June 25, 2022

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Development of allosteric drugs targeting calcium-sensitive receptors

Progress in research and development of allosteric drugs targeting calcium-sensitive receptors


Development of allosteric drugs targeting calcium-sensitive receptors.   CaSR (Calcium-Sensing Receptor) is widely expressed in the human body, especially in the parathyroid glands and kidneys. In these tissues, CaSR is responsible for the precise control of extracellular Ca2+ to keep the free Ca2+ concentration in the whole body within 1.2-1.4mM.

CaSR can negatively regulate the secretion of parathyroid hormone (PTH) due to the increase of Ca2+ concentration. In the kidney, CaSR will respond independently to PTH to the elevated concentration of Ca2+, further reducing the reabsorption of Ca2+. At present, CaSR has been found to play an important role in hyperparathyroidism, hypercalcemia, osteoporosis and other diseases.

It has screened and optimized a variety of active molecules that regulate the function of CaSR through allosteric. Some molecules have been used for the treatment of the above Diseases, and some molecules are in the preclinical and clinical research stages.

Development of allosteric drugs targeting calcium-sensitive receptors


From the perspective of signal pathways, CaSR inhibits adenylate cyclase (AC) by activating Gi/o protein, thereby reducing the level of cAMP. At the same time, the coupling of CaSR and Gq/11 protein will activate Phosphoinositide-specific phospholipase C (PI-PLC), thereby increasing Inositol Triphosphate (IP3) and Diacylglycerol ( Diacylglycerol, DAG) and trigger the release of stored Ca2+. Furthermore, the released Ca2+ activates phospholipase A2 (Phospholipase A2, PLA2) and PKC (Protein kinase C).

In addition, CaSR can increase the level of Ca2+ through L-type voltage gating and the influx of Ca2+ in the transient receptor potential ion channel (Ion channel, IC). In addition, CaSR also activates the MAPK signal cascade through Gq/11 protein-mediated PKC, Gi/o protein-mediated epidermal growth factor receptor (EGFR) activation and β inhibitory protein.

Development of allosteric drugs targeting calcium-sensitive receptors

From the structural point of view, the CaSR protein is encoded by 7 exons and expressed as a 7-pass transmembrane receptor protein of 1078 amino acids. Among them, the 2nd-6th exons encode the extracellular domain (ECD), and the 7th exon encodes the 7 transmembrane (7TM) region and the C-terminal tail. Studies have found that CaSR functions as a homodimer mediated by covalent and non-covalent interactions.

In ECD, it contains a two-leaf “Venus flytrap” (VFT) domain. It is so called because its two leaves (called LB1 and LB2 respectively) can open and close around the gap where the ligand binds. Like the Venus flytrap closes around the prey. VFT is connected to 7TM and its connected intracellular and extracellular loops (called ICL and ECL, respectively) through a Cysteine-rich domain (CRD).

The structural integrity of 7TM is partly maintained by the disulfide bond between C677 in ECL1 and C765 in ECL2. 7TM is connected to a large C-terminal tail, which facilitates cell surface expression, signal transduction, and binding to accessory proteins.

Development of allosteric drugs targeting calcium-sensitive receptors


The endogenous ligand of CaSR and its binding site

Ca2+ is the main physiological ligand of CaSR. X-ray crystallography combined with scattering analysis shows that Ca2+ will bind to the four sites in the VFT domain in a cooperative manner, that is, the binding of Ca2+ to one of the sites will promote the binding of Ca2+ to other sites. There is evidence that Ca2+ still retains agonist activity on CaSR lacking the entire ECD, so it can be speculated that Ca2+ also binds to at least one site in 7TM or ECL. Therefore, although Ca2+ is considered to be an orthostatic agonist, strictly speaking Ca2+ is its own allosteric modulator. In addition to Ca2+, CaSR can also respond to other divalent cations such as Mg2+, and trivalent cations such as Gd3+.

The VFT cleft of CaSR is the binding site of endogenous positive allosteric modulators (PAM), such as aromatic L-amino acids and γ-glutamine peptides, especially L-phenylpropanol Amino acid and L-tryptophan are the most effective PAM on CaSR. X-ray crystallographic analysis of the CaSR VFT structure shows that only when the gap is occupied by L-amino acids or similar entities, a fully active (closed state) VFT conformation can be produced. Other positively charged endogenous ligands will also return to activity through the allosteric site of CaSR, such as spermine, spermidine and putrescine.
The exogenous PAM molecule of CaSR and its allosteric site

Studies have found that calcium channel blockers fendiline and prenylamine can target CaSR. On this basis, new arylalkylamine PAMs that produce CaSR are optimized, including NPS R -467, NPS R-568, calcimimetics, evocalcet, calindol and its derivatives, etc. These arylalkylamine PAMs can enhance CaSR-mediated Ca2+ transfer in recombinant cells, or inhibit PTH secretion by parathyroid cells in the presence of physiological Ca2+ concentration, but their efficacy or affinity will be lower in the absence of Ca2+ reduce.

In 2010, Acadia Pharmaceuticals discovered the benzothiazole-based CaSR PAM, which is different in structure and chemical properties from the arylalkylamine PAM, and optimized the development of AC265347. AC265347 also exhibits allosteric agonist activity in the absence of cations, and its activity is more efficient than arylalkylamine PAM. In particular, AC265347 is also a biased CaSR allosteric modulator. Compared with Ca2+ transfer, it preferentially enhances CaSR-mediated ERK1/2 phosphorylation (pERK1/2). Docking studies have shown that AC265347 is deeper in the center of 7TM compared with arylalkylamine PAM, which allows AC265347 to stabilize different CaSR conformational states, thereby producing biased CaSR signal transduction.


CaSR’s polypeptide type PAM veracapeptide (Etelcalcetide)

In addition to small-molecular PAMs, peptide-based CaSR PAMs, such as Etelcalcetide, have also been discovered. As an octapeptide, it is produced by connecting a linear chain composed of seven D-amino acids to L-cysteine ​​through disulfide bonds. It is predicted that Etelcalcetide forms a disulfide bond with C482 near the “hinge” region of VFT-LB1, thereby binding to CaSR-VFT and mediating the closure of VFT. In the absence of Ca2+, Etelcalcetide activity in HEK293 cells is significantly reduced, but it still retains agonist activity, so it is a PAM agonist.
The exogenous NAM active molecule of CaSR and its allosteric site

NPS Pharmaceuticals has discovered the first CaSR NAM (Negative allosteric modulators)-NPS2143 through high-throughput screening and medicinal chemical modification, which has an arylalkylamine skeleton in its structure. Subsequent clinical studies on arylalkylamine NAM found several NAMs similar in structure and chemical properties to NPS2143, including ronacaleret, JTT305 (also known as MK3552) and NPSP795. According to the docking model, aralkylamine NAM and aralkylamine PAM are likely to act in the same 7TM cavity of CaSR, but the specific model requires further structural biology research to clarify the structure and function of CaSR. Differential adjustment.

In addition to arylalkylamine NAMs, Novartis also discovered a CaSR NAM compound containing quinazolinones, and further optimized them to obtain ATF936 and AXT914. Compared with NPS2143, ATF936 has a greater negative synergy, which means that it can better block CaSR activity. Mutation and docking studies have shown that NAMs containing quinazolinones can bind CaSR in the 7TM allosteric cavity in a different way from arylalkylamine PAMs and NAMs.


CaSR’s PAM/NAM mode switching allosteric active molecule

Calhex231 was discovered from a SAR study based on CaSR PAM calindol. Unexpectedly, calhex231 was reported as NAM in the past because it can inhibit the maximum effective concentration of Ca2+. However, recent research work has shown that the function of calhex231 can be switched between PAM and NAM, depending on whether it occupies one or two of the CaSR dimers. The binding site of calhex231 overlaps with the binding sites of cinacalcet, NPS2143 and other arylalkylamines PAM and NAM. The structural basis for its mode switching is mainly from the disubstituted cyclohexane ring.


Study on the clinical indications of CaSR allosteric modulators

1. CaSR PAM is used to treat hyperparathyroidism

CaSR plays a key role in negative regulation of PTH secretion. Primary hyperparathyroidism (Primary hyperparathyroidism, PHPT) is usually caused by parathyroid adenoma or cancer; secondary hyperparathyroidism (Secondary hyperparathyroidism, SHPT) is usually caused by phosphoric acid in chronic kidney disease Salt excretion and impaired synthesis of 1,25-dihydroxyvitamin D3 in the kidneys lead to a decrease in Ca2+, which subsequently leads to increased synthesis and secretion of PTH and hyperplasia of parathyroid glands. At present, there are 3 kinds of CaSR PAM for the treatment of hyperparathyroidism on the market.
In 2004, Cinacalcet (Sensipar) was the first CaSR targeted drug approved by the FDA for the treatment of chronic kidney disease caused by SHPT in hemodialysis patients. Cinacalcet is also the first GPCR allosteric modulator approved by the FDA. Although approximately 30% of patients experience gastrointestinal adverse reactions, including nausea, vomiting, or loss of appetite, in general, Cinacalcet is generally safe and well tolerated.

In 2017, the FDA approved eticacitide (Parsabiv) for intravenous CaSR PAM for the treatment of adult SHPT. Compared with oral cinacalcet, intravenous eterkaser can reduce gastrointestinal adverse events, but there is no significant difference in the self-reported nausea and vomiting symptoms of SHPT patients taking etercase or cinacalcet.

Recently, Evocalcet (alternative names MT-4580 and KHK7580) was approved for use in Japanese SHPT patients who have not responded to cinacalcet treatment. Compared with cinacalcet, Evocalcet has a higher bioavailability, so a lower dose is required to inhibit serum PTH levels. In humans, Evocalcet provides good short-term tolerance in upper gastrointestinal symptoms, while still providing similar therapeutic effects as cinacalcet.

Although all three clinically approved CaSR-PAMs can effectively reduce PTH levels in patients with hyperparathyroidism, the risk of hypocalcemia and the incidence of gastrointestinal side effects limit their clinical application. Therefore, there is still a need for new PAMs with fewer side effects.


2. CaSR PAMs are used to treat hypercalcemia

Familial hypocalciuric hypercalcaemia types 1, FHH1 and neonatal severe primary hyperparathyroidism (NSHPT) are caused by the inactivation of CaSR mutations. These mutations reduce the sensitivity of CaSR to Ca2+, or impair the biosynthesis and post-translational processing of CaSR in the endoplasmic reticulum or Golgi, leading to misfolding of CaSR and impaired cell surface expression. FHH1 is characterized by mild or moderately elevated serum calcium and magnesium, and mildly elevated or normal PTH levels. Although FHH1 patients are usually asymptomatic, up to 30% of patients develop symptomatic hypercalcemia, while others develop chondrocalcinosis, acute pancreatitis, and gallstones.

In patients with loss of function or expression loss caused by CaSR mutations, cinacalcet is increasingly successful in the treatment of NHSPT and FHH1 related complications. However, there are more and more reports that NSHPT patients do not respond adequately to cinacalcet. This may be due to homozygous mutations that cause CaSR to be truncated before the cinacalcet binding site in 7TM, or there may be missense mutations or in-frame deletions. , The expressed full-length CaSR has a single amino acid mutation or is shortened, thereby reducing the affinity of cinacalcet or enhancing the ability of Ca2+ by reducing allosteric synergy.
Interestingly, compared with cinacalcet, AC265347 is more effective in enhancing the Ca2+-mediated signal response of certain CaSR mutations. However, because AC265347 has not been clinically approved, it is still necessary to perform total parathyroidectomy for patients who do not respond to current drug interventions to normalize their serum parathyroid hormone levels.


3. CaSR NAMs are used to treat osteoporosis

NPS2143 is the first CaSR NAM evaluated in an ovariectomized rat model of postmenopausal osteoporosis. However, after 5 weeks of administration of NPS2143, no net increase in bone mass and density was observed. Moreover, the large distribution of NPS2143 leads to the prolonged exposure time of NPS2143 and the continuous increase of PTH level, which will simulate hyperparathyroidism, thereby stimulating bone resorption at the expense of bone formation.

Ronacaleret is the second CaSR NAM to be evaluated in osteoporosis. Although ronacaleret is similar in structure to NPS2143, its metabolism is more unstable. Due to the lack of effect of AXT914 on bone formation markers and the increased dose-limiting of serum calcium after four weeks of treatment, the clinical trial of AXT914 was also terminated early.

The reason why CaSR-NAMs do not stimulate bone formation is not fully understood, but it may be related to the targeted CaSR effect in extraparathyroid cells and tissues. For example, CaSR-NAMs may inhibit the important role of CaSR in osteoblasts, thereby counteracting the effects of transient release of PTH. In addition, although the pharmacokinetic characteristics of ronacaleret, JTT305, and AXT914 are more favorable than NPS2143, the NAM-mediated increase in serum PTH concentration in humans continues to exceed the baseline for 3.5 hours. Regardless of the cause of the clinical failure of CaSR-NAM, the development of NAMs for osteoporosis has stopped, and efforts have been focused on reusing CaSR-NAMs for alternative diseases.


4. CaSR NAMs are used to treat ADH and Bartter syndrome Ⅴ

Autosomal dominant hypocalcaemia type 1 (Autosomal dominant hypocalcaemia type 1, ADH1) is caused by CaSR mutation, and Bartter syndrome V is caused by CaSR mutation or ADH2 (GNA11 mutation). ADH is characterized by a mild or moderate decrease in serum calcium and PTH concentrations. In more severe cases, gain-of-function CaSR mutations promote the loss of renal sodium, potassium, magnesium, and chloride ions, leading to hypokalemic alkalosis and hyperrenin hyperaldosteronism. These symptoms are called Bartter syndrome V .

NPS2143 is expected to normalize the signal response related to CaSR and GNA11 mutations caused by ADH in vitro, increase Ca2+ and PTH concentrations in ADH1 and ADH2 mouse models, and prevent nephrocalcinosis in mice. However, NPS2143 is less effective in correcting the CaSR mutation that causes Bartter syndrome in vitro. In contrast, quinazolinone-derived NAMs (such as AXT914 and ATF936) can better correct Bartter syndrome V mutations in vitro, and may represent a class of NAMs derived from aralkylamines (such as NPS2143) , NAMs that are less prone to be affected by pharmacogenetic effects.

Although originally developed for osteoporosis, the NAM derived from arylalkylamine, NPSP795 has entered a phase II clinical trial for the treatment of ADH1. NPSP795 significantly increased PTH in 3 of 5 ADH1 trial patients, and caused a slight decrease in renal Ca2+ excretion. However, NPSP795 has no significant effect on the serum Ca2+ level, and the effect on PTH is also different. The reason remains to be determined.


5. CaSR NAMs are used to treat asthma

Asthma affects approximately 340 million people worldwide, and poses a serious health risk to approximately 10% of asthma patients (whose current drugs cannot effectively control asthma). CaSR is expressed in bronchial smooth muscle and epithelium. Recently, CaSR has been recognized as a potential target for the treatment of asthma. In bronchial biopsies of asthma patients, mouse asthma models, and airway smooth muscle cells exposed to asthma-related cytokines, the expression of CaSR is up-regulated, which may be achieved by the STAT and κB response elements in the CaSR gene promoter. CaSR NAM NPS2143 attenuates the release of Ca2+ in airway smooth muscle cells that respond to acetylcholine or histamine, suggesting the potential benefit of anti-CaSR signaling in asthma. In mouse models of allergic respiratory diseases, long-term treatment of CaSR-NAMs can reduce airway inflammation, fibrosis, and airway hyper-responsiveness to the muscarinic acetylcholine receptor agonist methacholine (Airway hyper-responsiveness, AHR). Recent research results show that in chronic airway inflammation models, NAMs ronacaleret, JTT-305, NPSP795 and AXT914 can all reduce airway inflammation and prevent goblet cell proliferation.


Summary and future outlook

CaSR is a multi-modal transmembrane chemical sensor that can respond to a variety of exogenous and endogenous stimuli through different allosteric sites.

Although people have made great efforts to target CaSR therapies, so far, only CaSR PAMs have entered the clinic. Although CaSR NAMs are promising as a treatment for ADH, it is still necessary to determine the reasons for the potential differences in patient response to NAMs in order to promote the development of such compounds.

Therefore, we still have a lot to understand about how CaSR NAMs bind to receptors and how naturally occurring mutations change the binding and function of NAMs.


(source:internet, reference only)

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