Structure of human spermine oxidase in complex with a highly selective allosteric inhibitor

Human spermine oxidase (hSMOX) plays a central role in polyamine catabolism. Due to its association with several pathological processes, including inflammation and cancer, hSMOX has garnered interest as a possible therapeutic target. Therefore, determination of the structure of hSMOX is an important step to enable drug discovery and validate hSMOX as a drug target. Using insights from hydrogen/deuterium exchange mass spectrometry (HDX-MS), we engineered a hSMOX construct to obtain the first crystal structure of hSMOX bound to the known polyamine oxidase inhibitor MDL72527 at 2.4 Å resolution. While the overall fold of hSMOX is similar to its homolog, murine N1-acetylpolyamine oxidase (mPAOX), the two structures contain significant differences, notably in their substrate-binding domains and active site pockets. Subsequently, we employed a sensitive biochemical assay to conduct a high-throughput screen that identified a potent and selective hSMOX inhibitor, JNJ-1289. The co-crystal structure of hSMOX with JNJ-1289 was determined at 2.1 Å resolution, revealing that JNJ-1289 binds to an allosteric site, providing JNJ-1289 with a high degree of selectivity towards hSMOX. These results provide crucial insights into understanding the substrate specificity and enzymatic mechanism of hSMOX, and for the design of highly selective inhibitors.

Colored boxes include part of the sequences that have been engineered using the color code of Figure 2c. Supplementary Fig. 2. Sequence coverage and H/D exchange heat map of relative deuterium incorporation. Level of deuteration for hSMOX Apo (a, c), in complex with MDL72527 (b) and JNJ-1289 (d). Thin lines indicate sequence coverage on top of the sequence. Green indicates confident peaks, yellow: medium-confident peaks (confidence based on theoretical isotope distribution of the chromatographic peak). The coverage for peptides used in HDX-MS analysis is 99.6% of the entire hSMOX. Under the protein sequence are heat maps in which each row represents the partially deuterated time points (10, 100 and 1000 sec). The color corresponds to the deuterated percentage of that area of protein per key on top right corner. Parts of the protein showing significant differences in deuterium uptake are highlighted in red boxes.

Supplementary Figure 3. Kinetic parameters of different engineered hSMOX constructs.
Steady-state kinetics of Spm for the different constructs employed in the HyPerBlu luminescence assay. The Spm concentrations were tested in the range of 0-500 M using 2.5 nM SMOX for constructs #6855, #7262, and #7181 or 1.76 nM for construct #6970. These results showed that kcat/Km values of all the constructs were within two-fold of one another. Overlay of ehSMOX, mPAOX in blue and green ribbon, respectively. a, S190 at the end of heSMOX S3 display orientation of the substrate-binding pocket loop different from that of mPAOX. Indeed, the heSMOX loop position clashes with N-AcSpm superimposed from mPAOX structure. b, Residues in the binding pocket different in ehSMOX and mPAOX are depicted in sticks (blue for ehSMOX and in green for mPAOX). FADs are in orange and green spheres for ehSMOX and mPAOX, respectively. and kcat values were determined to be 33.9 ± 2.7 µM and 4 s -1 , respectively. c, hPAOX enzyme activity was measured in the HyPerBlu luminescence assay using 10 µM N-AcSpm at a range of enzyme concentrations (0 -0.76 nM). Replots of rate profiles demonstrated linearity to 0.76 nM with an observed specific enzyme activity of 1350 min -1 . d, Steady-state kinetics of N-AcSpm oxidation was determined at 0.076 nM hPAOX. The N-AcSpm concentrations were tested in the range of 0 -200 µM and the resulting data fit and analyzed as described above. The corresponding Km and kcat values were determined to be 20.1 ± 0.63 µM and 57 s -1 , respectively. e, LSD1 enzyme activity was measured in the HyPerBlu luminescence assay using 50 µM peptide substrate representing residues 1-21 of histone H3 containing a dimethylated lysine at position 4 (H3K4me2) at a range of enzyme concentrations (0 -100 nM). Replot of LSD1 rate profile demonstrated linearity to 50 nM with an observed specific enzyme activity of 20 min -1 . f, Steady-state kinetics of H3K4me2 peptide oxidation was determined at 20 nM LSD1. The H3K4me2 peptide concentrations were tested in a range from 0 -100 µM and the resulting data fit and analyzed as described above. The corresponding Km and kcat values were determined to be 3.8 ± 0.35 µM and 0.2 s -1 , respectively. Progress curves were monitored for 1h and fit to Eqn S1 at each JNJ-1289 concentration to determine the observed rate constant, kobs.
[P] = Y0 + s + i − s obs (1 − − obs ) (S1) where i and s are the initial and steady state velocities, respectively, t is time and kobs represents the observed rate constant.
where I is the inhibitor concentration, 5 and 6 are the estimated forward and reverse rate constants, respectively, and iapp is the apparent value of the initial E·I complex.
c, Reaction progress curve analysis of 2 nM hSMOX and 312 nM JNJ-1289 with a range of Spm concentrations (7.8 -1000 µM). Progress curves were monitored for 1h and fit to Eqn S3 at each Spm concentration to determine the mode of inhibition. Pseudoatoms are depicted as spheres and color-coded by depth from the surface with blue (deepest) to red (surface exposed), while in dark gray are the atom positions (on the left). On the right, the protein surface is depicted in light gray and superimposed with its cartoon-loop representation of the protein and pseudoatoms represented as in the correspondent right figure. ehSMOX loop position in the bound form creates a cavity shape more similar in conformation (U shape depicted as a tunnel with the two exits) and depth to zPAOX than to mPAOX.  8, 152.9, 140.0, 137.0, 132.6, 132.5, 128.2, 122.1, 122.0, 120.0, 116.9, 115.6, 113.2, 106.5. c