Biotransformation-induced toxicity. Challenges in drug design M. Matilde Marques

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1 Biotransformation-induced toxicity. Challenges in drug design M. Matilde Marques Drug Discovery Session July 4, 2016

2 Drug Discovery Toxicology The Road to Safe Drugs Attrition in drug development remains high, with toxicity being the leading cause at all stages of the drug development process. This causes a problem for the pharmaceutical industry regarding patient welfare and monetary issues. Blomme & Will, Chem Res Toxicol 2016, 29, 473

3 Drug Cellular Accumulation Toxicity Phase I/Phase II metabolism Stable metabolites Bioactivation Reactive metabolites Quinone Hydroxylamine itroso Furan Thiophene Allylic Alcohol Epoxide Carbocation Quinone-imine Covalent modification of cellular macromolecules ucleic Acids Enzymes Transporters Signaling proteins Receptors Autologous proteins Excretion (urine, bile) Carcinogenicity Altered cellular function ecrosis Hypersensitivity Apoptosis

4 Covalent Adducts as Biomarkers of Toxicity Toxic (Reactive) Metabolites Difficult to monitor in vivo Stable Covalent DA and Protein Adducts Drugs Bioactivation Covalent Protein and DA adducts Biomarkers of toxicity that can be easily assessed Biomonitoring Correlation with toxic outcomes Biomarkers of Toxicity Identification of risk factors Establishment of risk/benefit relationships Toxicity Minimization of Toxic Events Personalized Medicine

5 Long-term toxicities associated with chronic exposures (eg., drugs) Biomonitoring of drug metabolites, DA and protein adducts as a key to risk assessment Synthetic Standards: Metabolites Protein adducts DA adducts Analytical tools: Development and validation In vivo Biomonitoring: Animal models Patients Correlations with: Biochemical changes Adverse clinical endpoints Individual characteristics Risk/Benefit relationships Minimization of dosedependent toxic events Personalized medicine Design of safer drugs

6 evirapine CH 3 H VP O First RTI approved by the FDA for use in combination therapy of HIV-1 infection (1996). Administered during labor and to infant in the first weeks of life. HIV transmission rates dropped from 25% to 13%. A more extended infant treatment period was proposed, to prevent HIV transmission via breast milk. Still first line choice for children younger than 3 years of age in developed countries. Most used antiretroviral in countries with limited resources. ew extended release formulation approved by the US FDA in 2011.

7 evirapine Side Effects Severe, life-threatening, sometimes fatal hepatotoxicity. Severe, life-threatening hypersensitivity reactions primarily characterized by skin rash. Genotoxicity o evidence of mutagenicity or clastogenicity in genetic toxicology tests. Increased incidence of hepatocellular adenomas and carcinomas in mice. Increased incidence of hepatocellular adenomas in rats. Carcinogenicity in humans currently unknown.

8 evirapine - Metabolism 6. 4-Carboxy-VP

9 evirapine Biomarkers Molecular biomarkers reflect: Exposure Individual susceptibility Early response Can we find biomarkers of metabolic VP activation? DA adducts are influenced by individual repair ability. If not repaired they induce mutations link to potential carcinogenicity Protein adducts are not repaired good markers of external exposure

10 12-Mesyloxy-nevirapine as a surrogate electrophile for nucleoside modification in vitro H O HO H O BuLi/ MoOPH Multiple DA Adducts MsO H Et 3, MsCl O BUT o evidence for DA adduct formation in rodents in vivo. dg, da, dc, dt or DA Antunes et al, Chem Res Toxicol 2008, 21, 1443

11 12-Mesyloxy-nevirapine as a surrogate electrophile for protein modification in vitro MALDI-TOF-MS/MS analyses of tryptic peptide digests VP adduct location HSA Hb-A Hb-B HSA Hb K214, W238, H362, K548/K549 H21 W38, S90 LC-ESI-MS/MS analyses VP adduct location H3, C34, H105 (trypsin/chymotrypsin digests) -terminal valine (after -alkyl Edman degradation) Antunes et al, Chem Res Toxicol 2010, 23, 1714

12 Protein modification by 12-Sulfoxy-nevirapine in vitro LC-MS/MS analyses VP adduct location HSA C34, H146, H242, H338 (trypsin digests) Meng et al, Chem Res Toxicol 2013, 26, 575 VP adducts in vivo Unequivocal evidence of VP bioactivation 12-MsO-VP validated as surrogate electrophile DA VP adduct location none reported to date Sample source Proteins Hb -terminal valine a human patients on VP HSA H146 b human patients on VP a Caixas et al, Toxicology 2012, 301, 33 b Meng et al, Chem Res Toxicol 2013, 26, 575

13 Blood proteins as models for toxicologically relevant proteins Advantages They are abundant and easily accessible. Reactive intermediates have to cross cell membranes to reach Hb -> Hb adducts are frequently assessed as surrogates for DA adducts of genotoxic carcinogens. HSA is well characterized and ca. 40% of extravascular HSA is located in the skin -> good tool to investigate protein haptenation mechanisms by skin allergens. Disadvantage The use of Hb and HSA protein adducts as surrogates for toxicologically relevant proteins precludes the establishment of direct correlations between the formation of covalent adducts in humans and the occurrence of drug-induced toxic events.

14 Histones as alternative model proteins Histones are lysine-rich nuclear proteins that regulate chromatin structure and gene expression. They are easily isolated from the buffy-coat fraction of peripheral blood -> potentially good models for biomonitoring purposes. Histone PTMs have a key role in the regulation of fundamental cellular processes -> effects on the etiology of diseases, including cancer. Increasing evidence is emerging of links between environmental exposure to toxicants, abnormal epigenetic marks and the development of human diseases.

15 Working Hypothesis Histones Reactive electrophiles Site-specific histone modifications Modification of chromatin structure Modification of the histone code Abnormal histone modifications by reactive electrophiles that reach the cell nuclei are conceivable. If refractory to "eraser" enzymes, these alterations may play a role in pathophysiological processes. Chemical Carcinogenesis?

16 Methodology MsO H O Bottom-up approach 1 2. Amicon filtration Recombinant full length human histones (H3, H4 and octamer) 3. Lyophilization 4. Digestion 5. LC-MS/MS analysis Mapping of VP modification sites

17 Comparison of the MS/MS spectra of a representative peptide from histone H4 VP-modified at H76 on-modified

18 Overview of H2A, H2B, H3 and H4 sequences with the VP-modified peptides All identified VPmodified residues are located on the exposed surface of the protein Lower protein coverage in H2A and H3 (higher Lys and Arg content)

19 Conclusions Multiple sites of VP-induced modification were identified in recombinant human histones: serine, histidine and (mostly) lysine residues. A significant proportion of histidine residues underwent modification by VP. This suggests a remarkable affinity of the imidazole ring for VP-derived electrophiles. -> Consistent with the identification of a modified histidine residue in HSA from VP-treated patients. In contrast with most known PTMs, the VP-derived modifications did not occur in the histone tails. -> Are there distinct regioselectivities for enzymatic and non-enzymatic histone modification?

20 Conclusions VP-induced modifications occurred in some positions known to undergo methylation (eg., K57 in H3), acetylation (eg., K78 in H4) and phosphorylation (eg., H76 in H4) Several of the VP-binding sites occurred in residues involved in histone-histone, histone-da or nucleosome-nucleosome interactions. Possible implications (if occurring in vivo): persistent modifications, inert to removal by eraser enzymes; prevention of key epigenetic modifications -> altered histone code -> influence on downstream gene expression. oteworthy: A preliminary study reported altered histone methylation patterns in rats administered VP (Pereira et al, Environ Mol Mutagen 2010, 5, 699).

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22 Ackowledgments PTDC/QUI-QUI/113910/2009 PTDC/SAU-TOX/111663/2009 RECI/QEQ-MED/0330/2012 UID/QUI/00100/2013 IF/ 01091/2013/CP1163/CT0001)