Discovery of a new class of potent pyrrolo[3,4-c]quinoline-1,3-diones based inhibitors of human dihydroorotate dehydrogenase: Synthesis, pharmacological and toxicological evaluation.

Twenty N-substituted pyrrolo[3,4-c]quinoline-1,3-diones 3a-t were synthesized by a cyclization reaction of Pfitzinger's quinoline ester precursor with the selected aromatic, heteroaromatic and aliphatic amines. The structures of all derivatives were confirmed by IR, 1H NMR, 13C NMR and HRMS spectra, while their purity was determined using HPLC techniques. Almost all compounds were identified as a new class ofpotent inhibitors against hDHODH among which 3a and 3t were the most active ones with the same IC50 values of 0.11 µM, about seven times better than reference drug leflunomide. These two derivatives also exhibited very low cytotoxic effects toward healthy HaCaT cells and the optimal lipophilic properties with logP value of 1.12 and 2.07 respectively, obtained experimentally at physiological pH. We further evaluated the comparative differences in toxicological impact of the three most active compounds 3a, 3n and 3t and reference drug leflunomide. The rats were divided into five groups and were treated intraperitoneally, control group (group I) with a single dose of leflunomide (20 mg/kg) group II and the other three groups, III, IV and V were treated with 3a, 3n and 3t (20 mg/kg bw) separately. The investigation was performed in liver, kidney and blood by examining serum biochemical parameters and parameters of oxidative stress.

Synthesis and biological evaluation of new quinoline-4-carboxylic acid-chalcone hybrids as dihydroorotate dehydrogenase inhibitors.

Fourteen novel quinoline-4-carboxylic acid-chalcone hybrids were obtained via Claisen-Schmidt condensation and evaluated as potential human dihydroorotate dehydrogenase (hDHODH) inhibitors. The ketone precursor 2 was synthesized by the Pfitzinger reaction and used for further derivatization at position 3 of the quinoline ring for the first time. Six compounds showed better hDHODH inhibitory activity than the reference drug leflunomide, with IC50 values ranging from 0.12 to 0.58 µM. The bioactive conformations of the compounds within hDHODH were resolved by means of molecular docking, revealing their tendency to occupy the narrow tunnel of hDHODH within the N-terminus and to prevent ubiquinone as the second cofactor from easily approaching the flavin mononucleotide as a cofactor for the redox reaction within the redox site. The results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that 4d and 4h demonstrated the highest cytotoxic activity against the A375 cell line, with IC50 values of 5.0 and 6.8 µM, respectively. The lipophilicity of the synthesized hybrids was obtained experimentally and expressed as logD7.4 values at physiologicalpH while the solubility assay was conducted to define physicochemical characteristics influencing the ADMET properties.

Polyphosphazene-Based Nanocarriers for the Release of Camptothecin and Epirubicin.

The design and study of efficient polymer-based drug delivery systems for the controlled release of anticancer drugs is one of the pillars of nanomedicine. The fight against metastatic and invasive cancers demands therapeutic candidates with increased and selective toxicity towards malignant cells, long-term activity and reduced side effects. In this sense, polyphosphazene nanocarriers were synthesized for the sustained release of the anticancer drugs camptothecin (CPT) and epirubicin (EPI). Linear poly(dichloro)phosphazene was modified with lipophilic tocopherol or testosterone glycinate, with antioxidant and antitumor activity, and with hydrophilic Jeffamine M1000 to obtain different polyphosphazene nanocarriers. It allowed us to encapsulate the lipophilic CPT and the more hydrophilic EPI. The encapsulation process was carried out via solvent exchange/precipitation, attaining a 9.2-13.6 wt% of CPT and 0.3-2.4 wt% of EPI. CPT-loaded polyphosphazenes formed 140-200 nm aggregates in simulated body physiological conditions (PBS, pH 7.4), resulting in an 80-100-fold increase of CPT solubility. EPI-loaded polyphosphazenes formed 250 nm aggregates in an aqueous medium. CPT and EPI release (PBS, pH 7.4, 37 °C) was monitored for 202 h, being almost linear during the first 8 h. The slow release of testosterone and tocopherol was also sustained for 150 h in PBS (pH 7.4 and 6.0) at 37 °C. The co-delivery of testosterone or tocopherol and the anticancer drugs from the nanocarriers was expected. Cells of the human breast cancer cell line MCF-7 demonstrated good uptake of anticancer-drug-loaded nanocarriers after 6 h. Similarly, MCF-7 spheroids showed good uptake of the anticancer-drug-loaded aggregates after 72 h. Almost all anticancer-drug-loaded polyphosphazenes exhibited similar or superior toxicity against MCF-7 cells and spheroids when compared to raw anticancer drugs. Additionally, cell-cycle arrest in the G2/M phase was increased in response to the drug-loaded nanocarriers. Almost no toxicity of anticancer-drug-loaded aggregates against primary human lung fibroblasts was observed. Furthermore, the aggregates displayed no hemolytic activity, which is in contrast to the parent anticancer drugs. Consequently, synthesized polyphosphazene-based nanocarriers might be potential nanomedicines for chemotherapy.

Backbone distortions in lactam-bridged helical peptides.

Side-chain-to-side-chain cyclization is frequently used to stabilize the α-helical conformation of short peptides. In a previous study, we incorporated a lactam bridge between the side chains of Lys-i and Asp-i+4 in the nonapeptide 1Y, cyclo-(2,6)-(Ac-VKRLQDLQY-NH2 ), an artificial ligand of the inhibitor of DNA binding and cell differentiation (ID) protein with antiproliferative activity on cancer cells. Herein, we show that only the cyclized five-residue segment adopts a helical turn whereas the C-terminal residues remain flexible. Moreover, we present nine 1Y analogs arising from different combinations of hydrophobic residues (leucine, isoleucine, norleucine, valine, and tyrosine) at positions 1, 4, 7, and 9. All cyclopeptides except one build a lactam-bridged helical turn; however, residue-4 reveals less helix character than the neighboring Arg-3 and Gln-5, especially with residue-4 being isoleucine, valine, and tyrosine. Surprisingly, only two cyclopeptides exhibit helix propagation until the C-terminus, whereas the others share a remarkable outward tilting of the backbone carbonyl of the lactam-bridged Asp-6 (>40° deviation from the orientation parallel to the helix axis), which prevents the formation of the H-bond between Arg-3 CO and residue-7 NH: As a result, the propagation of the helix beyond the lactam-bridged sequence becomes unfavorable. We conclude that, depending on the amino-acid sequence, the lactam bridge between Lys-i and Asp-i+4 can stabilize a helical turn but deviations from the ideal helix geometry are possible: Indeed, besides the outward tilting of the backbone carbonyls, the residues per turn increased from 3.6 (typical of a regular α-helix) to 4.2, suggesting a partial helix unwinding.

Self-Assembled Silk Fibroin-Based Aggregates for Delivery of Camptothecin.

A water-soluble hydrolysate of silk fibroin (SF) (~30 kDa) was esterified with tocopherol, ergocalciferol, and testosterone to form SF aggregates for the controlled delivery of the anticancer drug camptothecin (CPT). Elemental analysis and 1H NMR spectroscopy showed a degree of substitution (DS) on SF of 0.4 to 3.8 mol %. Yields of 58 to 71% on vitamins- and testosterone-grafted SF conjugates were achieved. CPT was efficiently incorporated into the lipophilic core of SF aggregates using a dialysis-precipitation method, achieving drug contents of 6.3-8.5 wt %. FTIR spectra and DSC thermograms showed that tocopherol- and testosterone-grafted SF conjugates predominantly adopted a ß-sheet conformation. After the esterification of tyrosine residues on SF chains with the vitamin or testosterone, the hydrodynamic diameters almost doubled or tripled that of SF. The zeta potential values after esterification increased to about -30 mV, which favors the stability of aggregates in aqueous medium. Controlled and almost quantitative release of CPT was achieved after 6 days in PBS at 37 °C, with almost linear release during the first 8 h. MCF-7 cancer cells exhibited good uptake of CPT-loaded SF aggregates after 6 h, causing cell death and cell cycle arrest in the G2/M phase. Substantial uptake of the CPT-loaded aggregates into MCF-7 spheroids was shown after 3 days. Furthermore, all CPT-loaded SF aggregates demonstrated superior toxicity to MCF-7 spheroids compared with parent CPT. Blank SF aggregates induced no hemolysis at pH 6.2 and 7.4, while CPT-loaded SF aggregates provoked hemolysis at pH 6.2 but not at pH 7.4. In contrast, parent CPT caused hemolysis at both pH tested. Therefore, CPT-loaded SF aggregates are promising candidates for chemotherapy.

Low cytotoxic quinoline-4-carboxylic acids derived from vanillin precursors as potential human dihydroorotate dehydrogenase inhibitors.

Twenty novel 2-substituted quinoline-4-carboxylic acids bearing amide moiety were designed and synthesized by Doebner reaction. Human dihydroorotate dehydrogenase (hDHODH) was recognized as a biological target and all compounds were screened as potential hDHODH inhibitors in an enzyme inhibition assay. The prepared heterocycles were also evaluated for their cytotoxic effects on the healthy HaCaT cell line while lipophilic properties were considered on the basis of experimentally determined logD values at physiological pH. The most promising compound 5j, with chlorine at para-position of terminal phenyl ring, showed good hDHODH inhibitory activity, low cytotoxicity, and optimal lipophilicity. The bioactive conformation of 5j on the hDHODH, determined by means of molecular docking, revealed the compound's pharmacology and provide guidelines for further lead optimization.

Potent human dihydroorotate dehydrogenase inhibitory activity of new quinoline-4-carboxylic acids derived from phenolic aldehydes: Synthesis, cytotoxicity, lipophilicity and molecular docking studies.

A series of novel 2-substituted quinoline-4-carboxylic acids was synthesized by Doebner reaction starting from freely available protocatechuic aldehyde and vanillin precursors. Human dihydroorotate dehydrogenase (hDHODH) was recognised as a clear molecular target for these heterocycles. All compounds were also tested for their antiproliferative potential against three cancer cells (MCF-7, A549, A375) and one normal cell line (HaCaT) to evaluate the selective cytotoxicity. Quinoline derivatives 3f and 3g were identified as potent hDHODH inhibitors while 3k and 3l demonstrated high cytotoxic activity against MCF-7 and A375 cells and good selectivity. In addition, the logD7.4 values obtained by the experimental method were found to be in the range from -1.15 to 1.69. The chemical structures of all compounds were confirmed by IR, NMR and elemental analysis. The compounds pharmacology on the molecular level was revealed by means of molecular docking, highlighting the structural differences that distinguish highly active from medium and low active hDHODH inhibitors.

Polyphosphazene-based nanocarriers for the release of agrochemicals and potential anticancer drugs.

The synthesis and characterisation of novel polyphosphazene nanocarriers, based on hydrophilic polyalkylene oxide Jeffamine M1000 and hydrophobic steroids with a glycinate linker for pH-controlled release of diosgenin and two brassinosteroids (DI31 and S7) with agrochemical and potential anticancer activity, is hereby described. Polyphosphazenes carrying approximately 17 wt% of DI31 or S7 self-assembled in water to form 120-150 nm nanoaggregates, which showed an excellent plant growth effect on radish cotyledons due to sustained delivery of approximately 30% of the agrochemicals after 4 days. Cytotoxic evaluation showed that all polymers carrying steroids and Jeffamine M1000 resulted in strong to moderate toxicity to MCF-7 cancer cells and were non-toxic to primary human lung fibroblast cells at 0.1 to 0.025 mg mL-1. Thus, DI31 and S7 bearing polymers applied at 10-4 to 10-6 mg mL-1 for delivery of recommended DI31 or S7 quantities to crops should be harmless to humans. Particularly, DI31 and S7 bearing polymers with strong cytotoxicity on MCF-7 and non-toxicity on primary human lung fibroblasts, good cell uptake after 6 hours, proper hydrodynamic sizes between 100 and 200 nm, and slow sustained release of cytotoxic drugs (DI31, S7) in acidic conditions might potentiate their accumulation in cancer tissues with good antitumour effects and minor side effects. These results demonstrated that preparation of brassinosteroid bearing polymers is a promising strategy for the preparation of better agrochemicals with reduced pollutant impact on sustainable agriculture and potential anticancer formulations based on analogues of brassinosteroids.

An explorative study towards the chemical synthesis of the immunoglobulin G1 Fc CH3 domain.

Monoclonal antibodies, fusion proteins including the immunoglobulin fragment c (Ig Fc) CH2-CH3 domains, and engineered antibodies are prominent representatives of an important class of drugs and drug candidates, which are referred to as biotherapeutics or biopharmaceuticals. These recombinant proteins are highly heterogeneous due to their glycosylation pattern. In addition, enzyme-independent reactions, like deamidation, dehydration, and oxidation of sensitive side chains, may contribute to their heterogeneity in a minor amount. To investigate the biological impact of a spontaneous chemical modification, especially if found to be recurrent in a biotherapeutic, it would be necessary to reproduce it in a homogeneous manner. Herein, we undertook an explorative study towards the chemical synthesis of the IgG1 Fc CH3 domain, which has been shown to undergo spontaneous changes like succinimide formation and methionine oxidation. We used Fmoc-solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL) to test the accessibility of large fragments of the IgG1 Fc CH3 domain. In general, the incorporation of pseudoproline dipeptides improved the quality of the crude peptide precursors; however, sequences larger than 44 residues could not be achieved by standard stepwise elongation with Fmoc-SPPS. In contrast, the application of NCL with cysteine residues, which were either native or introduced ad hoc, allowed the assembly of the C-terminal IgG1 Fc CH3 sequence 371 to 450. The syntheses reported here show advantages and limitations of the chemical approaches chosen for the preparation of the synthetic IgG1 Fc CH3 domain and will guide future plans towards the synthesis of both the native and selectively modified full-length domain.

The Recombinant Inhibitor of DNA Binding Id2 Forms Multimeric Structures via the Helix-Loop-Helix Domain and the Nuclear Export Signal.

The inhibitor of DNA binding and cell differentiation 2 (Id2) is a helix-loop-helix (HLH) protein that acts as negative dominant regulator of basic-HLH transcription factors during development and in cancer. The structural properties of Id2 have been investigated so far by using synthetic or recombinant fragments reproducing single domains (N-terminus, HLH, C-terminus): the HLH domain tends to dimerize into a four-helix bundle, whereas the flanking regions are flexible. In this work, the intact protein was expressed in E. coli, solubilized from inclusion bodies with urea, purified and dissolved in water at pH~4. Under these conditions, Id2 was obtained with both cysteine residues disulfide-bonded to ß-mercaptoethanol that was present during the solubilization process. Moreover, it existed in a self-assembled state, in which the N-terminus remained highly flexible, while the HLH domain and, surprisingly, part of the C-terminus, which corresponds to the nuclear export signal (NES), both were involved in slowly tumbling, rigid structures. The protein oligomers also formed twisted fibrils that were several micrometers long and up to 80 nm thick. These results show that self-assembly decreases the backbone flexibility of those two protein regions (HLH and NES) that are important for interaction with basic-HLH transcription factors or for nucleocytoplasmic shuttling.

Reduction of cancer cell viability by synergistic combination of photodynamic treatment with the inhibition of the Id protein family.

The inhibitor of DNA binding and cell differentiation (Id) proteins are dominant negative regulators of the helix-loop-helix transcription factor family and play a key role during development as well as in vascular disorders and cancer. In fact, impairing the Id-protein activity in cancer cells reduces cell growth and even chemoresistance. Recently, we have shown that a synthetic Id-protein ligand (1Y) consisting of a cyclic nonapeptide can reduce the viability of the two breast cancer cell lines MCF-7 and T47D and of the bladder cancer cells T24 to about 50% at concentrations ≥100µM. Moreover, the cyclopeptide displays both proapoptotic and antiproliferative effects on MCF-7 cells. Herein, we show that the cyclopeptide does not induce cell death at the dose of 5µΜ, but it still inhibits MCF-7 and T24 cell proliferation, which correlates with an increased protein level of the cell-cycle regulator p27Kip1. Furthermore, 1Y-pretreated MCF-7, T47D, and T24 cells are more susceptible than untreated cells to the phototoxic effects of the three photosensitizers meta-tetra(hydroxyphenyl)chlorin, porfimer sodium, and hypericin, which are applied in photodynamic therapy (PDT). The combination of the Id-protein ligand with each of the light-activated photosensitizers shows synergistic effects on the reduction of cell viability. In conclusion, an Id-protein ligand with moderate cancer cell killing activity at concentrations ≥100µM can be applied at a 20-fold lower and barely toxic dose to raise the sensitivity of cancer cells towards phototoxicity associated with photodynamic treatment. This suggests the potential benefit of targeting the Id proteins in combined drug approaches for cancer therapy.

Targeting of a Helix-Loop-Helix Transcriptional Regulator by a Short Helical Peptide.

The Id proteins (Id1-4) are cell-cycle regulators that play a key role during development, in cancer and vascular disorders. They contain a conserved helix-loop-helix (HLH) domain that folds into a parallel four-helix bundle upon self- or hetero-association with basic-HLH transcription factors. By using such protein-protein interactions, the Id proteins inhibit cell differentiation and promote cell-cycle progression. Accordingly, their supporting role in cancer has been convincingly demonstrated, which makes these proteins interesting therapeutic targets. Herein we present a short peptide containing an (i,i+4)-lactam bridge and a hydrophobic (Φ) three-residue motif Φ(i)-Φ(i+3)-Φ(i+6), which adopts a helical conformation in water, shows Id protein binding in the low-micromolar range, penetrates into breast (MCF-7 and T47D) and bladder (T24) cancer cells, accumulates in the nucleus, and decreases cell viability to ∼50 %. Thus, this cyclopeptide is a promising scaffold for the development of Id protein binders that impair cancer cell viability.

Impact of the amino acid sequence on the conformation of side chain lactam-bridged octapeptides.

Synthetic helical peptides are valuable scaffolds for the development of modulators of protein-protein interactions involving helical motifs. Backbone-to-side chain or side chain-to-side chain constraints have been and still are intensively exploited to stabilize short α-helices. Very often, these constraints have been combined with backbone modifications induced by Cα-tetrasubstituted, ß-, or γ-amino acids, which facilitate the α-peptide or α/ß/γ-peptide adopting an α-helical conformation. In this work, we investigated the helical character of octapeptides that were cyclized by a Lys-Asp-(i,i + 4)-lactam bridge. We started with two sequences extracted from the helix-loop-helix region of the Id proteins, which are inhibitors of cell differentiation during development and in cancer. Nineteen analogs containing the lactam bridge at different positions and displaying different amino acid core triads (i + 1,2,3) as well as outer residues were prepared by solid-phase methodology. Their conformation in water and water/2,2,2-trifluoroethanol mixtures was investigated by circular dichroism (CD) spectroscopy. The cyclopeptides could be grouped in helix-prone and non-helix-prone structures. Both the amino acid core triad (i + 1,2,3) and the pendant residues positively or negatively affected the formation of a helical structure. Computational studies based on the NMR-derived helical structure of a cyclopeptide containing Aib at position (i + 2) of the triad were generally in agreement with the secondary structure propensity of the cyclopeptides observed by CD spectroscopy. In conclusion, the Lys-Asp-(i,i + 4)-lactam bridge may succeed or fail in the stabilization of short helices, depending on the primary structure. Moreover, computational methods may be valuable tools to discriminate helix-prone from non-helix-prone peptide-based macrolactams. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

The Id-protein family in developmental and cancer-associated pathways.

Inhibitors of DNA binding and cell differentiation (Id) proteins are members of the large family of the helix-loop-helix (HLH) transcription factors, but they lack any DNA-binding motif. During development, the Id proteins play a key role in the regulation of cell-cycle progression and cell differentiation by modulating different cell-cycle regulators both by direct and indirect mechanisms. Several Id-protein interacting partners have been identified thus far, which belong to structurally and functionally unrelated families, including, among others, the class I and II bHLH transcription factors, the retinoblastoma protein and related pocket proteins, the paired-box transcription factors, and the S5a subunit of the 26 S proteasome. Although the HLH domain of the Id proteins is involved in most of their protein-protein interaction events, additional motifs located in their N-terminal and C-terminal regions are required for the recognition of diverse protein partners. The ability of the Id proteins to interact with structurally different proteins is likely to arise from their conformational flexibility: indeed, these proteins contain intrinsically disordered regions that, in the case of the HLH region, undergo folding upon self- or heteroassociation. Besides their crucial role for cell-fate determination and cell-cycle progression during development, other important cellular events have been related to the Id-protein expression in a number of pathologies. Dysregulated Id-protein expression has been associated with tumor growth, vascularization, invasiveness, metastasis, chemoresistance and stemness, as well as with various developmental defects and diseases. Herein we provide an overview on the structural properties, mode of action, biological function and therapeutic potential of these regulatory proteins.