Small-molecule A485 mobilizes white blood cells on demand

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White blood cells, or leukocytes, are the body’s first and second lines of defense against foreign organisms and particles. However, some drugs target the production and movement of these cells for clinically useful purposes. A new study published in the journal Immunity Explores the signaling molecular landscape to identify potential druggable targets for leukocyte migration in the bloodstream.

Study: Small-molecule CBP/p300 histone acetyltransferase inhibition activates bone marrow-derived leukocytes through the endocrine stress response.  Image credit: Rost9/ShutterstockStudy: Small-molecule CBP/p300 histone acetyltransferase inhibition activates bone marrow-derived leukocytes through the endocrine stress response.. Image credit: Rost9/Shutterstock

Leukocytes, including neutrophils, monocytes, and B lymphocytes, are formed as blood-forming precursor cells in the bone marrow and in several other specialized organs. They are stored in the bone marrow until they are released into the circulation.

There are two leukocyte compartments in blood and peripheral tissues, which show changes in size with different physical conditions. For example, when the body is injured, stressed or infected, the number of leukocytes in the affected tissue changes and returns to normal after containing the threat.

Multiple regulatory steps participate in leukocyte breakdown as well as movement to various sites where they are needed. They originate in the central nervous system (CNS) in response to peripheral signals, which are regulated by neural circuits in which both the sympathetic nervous system and the hypothalamo-pituitary-adrenal (HPA) axis participate.

These signals act to increase hemopoiesis in the bone marrow, recruit leukocytes to where they are needed in the blood and other tissues, and ensure that they return to normal levels after overcoming a challenge.

In some disease conditions, this homeostatic control is lost, thus leading to abnormal numbers, such as bone marrow failure on the one hand or acute leukemia on the other. So far, however, few drugs can help correct such abnormalities by altering the rate of leukocyte production, breakdown, or migration, whether in blood cancers, chronic inflammation, or acute hyperinflammatory conditions.

Available drugs include granulocyte colony-stimulating factor (G-CSF) family, CXC-motif chemokine receptor 4 (CXCR4) antagonists such as plerixafor/AMD3100), or inhibitors of integrin very late antigen 4 (VLA4). For example, G-CSF is used to correct neutropenia in patients on chemotherapy but is less effective in patients with acute febrile illness involving low neutrophil counts. Furthermore, G-CSF may cause adverse effects in some patients.

The need to learn more about this area of ​​pharmacology motivated the present study. It focuses on a small molecule called E1A-associated protein p300 (EP300 or p300), which is found to be newly acquired in the leukemic phase of a condition called severe congenital neutropenia (SCN).

Loss of function of this gene results in reduced blood cell production if deleted before birth, but high or leukemic leukocyte counts later in life. It has an ortholog, cyclic-adenosine-monophosphate-response-element-binding protein (CREBBP, also known as “CBP”), with 90% sequence homology. One of the 8 domains of this gene is responsible for histone acetyltransferase (HAT) activity. and has a mutation in SCN that causes leukemic transformation.

In this case, this domain may be druggable to produce”Leukocytosis on demand“Different leukocyte compartments change shape.

What does the study show?

The scientists found that inhibition of the CBP/p300 domain with HAT activity by the small molecule inhibitor A485 resulted in competitive inhibition of HAT enzyme activity, particularly for CBP and p300 compared to other HATs. As expected, this led to a rapid increase in acetyl CoA levels in bone marrow macrophages in mouse models. The result was rapid leukocytosis.

It was found to have a dose-dependent action and did not dissipate with repeated administration. When another type of CBP/p300 HAT inhibitor (C646) was used, the same effect was observed, confirming the mechanism of action. Conversely, inhibition of DNA binding by proteins or other HATs found in mammals fails to induce leukocytosis.

A485 levels rapidly increased in the blood when injected into rats, accumulating in the bone marrow, adipose tissue, liver, spleen, and kidney, but not in the brain. The number of leukocytes including neutrophils, lymphocytes and monocytes increased in parallel. After one week, no evidence of drug administration was observed, suggesting a transient effect.

The increase in leukocyte counts was comparable to that achieved by G-CSF, although slightly faster for neutrophils. When both were given, significantly higher neutrophil counts resulted. However, after 24 hours, all three blood cell types were raised with G-CSF versus A485.

This indicates a smaller and different action of A485 compared to G-CSF.

To extend the observations to human subjects, the researchers looked at data from a group of patients with a rare disease called Rubinstein-Taybi syndrome (RSTS), in which CREBBP And EP300 Mutation occurs. About two-thirds had high leukocyte counts, with 70% showing mutations in the HAT domain. As expected, this group was more likely to show leukocytosis than the other group, where HAT was avoided.

Does this observation have clinical utility? To find out, they tested the effect of A485 on a group of mice with myelodysplastic syndrome (MDS) and found that the small molecule kept leukocyte counts normal. Second, they induced severe neutropenia with a course of chemotherapy in a mouse model, showing that A485 led to an acute recovery of leukocyte counts.

Then, they introduce organisms Listeria monocytogenes At doses that induce sepsis in mice with chemotherapy-induced pancytopenia. Neutrophils are essential for the immune defense against these pathogens. After the infection set in, they injected A485 Vs. Vehicle under control.

While those treated with vehicle became ill and died of sepsis, A485 single dose led to improved survival, with less bacteria recovered from treated animals. A485 mobilizes leukocytes from the bone marrow, which is the process of leukocytosis. In contrast, there was no spontaneous hematopoiesis in the bone marrow.

Different subsets of leukocytes respond to distinct pathways triggered by A485. These involve both G-CSF-dependent and -independent pathways of neutrophilia, but also other pathways of lymphocytosis.

Furthermore, A485 uses the neurohumoral pathway, specifically the HPA axis, to induce leukocytosis, as shown by increased blood glucocorticoid levels after A485 administration. The leukocytosis response resulting from HPA activation does not depend on glucocorticoids, but occurs in response to CRHR1-regulated signals, including adrenocorticotropic hormone (ACTH), with loss of HPA feedback signaling.

While neutrophils increased with ACTH administration, lymphocyte numbers increased only with glucocorticoid blockade, indicating that both are differentially regulated.

What are the effects?

Competitive, reversible, small-molecule-mediated inhibition of the CBP/p300 HAT domain triggers acute and transient leukocyte mobilization from bone marrow.“Further research is needed to identify which clinical contexts are ideal for this drug. A485 may be better if only a rapid short-term increase in neutrophils is needed, while G-CSF is needed for long-term restoration of blood cell production in the bone marrow.

Timing of administration also needs to be defined for good results because patients with neutropenic sepsis present at different times and stages. Furthermore, the value of such drugs in bacterial or viral, rather than listerial, sepsis remains unexplored.

However, as reported by previous researchers, it has anti-tumor effects, which may make it valuable in adjuvant therapy for cancer patients. The present study focuses on the role of ACTH in leukocyte homeostasis and G-CSF activity, rather than its downstream product, glucocorticoids.



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