(2/2) Orjala Lecture: Chemistry of Antihypertensive I RAAS Biochemistry •Angiotensinogen (452AA) is a large protein constitutively released by the liver into the blood stream. •Renin is a protease that is released by the kidney upon reception of Hemodynamic signals

- Signal: ß Glom. filtration, ßIntravascular volume à Renin cleaves off a minute portion of Angiotensinogen, creating Angiotensin-I (10AA, AngI). à AngI is further catabolized by Angiotensin Converting Enzyme (ACE) to form the octa-peptide Angiotensin-II (8AA, AngII). •AngII: The major pressor of the RAAS system. It elicits its effects by binding to the Angiotensin Receptors (AT). AT1 is the most well characterized AT receptor, and has the most significant and diffuse effects upon activation. •AT1 Receptor activation will affect the:

- Kidney: ß GFR, Increased Na+/H2O reabsorption - Adrenal: Increased Aldosterone secretion - Vascular Smooth Muscle: Constrict, BPÝ - Heart: Myocardial Ý Contractility

RAAS Drug Targets - Renin, ACE, and the receptors of both aldosterone and AngII are ideal targets for drug therapy. Renin is the rate-

limiting enzyme (RLE), and is \ the optimal target site. Unfortunately, thus far it is the hardest druggable target - Bigger Picture: The RAAS Pathway is far bigger than previously described. AngI and AngII may be acted upon

by many more enzymes, such as Carboxypeptidase, Aminopeptidases, and even ACE-II… capable of forming products such as AngIII, AngI-9, AngI-7. But forget all of these, the only clinically relevant target is the ACE conversion of AngI à AngII. The resulting drugs have been titled ACE-Inhibitors.

Angiotensin Converting Enzyme (ACE) Structure: The ACE active site is located centrally. At the center is a Zinc Ion and 2 Chloride ions. The Zinc is essential for catalytic activity, and is precisely where ACE-I bind. Activity: ACE is a non-specific, Zinc-dependent, dipeptidyl carboxypeptidase • ‘Carboxypeptidase’ From the carboxy side… • ‘dipeptidyl’ It chops off 2 AA… • ‘Zinc-dependent’ Using Zn for its activity… • ‘relatively’ Unless penultimate Proline AngI Catalysis: ACE Cleaves at Phe8-His9

- Penultimate Proline: (Penultimate = 2nd to last). The only structural feature required by ACE is that the penultimate AA in the peptide substrate is not Proline. \, AngII is not further metabolized by ACE.

- Bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg): Vasodilator, Bronchoconstrictor. Metabolized by ACE. o ACE-I therapy can cause Bradykinin accumulation, bronchoconstriction, and a persistent dry cough!

The Discovery of Captopril and Beyond - Source: Bites by the South American Pit Viper (Bothrops jararaca) produced severe hypotension. So naturally we

find a bunch of snakes and milk them dry. We found the BP culprit, Teprotide (9AA). Turns out it inhibits ACE with a penultimate proline!

- Resources: Using what we knew about pancreatic enzyme Carboxipeptidase A (zinc-dependent monopeptidyl), we discovered that 1 carboxylate is for recognition (carboxypeptidase) and 1 carboxylate is to chelate Zinc

- Captopril: Thiol moieties are far stronger chelators than carboxylates, so boom we made captopril o (1) Specific Stereochemistry: Methyl group stereoconfiguration is important (L-AA) o (2) Sulfhydryl (R-SH): Essential for chelating to the Zinc ion… though many AE

- I say thiol, you think: Change in taste perception, produces a metallic taste. Thiols also have a high incidence of skin rashes and irritation. The AE and efficacy of thiols is largely linked to the sulfur metabolic interactions

- Metabolism of Captopril: Stomach acid may catalyze dimerization of captopril with other sulfhydryl groups – such as itself or ingested proteins. Take on an empty stomach! Disulfide dimers may induce allergic skin reactions

ACE Inhibitor Structure-Activity Relationships Dicarboxylate-Containing ACE-I

R1 : Methyl. (Lisinopril has (CH2)4NH2). Binds to S1’ site. R2 : Ethyl. ‘prodrug part – cleaved’ Binds to Zn upon bioactivation R3 : Phenyl group is the most potent. Binds to S1 site. Ring: Carbocyclic Acidic AA Binds to S2’ site.

- Binding of AngI to ACE o S1: Phe8 extends into the Aryl binding site (Pi-Pi) o S1’: His9 engages in dipole-dipole interactions with Glu162 o S2’: Pro7 carboxylate has ionic interactions with Lys511

- Binding of Lisinopril to ACE o S1: Phenethyl reaches further into the S1 aryl binding pocket

§ Phenyl ring is essential. To compensate for the SàCOO- substitution (CaptoprilàLisinopril), Phenyl ring support.

o S1’: Amine chain has ionic interactions at Glu162 o S2’: Proline Carboxylate has ionic interactions with Tyr520/Lys511 o Zinc: Terminal carboxylate forms an ionic bond with Zn

- Bioavailability: Dicarboxylate ACE-I used orally are formulated as ester prodrugs. They are bioactivated by esterases in the blood, yielding the COO-. Bioavailability is mostly determined by the S2’ group

o Unique Lisinopril: Lisinopril is dosed as the COO- formulation. Due to its structure, the ‘Lys’ amine and carboxyl side chains neutralize àß

- Proving R3 Phenyl’s Importance: Enalapril is bioactivated to Enalaprilat by hepatic esterases. Predictably less potent due to the Sulfhydryl to carboxylate switch, it is actually more potent due to the R3 phenyethyl side chain. The additional S1 binding activity greatly improves ACE-I potency

- Metabolism: Renal. Majority of dicarboxylate-containing ACE-I are excreted unchanged, though there are some metabolites. Renal elimination is either supported by (1) Glucuronidation, or (2) Cyclization

o (1) Glucuronidation depends on how extended the Ring group carboxylate is. Benazepril is glucuronidated as it is extended, whereas Ramipril is not.

o (2) Ramipril, Moexipril, and perindopril undergo cyclization to produce diketopiperazine metabolites

Phosphonate-Containing ACE-I - Pull up your socks and brace yourself, ‘Phosphorous binds better to Zinc than Sulfur.’ - MoA: The phosphinic acid moiety (POO-) mimics the tetrahedral intermediate produced during ACE peptide

hydrolysis. It has a higher affinity for ACE. Drug: Fosinopril (bioactivated by esterase to Fosinoprilat) Angiotensin II Receptor (AT) Antagonists (Blockers, ARBs)

- There are at least 4 known subtypes of AT receptors, though the only clinical relevant one is AT1. They are GPCRs. Natively, AngII controls peripheral vasoconstriction and aldosterone secretion.

AngII-Peptide Analogues: Synthesized to be antagonists, poor bioavailability limited their use as antihypertensive agents - MoA: A Sarcosine group, aka N-methylglycine, was substituted for AngII Asp1. They’re not really used.

-Sartan ARBs - Lead: S-8308 was the lead compound. It had poor antagonistic activity (IC = 15µM) - SARs:

o Acidic Group: Essential component, required to compensate for EITHER the Tyr4 or Asp1 of AngII

§ Ex: Eprosartan has Benzoic Acid to mimic the Tyr4 Phenol o Biphenyl Series: Two phenyl rings, acidic group must be in the ortho-

position for optimal activity § The acidic group is either carboxylate or tetrazole. A tetrazole

moiety is a bioisosteric replacement for a carboxylate group, but with superior metabolic stability, lipophilicity, and oral bioavailability.

o Thienyl methyl: A sulfur-based aromatic compound used to mimic Phe8, à Pi-pi stacking (Eprosartan)

- Losartan (Cozaar): The overall cardiovascular effects seen with losartan are due to the combined actions of the parent drug and the 14% of it oxidized to active metabolites that are 10-40x more potent

o Metabolism: CYP2C9/3A4 oxidizes the imidazole-methanol to carboxylic acid à more potent.

o Structure: This is a biphenyl series –sartan that has a tetrazole in the ortho-position as its ‘acidic group’ and a chlorine+methanol to hydrogen bond with the active site Phe8

- Candesartan Cilexetil (Atacand) and Olmesartan medoxomil (Benicar) are prodrugs, as conferred by their flamboyant Oxygen groups. These are highly reactive and are cleaved off quickly by esterases. They are completely metabolized to their active metaoblites in the intestinal walls.

- Azilisartan medoxomil (Edarbi): With similar prodrug behavior as to Candesartan, Azilisartan is completely metabolized to the active metabolite in the intestinal wall. In this compound, the tetrazole ring is replaced by an oxo-oxadiazole ring which is less acidic. Surprisingly, this binds more tightly to AT1 than other ARBs and dissociates more slowly.

o Shows better control of 24h systolic BP than the other ARBs. Renin Inhibitors: As discussed at the beginning of the lecture, Renin catalyzes the RLE of the RAAS system followed by secretion from the juxtaglomerular kidney cells. It is a proteolytic enzyme, converting Ang à AngI. Classic drug design would select Renin as the ideal target, however all efforts have failed.

- Enzyme Activity: Renin cleaves between Leu10 and Val11, two hydrophobic AA. - Aliskiren (Tekturna): Orally available selective inhibitor of renin, the first and only approved. It is contraX with

ARBs/ACE-I in patients with diabetes. It has a stacking group (NCCO) to bind in the active site. Thus far, the drug is not really working too well.

(2/5) Orjala Lecture: Chemistry of Antihypertensives II Calcium Channel Blockers (CCB): CCBs, or calcium antagonists, act by decreasing Ca2+ ion entry into excitable nerve and muscle cells through the Voltage-dependent and Time-dependent slow L-type Calcium Channels. As such, they affect muscular contraction, smooth muscle vasculature, and cardiac muscle contractility. Currently, there are 10 CCB available in the US for therapeutic use. They are grouped into 4 classes:

- 1,4-dihydro-pyridines (1,4-DHPs): Nifedipine + 7 others! Largest class of CCB - Phenylalkylamines (PAA): Verapamil only. Better affinity for the heart Ca2+ channels - Benzothiazepines (BTZ): Diltiazem only. Better affinity for the heart Ca2+ channels - Diaminopropranol ethers (DAPE): Bepridil only. AE. No longer marketed.

CCB Selectivity and MoA - CCB are selective for the L-type high-voltage-activated calcium channels (except Bepridil). Calcium influx is

influenced through binding to specific sites located within the channels. - What’s the deal? It’s just Ca2+!? Intracellular [Ca2+] is in the nanomolar range, whereas extracellular it is

millimolar. As such, Ca2+ is a significant second messenger. - General MoA: Ca2+ channels can exists in 3 different conformations: (1) Open, (2) Inactive, (3) Resting

Resting: Following a recovery period, the channel waits in the closed conformation for arrival of the next action potential (AP). à Arrival and propagation of an AP is modulated by Na2+, significantly changing the resting membrane potential and activating the voltage-dependent L-type Ca2+ channel Open: Ca2+ influx occurs only for a short duration before rapidly becoming inactivated Inactivated: The L-type Ca2+ channel will need to recover before being activated again Use-Dependency: CCB are more effective when the Ca2+ are used more frequently. Meaning, longer, more intense, and more frequent membrane depolarizations will make

them more effective. CCB preferentially interact with their receptors in the open or inactive state. - Sites of Drug-Action: On the a-1 subunit of the Ca2+ channel, there are 3 different binding sites

o These sites are allosterically-interacting, meaning activity at one site affects the others. § Negative Allosterism: If bound, Verapamil will prevent BTZ and 1,4DHP binding, +vice versa

• ContraX: \ these cannot be used together § Positive Allosterism: If bound, BTZ and 1,4DHP will enhance the binding of each other.

• This can be used together, however stringent therapeutic windows prevent safe use.