Discovery of TD-0212, an Orally Active Dual Pharmacology AT1 Antagonist and Neprilysin Inhibitor (ARNI).

Dual inhibition of angiotensin-converting enzyme (ACE) and neprilysin (NEP) by drugs such as omapatrilat produces superior antihypertensive efficacy relative to ACE inhibitors but is associated with a higher risk of life-threatening angioedema due to bradykinin elevations. We hypothesized that dual AT1 (angiotensin II type 1 receptor) blockade and NEP inhibition with a single molecule would produce similar antihypertensive efficacy to omapatrilat without the risk of angioedema since ACE (the rate limiting enzyme in bradykinin metabolism) would remain uninhibited. Merging the structures of losartan (an AT1 antagonist) and thiorphan (a NEP inhibitor) led to the discovery of a novel series of orally active, dual AT1 antagonist/NEP inhibitors (ARNIs) exemplified by compound 35 (TD-0212). In models of renin-dependent and -independent hypertension, 35 produced blood pressure reductions similar to omapatrilat and combinations of AT1 receptor antagonists and NEP inhibitors. Upper airway angioedema risk was assessed in a rat tracheal plasma extravasation (TPE) model. Unlike omapatrilat, 35 did not increase TPE at antihypertensive doses. Compound 35 therefore provides the enhanced activity of dual AT1/NEP inhibition with a potentially lower risk of angioedema relative to dual ACE/NEP inhibition.

Concomitant angiotensin AT1 receptor antagonism and neprilysin inhibition produces omapatrilat-like antihypertensive effects without promoting tracheal plasma extravasation in the rat.

Dual inhibition of angiotensin-converting enzyme (ACE) and neprilysin (NEP) by drugs such as omapatrilat produces superior antihypertensive efficacy but cause high incidence of angioedema. We examined whether dual inhibition of angiotensin AT1 receptor (ARB) and NEP (ARB-NEPI, valsartan-candoxatril) provides similar efficacy to omapatrilat without the risk of angioedema. Activity of test compounds at the targets was assayed using fluorescence-based enzyme assays (ACE, NEP, aminopeptidase P) or competition binding assays (AT1). Target engagement in vivo (ACE, AT1, and NEP) was quantified by measuring inhibition of angiotensin-pressor responses and potentiation of atrial natriuretic peptide-induced urinary cyclic guanosine monophosphate (cGMP) output in rats. Tracheal plasma extravasation (TPE) was used as a surrogate to assess propensity of compounds to promote upper airway angioedema. Antihypertensive efficacy in renin-dependent and -independent states was measured in spontaneously hypertensive rats and deoxycorticosterone acetate salt hypertensive rats, respectively. Administration of omapatrilat and coadministration of valsartan and candoxatril blocked angiotensin induced vasopressor responses and potentiated atrial natriuretic peptide-induced increase in urinary cGMP output. In spontaneously hypertensive rats, valsartan, omapatrilat, and valsartan-candoxatril combination all produced reduction in blood pressure to a similar extent, whereas candoxatril was ineffective. In deoxycorticosterone acetate rats, omapatrilat, candoxatril, and valsartan-candoxatril combination but not valsartan produced reduction in blood pressure. Antihypertensive doses of omapatrilat produced robust increases in TPE; by contrast, valsartan, candoxatril, or their combination did not increase TPE. Pretreatment with icatibant, a bradykinin B2 antagonist, abolished omapatrilat-induced TPE but not its antihypertensive effects. On the background of NEP inhibition, suppression of the renin-angiotensin system through ARB and ACE inhibition shows a similar antihypertensive efficacy but exerts differential effects on bradykinin metabolism and TPE indicative of reduced risk of angioedema. Thus, dual AT1 receptor blockade and NEP inhibition is potentially an attractive approach to retain the excellent antihypertensive effects of omapatrilat but with a superior safety profile.

Processing of DNA for nonhomologous end-joining is controlled by kinase activity and XRCC4/ligase IV.

Nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. To repair the broken ends, NHEJ processes noncompatible ends into a ligatable form but suppresses processing of compatible ends. It is not known how NHEJ controls polymerase and nuclease activities to act exclusively on noncompatible ends. Here, we analyzed processing independently of ligation by using a two-stage assay with extracts that recapitulated the properties of NHEJ in vivo. Processing of noncompatible ends required wortmannin-sensitive kinase activity. Since DNA-dependent protein kinase catalytic subunit (DNA-PKcs) brings the ends together before undergoing activation of its kinase, this suggests that processing occurred after synapsis of the ends. Surprisingly, all polymerase and most nuclease activity required XRCC4/Ligase IV. This suggests a mechanism for how NHEJ suppresses processing to optimize the preservation of DNA sequence.

Assays for nonhomologous end joining in extracts.

In mammalian cells, nonhomologous end-joining (NHEJ) repairs DNA double strand breaks created by ionizing radiation and V(D)J recombination. Using human whole cell extracts prepared by the method of Baumann and West (1998), we have described a cell-free system for NHEJ that joins both compatible and noncompatible DNA ends (Budman and Chu, 2005). To measure joining efficiency and assess the processing of DNA ends, we developed a quantitative polymerase chain reaction assay for the joining of two specific DNA ends. The in vitro NHEJ reaction recapitulates key features of NHEJ observed in vivo: end joining is dependent on DNA-PK and XRCC4/Ligase4, and noncompatible ends are processed by polymerase and nuclease activities that often stabilize the alignment of opposing ends by base pairing. This chapter describes methods for preparing whole cell extracts and for studying the NHEJ reaction in vitro.

Processing of DNA for nonhomologous end-joining by cell-free extract.

In mammalian cells, nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. We have developed a cell-free system capable of processing and joining noncompatible DNA ends. The system had key features of NHEJ in vivo, including dependence on Ku, DNA-PKcs, and XRCC4/Ligase4. The NHEJ reaction had striking properties. Processing of noncompatible ends involved polymerase and nuclease activities that often stabilized the alignment of opposing ends by base pairing. To achieve this, polymerase activity efficiently synthesized DNA across discontinuities in the template strand, and nuclease activity removed a limited number of nucleotides back to regions of microhomology. Processing was suppressed for DNA ends that could be ligated directly, biasing the reaction to preserve DNA sequence and maintain genomic integrity. DNA sequence internal to the ends influenced the spectrum of processing events for noncompatible ends. Furthermore, internal DNA sequence strongly influenced joining efficiency, even in the absence of processing. These results support a model in which DNA-PKcs plays a central role in regulating the processing of ends for NHEJ.