Modulation of lysyl oxidase-like 2 enzymatic activity by an allosteric antibody inhibitor.
In this report, we assessed the steady-state enzymatic activity of lysyl oxidase-like 2 (LOXL2) against the substrates 1,5-diaminopentane (DAP), spermine, and fibrillar type I collagen. We find that both DAP and spermine are capable of activating LOXL2 to the same extent and have similar Michaelis constants (K(m) approximately 1 mm) and catalytic rates (k(cat) approximately 0.02 s(-1)). We also show that LOXL2 is capable of being inhibited by a known suicide inhibitor of lysyl oxidase (LOX), beta-aminopropionitrile, which we find is a potent inhibitor of LOXL2 activity. The modality of inhibition of beta-aminopropionitrile was also examined and found to be competitive with respect to the substrates DAP and spermine. In addition, we identified an antibody inhibitor (AB0023) of LOXL2 enzymatic function and have found that the inhibition occurs in a non-competitive manner with respect to both spermine and DAP. The binding epitope of AB0023 was mapped to the scavenger receptor cysteine-rich domain four of human LOXL2. AB0023 binds to a region remote from the catalytic domain making AB0023 an allosteric inhibitor of LOXL2. This affords AB0023 several advantages, because it is specific for LOXL2 and inhibits the enzymatic function of LOXL2 in a non-competitive manner thereby allowing inhibition of LOXL2 regardless of substrate concentration. These results suggest that antibody allosteric modulators of enzymatic function represent a novel drug development strategy and, in the context of LOXL2, suggest that inhibitors such as these might be useful therapeutics in oncology, fibrosis, and inflammation.
Rodriguez
Hector M
HM
Arresto BioSciences, Palo Alto, CA 94304, USA. hrodriguez@arresto.com
Vaysberg
Maria
M
Mikels
Amanda
A
McCauley
Scott
S
Velayo
Arleene C
AC
Garcia
Carlos
C
Smith
Victoria
V
D000494:Allosteric Regulation; D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D000629:Aminopropionitrile / Q000494:pharmacology*; D000906:Antibodies / Q000494:pharmacology*; D001667:Binding, Competitive; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D010446:Peptide Fragments / Q000302:isolation & purification; D008249:Protein-Lysine 6-Oxidase / Q000378:metabolism; D011759:Pyrrolidines / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013096:Spermine / Q000378:metabolism; D020349:Surface Plasmon Resonance
D000596:Amino Acids; D000906:Antibodies; D017931:DNA Primers; D008670:Metals; D010446:Peptide Fragments; D011759:Pyrrolidines; D011994:Recombinant Proteins; C098461:dolaproine; D000629:Aminopropionitrile; D013096:Spermine; D000594:Amino Acid Oxidoreductases; C119091:LOXL2 protein, human; C435668:LOXL3 protein, human; C445682:LOXL4 protein, human; D008249:Protein-Lysine 6-Oxidase