ACE Human

What is the molecular structure of human ACE and how does it function in cardiovascular regulation?

Human Angiotensin-Converting Enzyme exists in two main forms: somatic ACE (found throughout the body, particularly in endothelial cells) and testis ACE (found in male germinal cells). Somatic ACE is a complex two-domain enzyme comprising an N-domain and C-domain, each containing an active site with similar but distinct substrate specificities and chloride-activation requirements .

The enzyme contains a zinc-binding site with the canonical HEXXH motif that is essential for its catalytic function. Structurally, somatic ACE consists of an N-terminal domain of about 612 amino acids, a 15-residue interdomain sequence, and a 650-residue C-terminal domain . A sequence of 22 hydrophobic amino acids near the carboxyl terminus serves as a transmembrane domain, creating a 28-residue cytosolic domain and a 1,227-residue glycosylated extracellular domain .

Functionally, ACE plays a central role in the renin-angiotensin system, controlling blood pressure, fluid and electrolyte homeostasis, renal and vascular function, and myocardial remodeling .

How does the gene structure of human ACE relate to its different isoforms?

Human endothelial ACE (somatic ACE) is encoded by a single gene consisting of 26 exons, with all but exon 13 being transcribed into the corresponding mRNA . The testis form of ACE is encoded by the same gene but uses an alternative promoter within intron 12 that is active only in adult male germinal cells .

Specifically, testis ACE mRNA begins before exon 13 and continues through exon 26. Translation of this mRNA results in a 701-amino-acid version of ACE that, except for the first 36 residues, is identical to the C-terminal domain of somatic ACE . The structure of the gene provides clear evidence of a gene duplication event in its evolutionary history, as a 357-amino-acid segment of the N-domain has more than 60% sequence identity to the corresponding segment of the C-domain .

This genetic organization explains why somatic ACE has two active sites while testis ACE has only one, and has significant implications for domain-selective inhibitor design.

What structural insights from the crystal structure of human testis ACE can inform domain-selective inhibitor design?

The 2.0 Å resolution crystal structure of human testis ACE (tACE) and its complex with lisinopril revealed essential structural features for inhibitor design . The enzyme is predominantly helical with the active site located in a deep central cavity. The catalytic Zn²⁺ ion, bound to the HEXXH sequence, is located approximately 10 Å from the entrance .

Key structural features relevant for inhibitor design include:

• The binding interactions of lisinopril with specific pockets: the phenyl ring interacts with the S₁ sub-site, the lysine with the S₁' sub-site, and the proline occupies the S₂' sub-site .

• Critical molecular interactions include the carboxyl group binding to Zn²⁺ and forming a hydrogen bond with Glu384, the side-chain amino group of lysine interacting with Glu162, and the C-terminal proline carboxyl group binding with Lys511 and Tyr520 .

• Two buried chloride ions located at 20.7 Å and 10.4 Å from the Zn²⁺ ion contribute to enzyme activation, with the first bound to Arg186, Arg489, and Trp485, and the second bound to Arg522 and Tyr224 .

The amino-terminal helices (α1-3) form a lid-like structure partially covering the active-site channel, with an aperture approximately 3 Å in diameter that requires conformational change for substrate entry . These features provide the foundation for structure-guided design of domain-selective inhibitors targeting either the N- or C-domain.

How do the N- and C-domains of ACE differ in structure and function, and what are the implications for selective inhibitor design?

Despite sharing ~60% amino acid sequence identity, the N- and C-domains of somatic ACE exhibit important differences that can be exploited for selective inhibitor design:

• The lid-like structure (helices α1-3) shows notable differences in hydrophobicity and charge between domains, affecting substrate specificity .

• Chloride activation requirements differ significantly: the C-domain has greater chloride dependence than the N-domain for substrate hydrolysis and inhibitor binding . This is partly explained by Arg186 (a key chloride-binding residue in tACE) being replaced by His164 in the N-domain, resulting in only one chloride-binding pocket in the N-domain compared to two in the C-domain .

• Homology modeling reveals that while both domains can bind lisinopril with similar affinity, Val518 and Ser516 in the N-domain (corresponding to Glu143 and Phe391 in the C-domain) could be targeted for developing N-domain selective inhibitors, as these residues might repulse certain chemical groups in potential inhibitors .

These structural and functional differences offer opportunities for developing domain-selective inhibitors using structure-guided drug design, potentially yielding next-generation ACE inhibitors with improved safety and efficacy profiles.

What technical challenges were overcome in determining the crystal structure of human ACE?

The determination of the crystal structure of human ACE represented a significant breakthrough after years of challenges. The main impediment was producing diffraction-quality crystals, which was overcome through two concurrent approaches:

• Targeted modification of glycosylation sites: Five of the N-linked glycosylation sites were disrupted by substituting glutamines for each of the asparagine residues in the glycosylation sequences .

• Expression with glycosylation control: A truncated form of ACE was expressed in the presence of a glucosidase inhibitor to control glycosylation patterns .

These approaches yielded crystals suitable for X-ray diffraction, enabling the three-dimensional structure determination of tACE and its complex with lisinopril at 2.0 Å resolution . This technological achievement provided unprecedented molecular insights into ACE structure and inhibitor binding, opening new avenues for rational drug design based on structural information rather than the serendipitous approaches that led to first-generation ACE inhibitors.

What are the most effective approaches for structure-guided design of domain-selective ACE inhibitors?

The crystal structure of tACE has enabled more rational approaches to ACE inhibitor design, particularly for developing domain-selective compounds:

• In silico modeling using existing inhibitors as scaffolds: Computational modeling based on the crystal structure allows for virtual screening and optimization of potential inhibitors before synthesis .

• Structure-guided modifications: By understanding the subtle differences between the N- and C-domains, researchers can modify existing inhibitors to preferentially interact with one domain. For example, targeting the differences in the S₁ and S₂' sub-sites between domains .

• Exploiting chloride-binding differences: Since the domains differ in their chloride dependence, designing inhibitors that leverage these differences could yield domain selectivity .

• Focus on the lid-like structure: Modifications that interact with the amino-terminal helices (α1-3) could potentially exploit the hydrophobicity and charge differences between domains .

• Iterative lead optimization: Using structural insights to guide synthetic chemistry efforts, allowing for progressive refinement of domain selectivity and potency .

These approaches represent a significant advancement over the development of current ACE inhibitors, which were designed without knowledge of the two-domain structure or three-dimensional architecture of the enzyme.

What potential therapeutic advantages could domain-selective ACE inhibitors offer compared to current non-selective inhibitors?

Current-generation ACE inhibitors, which inhibit both the N- and C-domains, have combined annual sales exceeding $6 billion but are hampered by common side effects . Domain-selective inhibitors could offer several advantages:

• Improved safety profile: Many side effects of current ACE inhibitors (such as persistent dry cough) may be related to non-selective inhibition. Domain-selective inhibitors could potentially reduce these adverse effects by targeting only the domain responsible for the therapeutic effect while preserving the function of the other domain .

• Enhanced efficacy: By specifically targeting the domain most relevant to a particular disease process, domain-selective inhibitors might achieve greater therapeutic efficacy at lower doses .

• Novel applications: Domain-selective inhibitors might enable new therapeutic applications beyond the current indications for ACE inhibitors, which include hypertension, heart failure, myocardial infarction, and kidney failure .

• Precision medicine approach: The ability to selectively target either the N- or C-domain could allow for more personalized treatment strategies based on individual patient characteristics and specific disease mechanisms .

The development of such domain-selective inhibitors represents a compelling example of revisiting an established drug target with modern drug discovery tools to create improved next-generation therapeutics.

What evidence supports the implementation of ACE enquiry in primary healthcare settings?

Research has established a strong and proportionate relationship between adverse childhood experiences (ACEs) and detrimental health and social outcomes later in life . Evidence supporting ACE enquiry implementation includes:

• Clinical impact: In San Diego, Kaiser Permanente's analysis of 135,000 patients showed that adding an ACE questionnaire with follow-up in exam rooms resulted in a 35% reduction in outpatient visits and an 11% reduction in emergency department visits over the following year compared to prior utilization .

• Acceptability: Contrary to practitioner concerns, evaluations indicate that service users appreciate being asked about ACEs, with the majority reporting that their appointment was improved as a result .

• Implementation feasibility: In England, routine or targeted ACE enquiry using the 'REACh' model has been shown to be feasible and acceptable to both staff and service users across various healthcare settings, including GP practices . Similar findings have been demonstrated in Wales and Scotland .

• Therapeutic value: ACE enquiry offers opportunities to mitigate the health impact of adversity by addressing the source of distress, potentially transforming treatment approaches for conditions with traditionally low success rates, such as addiction treatment (which has success rates as low as 10% for heroin addiction) .

These findings suggest that integrating ACE enquiry into primary care could significantly improve patient outcomes and healthcare resource utilization.

What methodological considerations are critical when implementing ACE enquiry in clinical research or practice?

Implementing ACE enquiry effectively requires careful methodological consideration:

These methodological considerations highlight the importance of a thoughtful, systematic approach to implementing ACE enquiry in research and clinical practice settings.