Hepatic Expression of the Na+-Taurocholate Cotransporting Polypeptide Is Independent from Genetic Variation.

The hepatic Na+-taurocholate cotransporting polypeptide NTCP/SLC10A1 is important for the uptake of bile salts and selected drugs. Its inhibition results in increased systemic bile salt concentrations. NTCP is also the entry receptor for the hepatitis B/D virus. We investigated interindividual hepatic SLC10A1/NTCP expression using various omics technologies. SLC10A1/NTCP mRNA expression/protein abundance was quantified in well-characterized 143 human livers by real-time PCR and LC-MS/MS-based targeted proteomics. Genome-wide SNP arrays and SLC10A1 next-generation sequencing were used for genomic analyses. SLC10A1 DNA methylation was assessed through MALDI-TOF MS. Transcriptomics and untargeted metabolomics (UHPLC-Q-TOF-MS) were correlated to identify NTCP-related metabolic pathways. SLC10A1 mRNA and NTCP protein levels varied 44-fold and 10.4-fold, respectively. Non-genetic factors (e.g., smoking, alcohol consumption) influenced significantly NTCP expression. Genetic variants in SLC10A1 or other genes do not explain expression variability which was validated in livers (n = 50) from The Cancer Genome Atlas. The identified two missense SLC10A1 variants did not impair transport function in transfectants. Specific CpG sites in SLC10A1 as well as single metabolic alterations and pathways (e.g., peroxisomal and bile acid synthesis) were significantly associated with expression. Inter-individual variability of NTCP expression is multifactorial with the contribution of clinical factors, DNA methylation, transcriptional regulation as well as hepatic metabolism, but not genetic variation.

An imidazole based H-Phe-Phe-NH2 peptidomimetic with anti-allodynic effect in spared nerve injury mice.

The dipeptide amide H-Phe-Phe-NH2 (1) that previously was identified as a ligand for the substance P 1-7 (SP1-7) binding site exerts intriguing results in animal models of neuropathic pain after central but not after peripheral administration. The dipeptide 1 is derived from stepwise modifications of the anti-nociceptive heptapeptide SP1-7 and the tetrapeptide endomorphin-2 that is also binding to the SP1-7 site. We herein report a strong anti-allodynic effect of a new H-Phe-Phe-NH2 peptidomimetic (4) comprising an imidazole ring as a bioisosteric element, in the spare nerve injury (SNI) mice model after peripheral administration. Peptidomimetic 4 was stable in plasma, displayed a fair membrane permeability and a favorable neurotoxic profile. Moreover, the effective dose (ED50) of 4 was superior as compared to gabapentin and morphine that are used in clinic.

Impact of N-methylation of the substance P 1-7 amide on anti-allodynic effect in mice after peripheral administration.

Substance P 1-7 (SP1-7, Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7) is the major bioactive metabolite formed after proteolytic degradation of the tachykinin substance P (SP). This heptapeptide often opposes the effects of the mother peptide. Hence, SP1-7 is having anti-inflammatory, anti-nociceptive and anti-hyperalgesic effects in experimental models. Despite all encouraging properties of SP1-7 its exact mode of action has not yet been elucidated which has hampered further development of this heptapeptide in drug discovery. Contrary to SP that mediates its biological activity via the NK-1 receptor, the N-terminal fragment SP1-7 acts through an unknown target that is distinct from all known opioid and tachykinin receptors. The SP1-7 amide 1 (Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-NH2) was previously shown to be superior to the endogenous SP1-7 in all experimental pain models where the two compounds were compared. Herein, we report that N-methylation scan of the backbone of the SP1-7 amide (1) results in peptides that are significantly less prone to undergo proteolysis in plasma from both mouse and human. However, with the two exceptions of the [MeLys3]SP1-7 amide (3) and the [MeGln5]SP1-7 amide (4), the peptides with a methyl group attached to the backbone are devoid of significant anti-allodynic effects after peripheral administration in the spared nerve injury (SNI) mouse model of neuropathic pain. It is suggested that the N-methylation does not allow these peptides to form the accurate bioactive conformations or interactions required for efficient binding to the macromolecular target. The importance of intact N-terminal Arg1 and C-terminal Phe7, anticipated to serve as address and message residues, respectively, for achieving the anti-allodynic effect is emphasized. Notably, the three heptapeptides: the SP1-7 amide (1), the [MeLys3]SP1-7 amide (3) amide and the [MeGln5]SP1-7 amide (4) are all considerably more effective in the SNI mouse model than gabapentin that is widely used in the clinic for treatment of neuropathic pain.

Variability in Mass Spectrometry-based Quantification of Clinically Relevant Drug Transporters and Drug Metabolizing Enzymes.

Many different methods are used for mass-spectrometry-based protein quantification in pharmacokinetics and systems pharmacology. It has not been established to what extent the results from these various methods are comparable. Here, we compared six different mass spectrometry-based proteomics methods by measuring the expression of clinically relevant drug transporters and metabolizing enzymes in human liver. Mean protein concentrations were in general quantified to similar levels by methods using whole tissue lysates. Methods using subcellular membrane fractionation gave incomplete enrichment of the proteins. When the enriched proteins were adjusted to levels in whole tissue lysates, they were on average 4-fold lower than those quantified directly in whole tissue lysates. The differences in protein levels were propagated into differences in predictions of hepatic clearance. In conclusion, caution is needed when comparing and applying quantitative proteomics data obtained with different methods, especially since membrane fractionation is common practice for protein quantification used in drug clearance predictions.

Importance of N- and C-terminal residues of substance P 1-7 for alleviating allodynia in mice after peripheral administration.

The heptapeptide SP1-7 (1, Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7) is the major bioactive metabolite formed after proteolytic processing of the neuropeptide substance P (SP, Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-Phe8-Gly9-Leu10-Met11-NH2). The heptapeptide 1 frequently exhibits opposite effects to those induced by SP, such as exerting antinociception, or attenuating thermal hyperalgesia and mechanical allodynia. The heptapeptide SP1-7 amide (2, Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-NH2) is often more efficacious than 1 in experimental pain models. We have now assessed the anti-allodynic outcome after systemic administration of 2 and a series of Ala-substituted and truncated analogues of 2, in the spared nerve injury (SNI) mice model and the results obtained were correlated with in vitro plasma stability and permeability measurements. It is herein demonstrated that an intact Arg1 in SP1-7 amide analogues is fundamental for retaining a potent in vivo effect, while Lys3 of 2 is less important. A displacement with Ala1 or truncation rendered the peptide analogues either inactive or with a significantly attenuated in vivo activity. Thus, the pentapeptide SP3-7 amide (7, t1/2=11.1 min) proven to be the major metabolite of 2, demonstrated an in vivo effect itself although considerably less significant than 2 in the SNI model. Intraperitoneal administration of 2 in a low dose furnished the most powerful anti-allodynic effect in the SNI model of all the analogous evaluated, despite a fast proteolysis of 2 in plasma (t1/2=6.4 min). It is concluded that not only the C-terminal residue, that we previously demonstrated, but also the N-terminal with its basic side chain, are important for achieving effective pain relief. This information is of value for the further design process aimed at identifying more drug-like SP1-7 amide related peptidomimetics with pronounced anti-allodynic effects.

Complete Knockout of Endogenous Mdr1 (Abcb1) in MDCK Cells by CRISPR-Cas9.

Madin-Darby canine kidney II cells transfected with one or several transport proteins are commonly used models to study drug transport. In these cells, however, endogenous transporters such as canine Mdr1/P-glycoprotein (Abcb1) complicate the interpretation of transport studies. The aim of this investigation was to establish a Madin-Darby canine kidney II cell line using CRISPR-Cas9 gene-editing technology to knock out endogenous canine Mdr1 (cMdr1) expression. CRISPR-Cas9-mediated Abcb1 homozygous disruption occurred at frequencies of around 20% and resulted in several genotypes. We selected 1 clonal cell line, cMdr1 KO Cl2, for further examination. Consistent with an on-target effect of CRISPR-Cas9 in specific regions of the endogenous canine Abcb1 gene, we obtained a cell clone with Abcb1 gene alterations and without any cMdr1 expression, as confirmed by genome sequencing and quantitative protein analysis. Functional studies of these cells, using digoxin and other prototypic MDR1 substrates, showed close to identical transport in the apical-to-basolateral and basolateral-to-apical directions, resulting in efflux ratios indistinguishable from unity.

Fast and sensitive total protein and Peptide assays for proteomic analysis.

The determination of total protein content is one of the most frequent analytical tasks in biochemistry and molecular biology. Here we evaluate measurements of tryptophan fluorescence (WF) for total protein determination in whole tissue lysates and for peptide quantification in protein digests. We demonstrate that the fluorescence spectrometry of tryptophan offers a simple, sensitive, and direct method for protein and peptide assays. The WF assay is fully compatible with SDS and other solutes that are commonly used for lysis of tissue and cells. We found that the content of tryptophan varies only a little between mouse tissues (1.16 ± 0.08% of total amino acids) and is similar in human cells (1.19 ± 0.06%). Therefore, free tryptophan can be used as a universal standard. We show that the assay can be carried out on a standard fluorescence spectrometer with cuvettes as well as in a 96-well format using a plate reader. The method is particularly suitable for determination of peptide content in diluted samples. Notably, the whole sample can be recovered after the measurement.

The impact of cyclopropane configuration on the biological activity of cyclopropyl-epothilones.

Two cis-12,13-cyclopropyl-epothilone B variants have been synthesized, differing only in the configuration of the stereocenters at C12 and C13. The syntheses were based on a common allylic alcohol intermediate that was converted into the corresponding diastereomeric hydroxymethyl-cyclopropanes by means of stereoselective Charette cyclopropanations. Macrocyclizations were accomplished through ring-closing metathesis (RCM). Substantial differences between the two compounds were found with regard to microtubule binding affinity, antiproliferative activity and their effects on the cellular microtubule network. While the analogue with the cyclopropane moiety oriented in a corresponding way to the epoxide configuration in natural epothilones was almost equipotent with epothilone A, the other was significantly less active. Based on these findings, natural epothilone-like activity of cis-fused 12,13-cyclopropyl-epothilone analogues is tightly linked to the natural orientation of the cyclopropane moiety.

MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool.

Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.

Diversity through semisynthesis: the chemistry and biological activity of semisynthetic epothilone derivatives.

Epothilones are myxobacterial natural products that inhibit human cancer cell growth through the stabilization of cellular microtubules (i.e., a "taxol-like" mechanism of action). They have proven to be highly productive lead structures for anticancer drug discovery, with at least seven epothilone-type agents having entered clinical trials in humans over the last several years. SAR studies on epothilones have included a large number of fully synthetic analogs and semisynthetic derivatives. Previous reviews on the chemistry and biology of epothilones have mostly focused on analogs that were obtained by de novo chemical synthesis. In contrast, the current review provides a comprehensive overview on the chemical transformations that have been investigated for the major epothilones A and B as starting materials, and it discusses the biological activity of the resulting products. Many semisynthetic epothilone derivatives have been found to exhibit potent effects on human cancer cell growth and several of these have been advanced to the stage of clinical development. This includes the epothilone B lactam ixabepilone (Ixempra(®), which has been approved by the FDA for the treatment of advanced and metastatic breast cancer.