Supplementary Materials
Engineering of neprilysin for enhanced A activity
A cleavage assays
Peptide cleavage activity of neprilysin (NEP) was determined using a fluorescence
polarisation assay based on a previously described method (Leissring et al., 2003b).
A peptides were synthesised by Bachem (Bubendorf, Switzerland) and were labelled
with a N-terminal 5(6)FAM and a C-terminal biotin.
The biotin facilitates the
increase in the molecular size of uncleaved molecules after addition of avidin, thereby
increasing the assay sensitivity.
The assay was performed in a black 96-well
microtitre plate containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 0.05% (w/v)
bovine serum albumin (BSA), 1-200 µM peptide substrate and 1-500 nM NEP
protein. Samples were incubated at 37°C before the reaction was stopped at various
timepoints between 2 and 360 min by transferring 5 µL aliquots to 245 µL 50 mM
HEPES buffer containing 2 mM 1,10-phenanethroline monohydrate and 2 µM avidin.
The fluorescence polarisation of the resulting solution was measured on a Victor plate
reader and the amount of substrate cleaved was determined with reference to
substrate-only controls with and without avidin. Initial rates were obtained by linear
regression of the linear regions of time courses. Enzyme velocity was plotted as a
function of substrate concentration and the Michaelis-Menten equation was used to fit
the data, giving the parameters kcat and KM.
Degradation of A in buffer and plasma
Wild-type NEP or HSA-fused Neprilysin variant (NEPv) were diluted in HEPES
buffer (50 mM HEPES, 100 mM NaCl, 0.05% BSA, pH7.4) or human plasma in a
step-wise fashion starting with a concentration of 200-500 µg/ml. Degradation of
A1-40 and A1-42 was performed by incubating peptides and enzyme for 1 hour at
room temperature. The reaction was stopped by the addition of 1.10-phenanthroline to
a final concentration of 10 M. 50 µl (A40 analysis) or 100 l (A42 analysis) of
the reaction mix was transferred to the ELISA plate. A40 and A42 concentrations
after degradation were determined using Invitrogen human Aβ40 ELISA kit
(Invitrogen, California, US), and Innotest β-amyloid (1-42) ELISA (Innogenetics,
Gent, Belgium).
Degradation of A in brain homogenate
Degradation of Aβ1-40 by NEP was determined in homogenised brain from control
male Wistar rats. Brains were homogenised in a 2:5 (w/v) ratio of 50 mM HEPES
(pH 7.4) containing 150 mM NaCl. HSA-fused human NEP wild-type or human NEP
variant (hNEPv) was added to the homogenate at 0.06-250 µg/mL and incubated for
1 h. Reactions were stopped by the addition of 1 mM 1,10-phenanthroline and Aβ
was quantified using an ELISA kit as described in Materials and Methods.
Results
The enzyme neprilysin is well characterised for its ability to degrade Aβ (Shirotani et
al., 2001, Takaki et al., 2000, Roques et al., 1993, Turner et al., 2001) and has
previously been shown to be amenable to protein engineering to alter activity and
specificity (Sexton et al., 2012). The active site is located within a solvent accessible
core surrounded by a protein shell (Oefner et al., 2000, Oefner et al., 2004, Oefner et
al., 2007). By mutagenesis of the solvent exposed amino acids lining this central
cavity, we identified a panel of neprilysin variants with altered activity on Aβ. To
further improve the activity, changes to amino acids that increased activity on A in
this first round were combined and the activity was rescreened. A third round of
combinations and screening was performed, and whilst further improvements in
activity were obtained this was at the expense of protein thermodynamic stability
which declined rapidly as more mutations were introduced, resulting in rapid
aggregation and degradation of the protein (data not shown).
The lead variant after two rounds of mutation and screening contained two amino acid
substitutions, valine for glycine at position 399 and lysine for glycine at position 714.
This neprilysin variant (NEPv) was characterised for changes in its Michaelis Menten
kinetics and ability to degrade Aβ in plasma (Supplementary Figures 1A and 1B). In
plasma approximately 10% of the amount of NEPv was required to maximally
degrade Aβ in 1 hour compared to the wild-type enzyme. This could be
predominantly attributed to an increase in the Kcat.
HSA-hNEPv was also able to degrade Aβ1-40 in rat brain extract in a concentrationdependent manner, giving an IC50 of 4.0 nM (Supplementary Figure 1C). Wild-type
HSA-NEP degraded the peptide with a lower potency (IC50 of 230 nM).
To facilitate prolonged reduction of peripheral in vivo, NEPv was fused to protein
domains that prolong serum half-life. As the C-terminus of NEP is buried within the
structure, fusion was made to the N-terminus. Attempts were made to fuse to Fc and
HSA. Whilst fusion of NEPv to both was straight forward to express and purify, the
Fc fusion had a high propensity to aggregate, proved difficult to work with and could
not be reliably produced at the scale required to supply the studies described in this
paper. Therefore, HSA was chosen as a means of extending the serum half-life.
An equivalent mouse variant was also generated (MSA-mNEPv) to reduce the
potential for immunogenicity and therefore facilitate repeated dosing of mice over
prolonged periods. Whilst not having the same level of activity as the human
molecule (Supplementary Figure 1A) the engineered variant was demonstrated to
have enhanced Aβ1-40 and Aβ1-42 degradation activity compared to the wild-type
mouse enzyme.
Pharmacokinetics of MSA-mNEPv and HSA-hNEPv after single dosing in mice,
rats and monkeys
Data Analysis
Pharmacokinetic parameters were estimated by non-compartmental analysis using
WinNonlin Professional (version 5.2, Pharsight Corp., Mountain View, California,
US).
Results
A single dose study, carried out by administration of MSA-mNEPv (25 mg/kg) to
female Tg2576 mice by intravenous bolus or intraperitoneal injection indicated that
the serum clearance was 94 mL/day/kg, the volume of distribution at steady-state
(Vss) was 127 mL/kg and the terminal half-life (t1/2) was about 23 hours
(Supplementary Figure 2).
A single dose study in male Sprague-Dawley rats administered HSA-hNEPv by
intravenous bolus injection at dose levels of 5 and 25 mg/kg indicated that the serum
clearance was 209 mL/day/kg, the volume of distribution at steady-state (Vss) was
272 mL/kg and the terminal half-life (t1/2) was about 22 hours (Supplementary Figure
3).
A single dose PK study in male cynomolgus monkeys at dose levels of 5 and 23
mg/kg indicated that HSA-hNEPv exhibited linear and dose proportional
pharmacokinetics following a single intravenous bolus dose with serum clearance of
about 68 mL/day/kg, volume of distribution at steady-state (Vss) of about 194 mL/kg
and terminal half-life (t1/2) was 2.8 days (Supplementary Figure 4).
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Supplementary Figure Legends
Supplementary Figure 1. Activity of NEP and variants in vitro
(A) Michalis-Menten plots for human wild-type (WT-NEP) and variant NEP (NEPv;
left) and mouse wild-type (WT) and mouse variant NEP (NEPv; right) for the
cleavage of human 1-40. Mean (± SEM).
(B) Degradation of  by human NEP and human NEPv. Top left degradation of
1-40 in buffer, top right degradation of 1-42 in buffer, bottom left degradation
of 1-40 in human plasma, bottom right degradation of 1-42 in human plasma.
Mean (± SEM)
(C) Degradation of Aβ1-40 in control rat brain homogenate by HSA-hNEP wild-type
(HSA-NEP WT) or HSA-hNEPv (HSA-NEPv) Mean (± SEM).
Supplementary Figure 2. Serum concentration (mean ± SD)-time profiles for MSAmNEPv in female Tg2576 mice after a single dose at 25 mg/kg via the intravenous
(iv) and intraperitoneal (ip) routes (n=6 animals/timepoint).
Supplementary Figure 3. Serum concentration (mean ± SD)-time profiles for HSAhNEPv in male rats after a single intravenous dose at 5 or 25 mg/kg (n= 4
animals/timepoint).
Supplementary Figure 4. Serum concentration (mean ± SD)-time profiles for HSAhNEPv in male cynomolgus monkeys after a single intravenous dose at 5 or 23 mg/kg
(n=3 animals/timepoint).