New Renin Inhibitor VTP-27999 Alters Renin Immunoreactivity and Does Not Unfold Prorenin

Abstract

R enin belongs to the A1 family of aspartic proteases. Its 3-dimensional (3D) structure consists of 2 β-sheet domains (N and C domain) related by an ≈2-fold axis. The active site is a deep cleft between the N and C domains that extends over 8 residues of renin's substrate, angiotensinogen. Each domain supplies 1 catalytic aspartic acid residue in the center of the binding cleft. A long β-hairpin loop structure called the flap in the N-terminal domain covers the central part of the angiotensinogen-binding site, and the conformation with the tip of the flap open allows the entrance of the substrate and removal of hydrolytic products during the catalytic turnover, while the tip closes on substrate binding. 1 In the case of renin's inactive precursor, prorenin, the flap is open to accommodate the 43-amino acid prosegment.1,2 Morales et al 2 recently proposed that in prorenin, the catalytic binding site is covered by the N-terminal part of mature renin that, in turn, is covered by the prosegment. After removal of the prosegment, this N-terminal part becomes part of a 6-stranded β-sheet on the back of the mature renin molecule, previously occupied by the prosegment. This requires a conformational change that fully exposes the active cleft. 2Prosegment unfolding occurs in a pH-and temperaturedependent manner and, if not followed by cleavage, results in 2 prorenin conformations as follows: a closed, inactive form, and an open form that displays full enzymatic activity (Figure 1). 3,4 In addition, an intermediate form exists where the prosegment has moved away from the cleft, but where the renin part still has to undergo the above-mentioned conformational changes. Under physiological conditions, <2% of prorenin is in the open conformation. The recently introduced renin inhibitor, aliskiren ( Figure S1A in the online-only Data Supplement), binds to prorenin in the open conformation, as well as to the intermediate form of prorenin. 3,4 Binding to the intermediate form induces prorenin unfolding. Because of the tight binding of the renin inhibitor, the refolding step (ie, the return to the closed conformation) is no longer possible, and thus the equilibrium between the closed and open conformation will shift in favor of the open conformation. Eventually, depending on the concentration of aliskiren, a significant proportion of prorenin may be open (nonproteolytic activation), allowing its recognition by the active site-directed antibodies used in renin immunoradiometric assays (IRMAs), despite the fact that the Abstract-Renin inhibitors like aliskiren not only block renin but also bind prorenin, thereby inducing a conformational change (like the change induced by acid) allowing its recognition in a renin-specific assay. Consequently, aliskiren can be used to measure prorenin. VTP-27999 is a new renin inhibitor with an aliskiren-like IC 50 and t 1/2 , and a much higher bioavailability. This study addressed (pro)renin changes during treatment of volunteers with VTP-27999 or aliskiren. Both drugs increased renin immunoreactivi...

Association Studies Suggest a Key Role for Endothelin-1 in the Pathogenesis of Preeclampsia and the Accompanying Renin–Angiotensin–Aldosterone System Suppression

“…4 Here, it should be considered that pregnant women display elevated prorenin and angiotensinogen levels. 2 Prorenin may interfere in the immunoreactive renin assay by cross-reacting with the antibody that recognizes renin's active site, 20 and the 4-to 5-fold elevated angiotensinogen levels will lead to a higher PRA (and, consequently, aldosterone) level for a given PRC as compared with nonpregnant women. From this point of view, it is better to compare aldosterone with PRA because this parameter takes into account the changes in angiotensinogen and is not affected by prorenin cross-reactivity.…”

Renin inhibitor VTP-27999 differs from aliskiren

“…In human plasma, VTP-27999 is approximately 75% protein-bound (R. Gregg, Vitae Pharmaceuticals, unpublished observations), which is comparable to or somewhat higher than the protein binding described for aliskiren [2]. Unexpectedly, VTP-27999 was found to alter the affinity of the active sitedirected antibodies applied in renin imunoassays, thereby increasing the outcome of such assays [3]. Such effects on immunoreactivity might also alter renin's binding to clearance receptors and/or the (pro)renin receptor ((P)RR) [4].…”

“…Such effects on immunoreactivity might also alter renin's binding to clearance receptors and/or the (pro)renin receptor ((P)RR) [4]. In addition, although VTP-27999 did bind to prorenin, it did not, in contrast to aliskiren and other renin inhibitors, subsequently 'unfold' prorenin [3]. Prorenin unfolding normally allows the inactive enzyme to be detected in a renin-specific assay.…”

Drugs Targeting RAAS in the Treatment of Hypertension and Other Cardiovascular Diseases

“…This prompted the development of newer renin inhibitors with improved therapeutic profi les. Currently, a number of them are under different phases of clinical trials, the most prominent among them is VTP-27999 [ 22 ].…”