Effects of Conformational Changes in Peptide-CRM197 Conjugate Vaccines.

Conjugate vaccines prepared with the cross-reactive material 197 (CRM197) carrier protein have been successful in the clinic and are of great interest in the field of immunotherapy. One route to preparing peptide-CRM197 conjugate vaccines involves an activation-conjugation strategy, effectively coupling lysine residues on the protein to cysteine thiolate groups on the peptide of interest using a heterobifunctional linker as an activation agent. This method has been found to result in two distinct populations of conjugates, believed to be the result of a conformational change of CRM197 during preparation. This report explores the factors that lead to this conformational change, pointing to a model in which the unintentional alkylation of histidine-21 by the activating agent promotes the "opening" of the monomeric protein. This exposes a new set of lysine residues that are modified by additional activation agents. Subsequent peptide ligation to these sites results in the two conformers. This is the first time that a specific chemical modification is demonstrated to induce a defined conformational change for this carrier protein. Importantly, alternative conditions and reagents have been found to minimize this effect, improving the conformational homogeneity of peptide-CRM197 conjugates.

Systematic Investigation of EDC/sNHS-Mediated Bioconjugation Reactions for Carboxylated Peptide Substrates.

1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) bioconjugations have been utilized in preparing variants for medical research. While there have been advances in optimizing the reaction for aqueous applications, there has been limited focus toward identifying conditions and side reactions that interfere with product formation. We present a systematic investigation of EDC/N-hydroxysulfosuccinimide (sNHS)-mediated bioconjugations on carboxylated peptides and small proteins. We identified yet-to-be-reported side products arising from both the reagents and substrates. Model peptides used in this study illustrate particular substrates are more susceptible to side reactions than others. From our studies, we found that bioconjugations are more efficient with high concentrations of amine nucleophile but not sNHS. Performing bioconjugations on a model affibody protein show that the trends established with model peptides hold for more complex systems.

Discovery of N-(4-Fluoro-3-methoxybenzyl)-6-(2-(((2S,5R)-5-(hydroxymethyl)-1,4-dioxan-2-yl)methyl)-2H-tetrazol-5-yl)-2-methylpyrimidine-4-carboxamide. A Highly Selective and Orally Bioavailable Matrix Metalloproteinase-13 Inhibitor for the Potential Treatment of Osteoarthritis.

Matrix metalloproteinase-13 (MMP-13) is a zinc-dependent protease responsible for the cleavage of type II collagen, the major structural protein of articular cartilage. Degradation of this cartilage matrix leads to the development of osteoarthritis. We previously have described highly potent and selective carboxylic acid containing MMP-13 inhibitors; however, nephrotoxicity in preclinical toxicology species precluded development. The accumulation of compound in the kidneys mediated by human organic anion transporter 3 (hOAT3) was hypothesized as a contributing factor for the finding. Herein we report our efforts to optimize the MMP-13 potency and pharmacokinetic properties of non-carboxylic acid leads resulting in the identification of compound 43a lacking the previously observed preclinical toxicology at comparable exposures.

Does training of the nondominant upper extremity reduce the surgeon's muscular strain during laparoscopy?: Results from a randomized controlled trial.

INTRODUCTION: In laparoscopy, suboptimal ergonomics frequently lead to morbidity for surgeons. Physical complaints are more commonly reported on the dominant upper extremity. This may be the consequence of challenging laparoscopic tasks being easier to perform with the dominant side. The authors hypothesized that specific training of the nondominant upper extremity may equip this side better and lead to a more equal distribution of physical load. MATERIALS AND METHODS: Participants (medical doctors) were randomized to a 3-week training schedule or no training. The training program consisted of training the nondominant upper extremity. Participants were not allowed to train on a laparoscopic box or virtual reality trainer during the study period. Baseline and outcome measurements after 3 weeks were examined with the use of EMG measurements during a validated task on a laparoscopic box trainer. Muscle strain of the trapezius and deltoid muscles and effective alternation of brachioradial and abductor pollicis brevis muscles were used as outcome variables. RESULTS: In all, 26 participants were included. EMG analysis revealed that participants in both intervention and control groups showed a decrease in muscle strain of trapezius and deltoid muscles. However, there were no significant differences between groups. Those in the intervention group showed significantly better alternation in the brachioradial muscle. CONCLUSION: Training the nondominant upper extremity leads to better alternated use of lower-arm muscles during a validated box trainer task. Repeating the task after 3 weeks led to less muscle tension in the trapezius and deltoid muscles.

Enhanced physiological tremor deteriorates plantar flexor torque steadiness after bed rest.

This study evaluated the effectiveness of resistance training to preserve submaximal plantar flexor (PF) torque steadiness following 60 days of bed rest (BR). Twenty-two healthy male subjects underwent either BR only (CTR, n=8), or BR plus resistance training (RT, n=14). The magnitude of torque fluctuations during steady submaximal isometric PF contractions (20%, 40%, 60% and 80% of maximum) were assessed before and after BR. Across contraction intensities, torque fluctuations (coefficient of variation, CV) increased more (P<0.05) after BR for CTR (from 0.31±0.10 to 0.92±0.63; P<0.001), than for RT (from 0.30±0.09 to 0.54±0.27; P<0.01). A shift in the spectral content of torque fluctuations towards increased rhythmic activity between 6.5 and 20Hz was observed in CTR only (P<0.05). H-reflex amplitude (H(max)/M(max) ratio) declined across groups from 0.57±0.18 before BR to 0.44±0.14 following BR (P<0.01) without correlation to CV. The present study showed that increased torque fluctuation after BR resulted from enhanced physiological tremor. Resistance training prevented the spectral shift in isometric PF torque fluctuation and offset ∼50% of the decline in performance associated with long-term BR.

Motor unit characteristics in healthy subjects and those with postpoliomyelitis syndrome: a high-density surface EMG study.

The purpose of this study was to identify optimal ways to detect neurogenic changes with high-density surface electromyography (HD-sEMG). For this purpose, we searched for the variables that most clearly discriminated between postpoliomyelitis and healthy subjects. We obtained HD-sEMG from the quadriceps muscle at different force levels in nine subjects with postpoliomyelitis syndrome and in matched healthy controls. Single motor unit action potentials (MUAPs), extracted from the HD-sEMG signal and the raw signal itself, were analyzed. Areas under the curve of the extracted MUAP waveform, indicating motor unit size, perfectly separated both groups. Raw signal analysis showed significant differences between groups for the monopolarly recorded amplitude up to 60% of maximal force and for the level of interference at higher force levels (40-100% force). We conclude that with HD-sEMG it is possible to detect neurogenic motor unit changes noninvasively, both by analysis of the raw signal itself and by analysis of extracted single MUAPs. The diagnostic yield of the single MUAP analysis is clearly higher. These findings point toward applications for clinical practice and invite further studies exploring the diagnostic value of HD-sEMG.

Inhibitors of lipoprotein(a) assembly.

Compounds of the general structure A and B were investigated for their activity as lipoprotein(a), [Lp(a)], assembly (coupling) inhibitors. SAR around the amino acid derivatives (structure A) gave compound 14-6 as a potent coupling inhibitor. Oral dosing of compound 14-6 to Lp(a) transgenic mice and cymologous monkeys resulted in a>30% decrease in plasma Lp(a) levels after 1-2 weeks of treatment at 100 mg/kg/day.

Discovery of a simple picomolar inhibitor of cholesteryl ester transfer protein.

A novel series of substituted N-[3-(1,1,2,2-tetrafluoroethoxy)benzyl]-N-(3-phenoxyphenyl)-trifluoro-3-amino-2-propanols is described which potently and reversibly inhibit cholesteryl ester transfer protein (CETP). Starting from the initial lead 1, various substituents were introduced into the 3-phenoxyaniline group to optimize the relative activity for inhibition of the CETP-mediated transfer of [3H]-cholesteryl ester from HDL donor particles to LDL acceptor particles either in buffer or in human serum. The better inhibitors in the buffer assay clustered among compounds in which the phenoxy group was substituted at the 3, 4, or 5 positions. In general, small lipophilic alkyl, haloalkyl, haloalkoxy, and halogen moieties increased potency relative to 1, while analogues containing electron-donating or hydrogen bond accepting groups exhibited lower potency. Compounds with polar or strong electron-withdrawing groups also displayed lower potency. Replacement of the phenoxy ring in 1 with either simple aliphatic or cycloalkyl ethers as well as basic heteroaryloxy groups led to reduced potency. From the better compounds, a representative series 4a-i was prepared as the chirally pure R(+) enantiomers, and from these, the 4-chloro-3-ethylphenoxy analogue was identified as a potent inhibitor of CETP activity in buffer (4a, IC50 0.77 nM, 59 nM in human serum). The simple R(+) enantiomer 4a represents the most potent acyclic CETP inhibitor reported. The chiral synthesis and biochemical characterization of 4a are reported along with its preliminary pharmacological assessment in animals.

Chiral N,N-disubstituted trifluoro-3-amino-2-propanols are potent inhibitors of cholesteryl ester transfer protein.

A novel series of substituted N-benzyl-N-phenyl-trifluoro-3-amino-2-propanols are described that reversibly inhibit cholesteryl ester transfer protein (CETP). Starting with screening lead 22, various structural features were explored with respect to inhibition of the CETP-mediated transfer of [(3)H]cholesterol from high-density cholesterol donor particles to low-density cholesterol acceptor particles. The free hydroxyl group of the propanol was required for high potency, since acylation or alkylation reduced activity. High inhibitory potency was also associated with 3-ether moieties in the aniline ring, and the highest potencies were exhibited by 3-phenoxyaniline analogues. Activity was substantially reduced by oxidation or substitution in the methylene of the benzylic group, implying that the benzyl ring orientation was important for activity. In the benzylic group, substitution at the 3-position was preferred over either the 2- or the 4-positions. Highest potencies were observed with inhibitors in which the 3-benzylic substituent had the potential to adopt an out of plane orientation with respect to the phenyl ring. The best 3-benzylic substituents were OCF(2)CF(2)H (42, IC(50) 0.14 microM in buffer, 5.6 microM in human serum), cyclopentyl (39), 3-iso-propoxy (27), SCF(3) (67), and C(CF(3))(2)OH (36). Separation of 42 into its enantiomers unexpectedly showed that the minor R(+) enantiomer 1a was 40-fold more potent (IC(50) 0.02 microM in buffer, 0.6 microM in human serum) than the major S(-) enantiomer 1b, demonstrating that the R-chirality at the propanol 2-position is key to high potency in this series. The R(+) enantiomer 1a represents the first reported acyclic CETP inhibitor with submicromolar potency in plasma. A chiral synthesis of 1a is reported.