Human Pharmacokinetics of LYS006, an Oral Leukotriene A4 Hydrolase Inhibitor Displaying Target-Mediated Drug Disposition.

LYS006 is a potent leukotriene A4 hydrolase inhibitor currently in clinical development for long-term treatment of various neutrophil-driven inflammatory conditions. Here, we present pharmacokinetics from the first-in-human study with complementary metabolism and transporter profiling data. The randomized first-in-human study included nine cohorts receiving 5-2*100 mg of LYS006 or placebo, a crossover food-effect part, and a multiple-dose part consisting of two fasted (5 mg and 15 mg once daily) and three fed cohorts (20-80 mg twice a day) of LYS006 or placebo. LYS006 and metabolites were assessed in plasma and urine, and transporters involved in LYS006 disposition were analyzed in vitro. Systemic plasma exposure increased with dose; steady-state exposure was dose proportional up to 40 mg twice a day. Steady state was achieved after ∼3 days, with mean accumulation of 2.1-fold for 5 mg once daily and ≤1.4-fold for all higher doses. Despite limited accumulation, a long terminal half-life (T1/2) was observed. The long T1/2 and saturable binding to blood cells, which causes a highly nonlinear blood-to-plasma distribution, reflect a strong impact of target binding on drug distribution at lower concentrations. Skin biopsy and blister fluid concentration data indicated saturable binding in the former but not the latter, suggesting saturable binding in tissues beyond blood. Major excretion of LYS006 (∼90% of dose) through urine at steady state triggered renal transporter investigations that identified LYS006 as a substrate of organic anion transporter (OAT)3, OAT4, breast cancer resistance protein, and multidrug resistance-associated protein 4. Seven metabolites were identified in human plasma and urine, comprising only 4% of the dose recovered in urine at steady state. SIGNIFICANCE STATEMENT: Pharmacokinetic data from a first-in-human study combined with in vitro work support dose and regimen selection for patient studies with LYS006 and provide guidance on drug interaction investigations and other clinical pharmacology work needed for further development. Mass balance information at steady state without the use of a radiolabel, skin concentrations, and identification of the major clearance pathway, as well as the transporters driving elimination, make this a particularly conclusive early study despite nonlinear pharmacokinetics impacted by target binding.

A Systematic In Vitro Investigation of the Inhibitor Preincubation Effect on Multiple Classes of Clinically Relevant Transporters.

Preincubation of a drug transporter with its inhibitor in a cell-based assay may result in the apparent enhancement of the inhibitory potency. Currently, limited data are available on potentiation of transporter inhibition by preincubation (PTIP) for clinically relevant solute-carrier transporters other than OATP1B1 and OATP1B3. Therefore, PTIP was examined systematically using OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, and MATE2-K cell lines. IC50 values of 30 inhibitors were determined with or without 3 hours of preincubation, and compounds with a PTIP ≥2.5× were further characterized by assessing the time course of transport inhibition potency and cellular concentration. For each compound, correlations were calculated between highest observed PTIP and physicochemical properties. PTIP was prevalent among organic cation transporters (OCTs) and organic anion-transporting polypeptides (OATPs) but not among organic anion transporters (OATs) or multidrug and toxin extrusion transporters (MATEs), and most instances of PTIP persisted after controlling for toxicity and nonspecific binding. Occasionally, preincubation in excess of 2 hours was required to attain full inhibitory potency. For four drugs examined, preincubation had the potential to change the in vitro drug-drug interaction risk prediction from "no risk" to "risk" on the basis of current regulatory criteria. Molecular weight and LogD7.4, as well as the ratio of passive cellular accumulation and cellular uptake rate correlated with PTIP; thus, low cellular permeation and a slow build-up of unbound intracellular inhibitor concentration may contribute to PTIP. Taken together, our data suggest that PTIP is partly determined by the physicochemical properties of the perpetrator drug, and preincubation may affect the in vitro predicted drug-drug interaction risk for OCTs as well as OATPs. SIGNIFICANCE STATEMENT: During the development of a novel pharmaceutical drug, in vitro studies are conducted to assess the risk of potential adverse interactions between existing medications a patient may already be taking and the novel compound. The exact way these in vitro assays are performed may influence the outcome of risk assessment. Here we suggest that the interaction risk may be underestimated unless specific assay protocols are modified to include an additional incubation step that allows the test drug to accumulate inside the cells, and demonstrate that adding this step is particularly important for large and hydrophobic drug molecules.

In-cell selectivity profiling of serine protease inhibitors by activity-based proteomics.

Activity-based proteomics is a methodology that is used to quantify the catalytically active subfraction of enzymes present in complex mixtures such as lysates or living cells. To apply this approach for in-cell selectivity profiling of inhibitors of serine proteases, we designed a novel activity-based probe (ABP). This ABP consists of (i) a fluorophosphonate-reactive group, directing the probe toward serine hydrolases or proteases and (ii) an alkyne functionality that can be specifically detected at a later stage with an azide-functionalized reporter group through a Cu(I)-catalyzed coupling reaction ("click chemistry"). This novel ABP was shown to label the active site of several serine proteases with greater efficiency than a previously reported fluorophosphonate probe. More importantly, our probe was cell-permeable and achieved labeling of enzymes within living cells with efficiency similar to that observed for the corresponding lysate fraction. Several endogenous serine hydrolases whose activities were detected upon in-cell labeling were identified by two-dimensional gel and MS analyses. As a proof of principle, cell-permeable inhibitors of an endogenous serine protease (prolyl endopeptidase) were assessed for their potency and specificity in competing for the in situ labeling of the selected enzyme. Altogether these results open new perspectives for safety profiling studies in uncovering potential cellular "side effects" of drugs (unanticipated off-target inhibition or activation) that may be overlooked by standard selectivity profiling methods.

Examining P-gp efflux kinetics guided by the BDDCS - Rational selection of in vitro assay designs and mathematical models.

The generation of reliable kinetic parameters to describe P-glycoprotein (P-gp) activity is essential for predicting the impact of efflux transport on gastrointestinal drug absorption. The compound-specific selection of in vitro assay designs and ensuing data analysis methods is explored in this manuscript. We measured transcellular permeability and cellular uptake of five P-gp substrates in Caco-2 and LLC-PK1 MDR1 cells. Kinetic parameters of P-gp-mediated efflux transport (Km, Vmax) were derived from conventional and mechanistic compartmental models. The estimated apparent Km values based on medium concentrations in the conventional permeability model indicated significant differences between the cell lines. The respective intrinsic Km values based on unbound intracellular concentrations in the mechanistic compartmental models were significantly lower and comparable between cell lines and assay formats. Non-specific binding or lysosomal trapping were shown to cause discrepancies in the kinetic parameters obtained from different assay formats. A guidance for the selection of in vitro assays and kinetic assessment methods is proposed in line with the Biopharmaceutics Drug Disposition Classification System (BDDCS). The recommendations are expected to aid the acquisition of robust and reproducible kinetic parameters of P-gp-mediated efflux transport.

Fragment-Based Protein-Protein Interaction Antagonists of a Viral Dimeric Protease.

Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose-response determination was performed as a confirmation screen, and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed by NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80 % of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogues. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces.

The paracaspase MALT1 cleaves HOIL1 reducing linear ubiquitination by LUBAC to dampen lymphocyte NF-κB signalling.

Antigen receptor signalling activates the canonical NF-κB pathway via the CARD11/BCL10/MALT1 (CBM) signalosome involving key, yet ill-defined roles for linear ubiquitination. The paracaspase MALT1 cleaves and removes negative checkpoint proteins, amplifying lymphocyte responses in NF-κB activation and in B-cell lymphoma subtypes. To identify new human MALT1 substrates, we compare B cells from the only known living MALT1(mut/mut) patient with healthy MALT1(+/mut) family members using 10-plex Tandem Mass Tag TAILS N-terminal peptide proteomics. We identify HOIL1 of the linear ubiquitin chain assembly complex as a novel MALT1 substrate. We show linear ubiquitination at B-cell receptor microclusters and signalosomes. Late in the NF-κB activation cycle HOIL1 cleavage transiently reduces linear ubiquitination, including of NEMO and RIP1, dampening NF-κB activation and preventing reactivation. By regulating linear ubiquitination, MALT1 is both a positive and negative pleiotropic regulator of the human canonical NF-κB pathway-first promoting activation via the CBM--then triggering HOIL1-dependent negative-feedback termination, preventing reactivation.