and transport assays in comparison to AAP1-5 characterize AAP8 and AAP6 as high affinity amino acid transport systems from Arabidopsis. Histochemical promoter-beta-glucuronidase (GUS) studies identified AAP6 expression in xylem parenchyma, cells requiring high affinity transport due to the low amino acid concentration in xylem sap. AAP6 may thus function in uptake of amino acids from xylem. Histochemical analysis of AAP8 revealed stage-dependent expression in siliques and developing seeds. Thus AAP8 is probably responsible for import of organic nitrogen into developing seeds. The only missing transporter of the family AAP7 was nonfunctional in yeast with respect to amino acid transport, and expression was not detectable. Therefore, AAP6 and -8 are the only members of the family able to transport aspartate with physiologically relevant affinity. AAP1, -6 and -8 are the closest AAP paralogs. Although AAP1 and AAP8 originate from a duplicated region on chromosome I, biochemical properties and expression pattern diverged. Overlapping substrate specificities paired with individual properties and expression patterns point to specific functions of each of the AAP genes in nitrogen distribution rather than to mere redundancy.