Angiotensin converting enzyme versus angiotensin converting enzyme-2 selectivity of MLN-4760 and DX600 in human and murine bone marrow-derived cells

Abstract

Angiotensin-converting enzymes, ACE and ACE2, are key members of renin angiotensin system. Activation of ACE2/Ang-(1-7) pathway enhances cardiovascular protective functions of bone marrow-derived stem/progenitor cells. The current study evaluated the selectivity of ACE2 inhibitors, MLN-4760 and DX-600, and ACE and ACE2 activities in human (hu) and murine (mu) bone marrow cells. Assays were carried out in hu and mu mononuclear cells (MNCs) and huCD34+ cells or mu-lineage-depleted (muLin-) cells, human-recombinant (rh) enzymes, and mu-heart with enzyme-specific substrates. ACE or ACE2 inhibition by racemic MLN-4760, its isomers MLN-4760-A and MLN-4760-B, DX600 and captopril were characterized. MLN-4760-B is relatively less efficacious and less-selective than the racemate or MLN-4760-A at hu-rhACE2, and all three of them inhibited 43% rhACE. In huMNCs, MLN-4760-B detected 63% ACE2 with 28-fold selectivity over ACE. In huCD34+ cells, MLN-4760-B detected 38% of ACE2 activity with 63-fold selectivity. In mu-heart and muMNCs, isomer B was 100- and 228-fold selective for ACE2, respectively. In muLin- cells, MLN-4760-B detected 25% ACE2 activity with a pIC50 of 6.3. The racemic mixture and MLN-4760-A showed lower efficacy and poor selectivity for ACE2 in MNCs and mu-heart. ACE activity detected by captopril was 32 and 19%, respectively, in huCD34+ and muLin- cells. DX600 was less efficacious, and more selective for ACE2 compared to MLN-4760-B in all samples tested. These results suggest that MLN-4760-B is a better antagonist of ACE2 than DX600 at 10μM concentration in human and murine bone marrow cells, and that these cells express more functional ACE2 than ACE.

The Yin and Yang of ACE/ACE2 Pathways: The Rationale for the Use of Renin-Angiotensin System Inhibitors in COVID-19 Patients

“…Moreover, all three inhibitors exerted a significantly higher inhibitory activity against soluble than membrane-bound forms of (m)ACE2 (near maximal mACE2 inhibition at 10 −6 M), being the isomer B more selective and effective than the racemate or the isomer A for mACE2 vs. mACE expressed on the surface of mononuclear cells (or CD34 hematopoietic progenitors) [114]. A second ACE2 inhibitor, Dx600, produced similar results to those obtained with the isomer B [114]. Another study evaluated the inhibitory activity of both MLN-4760 and DX600 (either the linear conformational form or the disulfide bridged cyclic variant) inhibitors.…”

“…Actually, several reports describing Dx600 inhibitor administration in mice suggest its safe use. Of interest, a report described its (safe) use alone (1 mg/kg per day) by nasal inhalation for 3 days in a mouse model of endotoxin-induced lung inflammation [121]; however, this inhibitor is less efficacious than MLN-4760 in inhibiting the soluble forms of human rACE2 [114,115]. Altogether these reports on administration of ACE2 inhibitors do not reveal significant adverse impacts or mortality in experimental animals, which suggests their safety in chronic administration.…”

“…MLN4760 is a potent and selective human ACE2 inhibitor (IC50 = 0.44 nM against soluble human ACE2) whose synthesis produces a racemic mixture of two diastereomers that showed 75:25 ratio for Isomer A: Isomer B [ 113 , 114 ] and the purified isomer B is the isomer commercially available from Merck Millipore [ 114 ]. Testing MLN-4760 racemic mixture and its isomers, it was observed a concentration-dependent inhibition of recombinant human (rh)ACE or rhACE2 activities with all three inhibitors [ 114 ].…”

Natural Flavonoids as Potential Angiotensin-Converting Enzyme 2 Inhibitors for Anti-SARS-CoV-2

“…In the past 20 years, MLN-4760 (imidazole) [ 79 , 80 , 81 ], captopril derivative [ 82 , 83 ], DX600 and TAPI-2 peptide [ 84 , 85 ], losartan and its derivatives (benzimidazole [ 56 , 82 , 86 , 87 ], chloroquine and its derivatives (quinolone) [ 88 ], diminazene aceturate [ 89 ], cepharanthine (alkaloid) [ 75 ], thiorphan (palmitoyl) [ 87 ], and N -(2-aminoethyl)-1 aziridineethanamine (amino ethyl) [ 90 ] were discovered to have potential as ACE2 inhibitors. However, caution should be taken because, although ACE1 inhibitors (such as captopril, enalapril, and lisinopril) and angiotensin II receptor blockers (ARB) (such as olmesartan, losartan, candesartan, and valsartan) do have inhibitory effects on ACE2 [ 91 ], several studies showed that these drugs can increase the ACE2 blood level [ 86 ], which will likely increase the risk of contracting SARS [ 92 ].…”

Bone biology and COVID-19 infection: Is ACE2 a potential influence factor?

“…Evidence has shown that ACE2 locally expresses in human bone marrow-derived stem/progenitor cells (BMSPCs) and manipulates cytokine-sensing to promote skeletal repair [4] . ACE2 promotes AngII degradation and synthetizes Ang1-7, which perform functions by way of Mas receptor.…”