Design,synthesis, in vitro and in vivo evaluation,and structure-activity relationship (SAR) discussion of novel dipeptidyl boronic acid proteasome inhibitors as orally available anti-cancer agents for the treatment of multiple myeloma and mechanism studies

A series of novel dipeptidyl boronic acid inhibitors of 20S proteasome were designed and synthesized. Aliphatic groups at R¹ position were designed for the first time to fully understand the SAR (structure–activity relationship). Among the screened compounds, novel inhibitor 5c inhibited the CT-L (chymotrypsin-like) activity with IC₅₀ of 8.21?nM and the MM (multiple myeloma) cells RPMI8226, U266B and ARH77 proliferations with the IC₅₀ of 8.99, 6.75 and 9.10?nM, respectively, which showed similar in vitro activities compared with the compound MLN2238 (biologically active form of marketed MLN9708). To investigate the oral availability, compound 5c was esterified to its prodrug 6a with the enzymatic IC₅₀ of 6.74?nM and RPMI8226, U266B and ARH77 cell proliferations IC₅₀ of 2.59, 4.32 and 3.68?nM, respectively. Furthermore, prodrug 6a exhibited good pharmacokinetic properties with oral bioavailability of 24.9%, similar with MLN9708 (27.8%). Moreover, compound 6a showed good microsomal stabilities and displayed stronger in vivo anticancer efficacy than MLN9708 in the human ARH77 xenograft mouse model. Finally, cell cycle results showed that compound 6a had a significant inhibitory effect on CT-L and inhibited cell cycle progression at the G2M stage.     

Probing the self-assembly and the accompanying structural changes of hydrophobin SC3 on a hydrophobic surface by mass spectrometry

The fungal class I hydrophobin SC3 self-assembles into an amphipathic membrane at hydrophilic-hydrophobic interfaces such as the water-air and water-Teflon interface. During self-assembly, the water-soluble state of SC3 proceeds via the intermediate alpha-helical state to the stable end form called the beta-sheet state. Self-assembly of the hydrophobin at the Teflon surface is arrested in the alpha-helical state. The beta-sheet state can be induced at elevated temperature in the presence of detergent. The structural changes of SC3 were monitored by various mass spectrometry techniques. We show that the so-called second loop of SC3 (C39-S72) has a high affinity for Teflon. Binding of this part of SC3 to Teflon was accompanied by the formation of alpha-helical structure and resulted in low solvent accessibility. The solvent-protected region of the second loop extended upon conversion to the beta-sheet state. In contrast, the C-terminal part of SC3 became more exposed to the solvent. The results indicate that the second loop of class I hydrophobins plays a pivotal role in self-assembly at the hydrophilic-hydrophobic interface. Of interest, this loop is much smaller in case of class II hydrophobins, which may explain the differences in their assembly.     

Enhanced antimicrobial activity of novel synthetic peptides derived from vejovine and hadrurin

Background

Microbial antibiotic resistance is a challenging medical problem nowadays. Two scorpion peptides displaying antibiotic activity: hadrurin and vejovine were taken as models for the design of novel shorter peptides with similar activity.

Methods

Using the standard Fmoc-based solid phase synthesis technique of Merrifield twelve peptides (18 to 29 amino acids long) were synthesized, purified and assayed against a variety of multi-drug resistant Gram-negative bacteria from clinical isolates. Hemolytic and antiparasitic activities of the peptides and their possible interactions with eukaryotic cells were verified. Release of the fluorophore calcein from liposomes treated with these peptides was measured.

Results

A peptide with sequence GILKTIKSIASKVANTVQKLKRKAKNAVA), and three analogs: Δ(Α29), Δ(K12-Q18; Ν26−Α29), and K4N Δ(K12-Q18; Ν26−Α29) were shown to inhibit the growth of Gram-negative (E. coli ATCC25922) and Gram-positive bacteria (S. aureus), as well as multi-drug resistant (MDR) clinical isolated. The antibacterial and antiparasitic activities were found with peptides at 0.78 to 25 μM and 5 to 25 μM concentration, respectively. These peptides have low cytotoxic and hemolytic activities at concentrations significantly exceeding their minimum inhibitory concentrations (MICs), showing values between 40 and 900 μM for their EC₅₀, compared to the parent peptides vejovine and hadrurin that at the same concentration of their MICs lysed more than 50% of human erythrocytes cells.

Conclusions

These peptides promise to be good candidates to combat infections caused by Gram-negative bacteria from nosocomial infections.

General significance

Our results confirm that well designed synthetic peptides can be an alternative for solving the lack of effective antibiotics to control bacterial infections.     

Design,synthesis and biological activity of 3-oxoamino-benzenesulfonamides as selective and reversible LSD1 inhibitors

Lysine specific demethylase 1 (LSD1) is a flavin-dependent amine oxidase that selectively removes one or two methyl groups from H3 at Lys4 and is recognized as a promising therapeutic target for cancer and other diseases. Here, a series of 3-oxoamino-benzenesulfonamides were synthesized and evaluated for their inhibitory activity against LSD1. Compounds 7b and 7h showed the most potent inhibition with the IC₅₀ values of 9.5 and 6.9 μM, respectively. Furthermore, the LSD1 inhibition of 7b and 7h were reversible and selective. Docking study presented the possible binding mode between 7b, 7h and the LSD1 active site. Taken together, 3-oxoamino-benzenesulfonamides may represent a new class of reversible LSD1 inhibitors and 7b and 7h were two hit compounds deserved further structural optimization.     

Synthesis and evaluation of Re/99mTc(I) complexes bearing a somatostatin receptor-targeting antagonist and labeled via a novel [N,S,O] clickable bifunctional chelating agent

The somatostatin receptor subtype 2 (SSTR2) is often highly expressed on neuroendocrine tumors (NETs), making it a popular in vivo target for diagnostic and therapeutic approaches aimed toward management of NETs. In this work, an antagonist peptide (sst₂-ANT) with high affinity for SSTR2 was modified at the N-terminus with a novel [N,S,O] bifunctional chelator (2) designed for tridentate chelation of rhenium(I) and technetium(I) tricarbonyl cores, [Re(CO)₃]⁺ and [^99mTc][Tc(CO)₃]⁺. The chelator-peptide conjugation was performed via a Cu(I)-assisted click reaction of the alkyne-bearing chelator (2) with an azide-functionalized sst₂-ANT peptide (3), to yield NSO-sst₂-ANT (4). Two synthetic methods were used to prepare Re-4 at the macroscopic scale, which differed based on the relative timing of the click conjugation to the [Re(CO)₃]⁺ complexation by 2. The resulting products demonstrated the expected molecular mass and nanomolar in vitro SSTR2 affinity (IC₅₀ values under 30?nM, AR42J cells, [¹²⁵I]iodo-Tyr¹¹-somatostatin-14 radioligand standard). However, a difference in their HPLC retention times suggested a difference in metal coordination modes, which was attributed to a competing N-triazole donor ligand formed during click conjugation. Surprisingly, the radiotracer scale reaction of [^99mTc][Tc(OH₂)₃(CO)₃]⁺ (^99mTc; t_½?=?6?h, 141?keV γ) with 4 formed a third product, distinct from the Re analogues, making this one of the unusual cases in which Re and Tc chemistries are not well matched. Nevertheless, the [^99mTc]Tc-4 product demonstrated excellent in vitro stability to challenges by cysteine and histidine (≥98% intact through 24?h), along with 75% stability in mouse serum through 4?h. In vivo biodistribution and microSPECT/CT imaging studies performed in AR42J tumor-bearing mice revealed improved clearance of this radiotracer in comparison to a similar [^99mTc][Tc(CO)₃]-labeled sst₂-ANT derivative previously studied. Yet despite having adequate tumor uptake at 1?h (4.9% ID/g), tumor uptake was not blocked by co-administration of a receptor-saturating dose of SS-14. Aimed toward realignment of the Re and Tc product structures, future efforts should include distancing the alkyne group from the intended donor atoms of the chelator, to reduce the coordination options available to the [M(CO)₃]⁺ core (M?=?Re, ^99mTc) by disfavoring involvement of the N-triazole.     

Skin fibroblast metabolomic profiling reveals that lipid dysfunction predicts the severity of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a triplet guanine-adenine-adenine (GAA) repeat expansion in intron 1 of the FXN gene, which leads to decreased levels of the frataxin protein. Frataxin is involved in the formation of iron-sulfur (Fe-S) cluster prosthetic groups for various metabolic enzymes. To provide a better understanding of the metabolic status of patients with FRDA, here we used patient-derived fibroblast cells as a surrogate tissue for metabolic and lipidomic profiling by liquid chromatography-high resolution mass spectrometry. We found elevated HMG-CoA and β-hydroxybutyrate-CoA levels, implying dysregulated fatty acid oxidation, which was further demonstrated by elevated acyl-carnitine levels. Lipidomic profiling identified dysregulated levels of several lipid classes in FRDA fibroblast cells when compared with non-FRDA fibroblast cells. For example, levels of several ceramides were significantly increased in FRDA fibroblast cells; these results positively correlated with the GAA repeat length and negatively correlated with the frataxin protein levels. Furthermore, stable isotope tracing experiments indicated increased ceramide synthesis, especially for long-chain fatty acid-ceramides, in FRDA fibroblast cells compared with ceramide synthesis in healthy control fibroblast cells. In addition, PUFA-containing triglycerides and phosphatidylglycerols were enriched in FRDA fibroblast cells and negatively correlated with frataxin levels, suggesting lipid remodeling as a result of FXN deficiency. Altogether, we demonstrate patient-derived fibroblast cells exhibited dysregulated metabolic capabilities, and their lipid dysfunction predicted the severity of FRDA, making them a useful surrogate to study the metabolic status in FRDA.     

Structure-function relationship of conotoxin lt14a, a potential analgesic with low cytotoxicity

A novel conotoxin lt14a containing 13 amino acid residues with an amidated C-terminus derived from Conus litteratus, belongs to C-C-C-C cysteine pattern. As the smallest peptide of conotoxin framework 14, lt14a could inhibit nicotinic acetylcholine receptor and suppress pain. To elucidate structure-function relationship, we determine the solution structure by NMR and find that lt14a comprises a short duple β-strand region and β-turn motif. An analog [K7A]-lt14a of Ala substitution for Lys in position 7 is designed. Interestingly, [K7A]-lt14a exhibits higher activity than lt14a as long-lasting analgesic in the hotplate pain model in mice. Additionally, MTT assay reveals that the two peptides have low toxicity to human cells. The studies suggest that positively charged residue may not be involved in the blocking mechanism. However, due to the Ala substitution, hydrophobic residues’ patch expansion strengthens the binding ability. A hypothesis is given that in conotoxin lt14a, hydrophobic residues rather than charged residues play a key role during target binding.     

Synthesis and characterization of de novo designed peptides modelling the binding sites of [4Fe-4S] clusters in photosystem I

Photosystem I (PS I) converts the energy of light into chemical energy via transmembrane charge separation. The terminal electron transfer cofactors in PS I are three low-potential [4Fe-4S] clusters named F_X, F_A and F_B, the last two are bound by the PsaC subunit. We have modelled the F_A and F_B binding sites by preparing two apo-peptides (maquettes), sixteen amino acids each. These model peptides incorporate the consensus [4Fe-4S] binding motif along with amino acids from the immediate environment of the iron-sulfur clusters F_A and F_B. The [4Fe-4S] clusters were successfully incorporated into these model peptides, as shown by optical absorbance, EPR and Mössbauer spectroscopies. The oxidation-reduction potential of the iron-sulfur cluster in the F_A-maquette is − 0.44 ± 0.03 V and in the F_B-maquette is − 0.47 ± 0.03 V. Both are close to that of F_A and F_B in PS I and are considerably more negative than that observed for other [4Fe-4S] model systems described earlier (Gibney, B. R., Mulholland, S. E., Rabanal, F., and Dutton, P. L. Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 15041-15046). Our optical data show that both maquettes can irreversibly bind to PS I complexes, where PsaC-bound F_A and F_B were removed, and possibly participate in the light-induced electron transfer reaction in PS I.