EP3

Short-chain fatty acids (SCFAs), which are produced by gut bacteria from dietary fiber, regulate our immune system, but the mechanism of their action remains unknown. In a recent study, researchers investigated how SCFAs interact with mast cells, a type of white blood cell that plays a central role in allergic reactions. Their findings and insights could lead to innovative and effective anti-allergy medications, supplements, and diets, paving the way for healthier lives. The intricate relationship that exists between humans and the gut microbiome has become a hot research topic, and scientists are constantly uncovering new reasons why a healthy diet can lead to a healthier life. Dietary fibers are a particularly important aspect of this connection. When we ingest these compounds, which are mainly found in plant-based foods, our gut bacteria break them down into small molecules, called short-chain fatty acids (SCFAs). Over the past few years, studies have revealed various important anti-inflammatory and immunomodulating effects of SCFAs. One of the ways SCFAs interact with our immune system is by mediating the activation of mast cells (MCs). These white blood cells are loaded with small sacs called "granules," which are full of enzymes and signaling molecules like histamine. When an MC detects an antigen (foreign body), it becomes active and undergoes degranulation, releasing these substances to nearby tissue and triggering a fast immune response. Usually, MCs play a central role in allergic diseases, including pollinosis and severe food allergies. While there is considerable evidence that SCFAs have anti-allergic properties, the precise mechanisms by which they regulate the function of MCs remain unclear. In a recent study, a research team led by Professor Chiharu Nishiyama from Tokyo University of Science, Japan, decided to address this knowledge gap through extensive experiments on mouse MCs and SCFAs. Their findings were published in The Journal of Immunologyon January 10, 2024. The researchers initially found that feeding mice with butyric acid and valeric acid, two representative SCFAs, significantly suppressed passive cutaneous anaphylaxis (a type of well-studied allergic reaction induced artificially in laboratory settings). Using MC cultures, the team then showed that treating MCs with various SCFAs suppressed Immunoglobulin E (IgE)-mediated activation, a crucial pathway in allergic reactions. Through subsequent experiments involving genetically modified cells and precise RNA inhibitors, they managed to further piece together the puzzle. The researchers found that SCFAs mediated MC activation in mainly two ways. The first was through the recognition and interaction with the GPR109A receptor. Upon binding with SCFA, a chemical cascade ensues, culminating with the synthesis and secretion of prostaglandins. These substances interact with EP3 receptors in MCs and prevent degranulation, limiting the release of histamine and minimizing allergic response. The second was through epigenetics (or reversibly altering specific genes). The researchers confirmed that SCFAs affected histone deacetylase inhibitory activity, which regulates epigenetic modifications. This led to changes in the expression levels of the IgE-receptor, which ultimately inhibited degranulation in MCs. Additional experiments revealed that non-steroidal anti-inflammatory drugs suppressed the anti-allergic effects of SCFAs, and that vitamin B-3, which interacts with the GPR109A receptor, also inhibited MC degranulation. Together, the findings of this study can help scientists navigate the intricacies of how our bodies regulate our immune system with the help of gut bacteria. However, people with allergies stand to gain from these insights, as Prof. Nishiyama points out: "The activation of mast cell is a common cause of various allergic diseases and not limited to anaphylaxis. In addition, I think that the increasing frequency of allergic patients is associated with changes in diet in these decades. Allergies are so common that one in two Japanese people is said to have some kind of allergic disease, and the results of our study showcase dietary fibers as an effective way of treating them." Considering that about 200,000 visits to the emergency room per year are due to anaphylaxis in the United States alone, this work could not only save lives from by preventing this dangerous condition, but also help ameliorate the burden on healthcare systems. Satisfied with the results, Prof. Nishiyama concludes by noting the importance of eating well. "It is important to demonstrate with scientific evidence how dietary conditions affect health," she says. "This research has revealed part of the complex regulatory mechanisms involving various food-related components, including dietary fiber, SCFAs, polyunsaturated fatty acids, and vitamins." Notably, Prof. Nishiyama and her colleagues now intend to investigate the relationship between the mucosal environment and immunoregulation by focusing on specific SCFAs, which may lead to the uncovering of the health benefits of various foods soon.

In vitro and in vivo data show significant increased potency by blocking prostaglandin PGE2 signaling through both EP2 and EP4 receptors compared to celecoxib or single EP4 antagonists TPST-1495 anti-tumor response shown to be both immune dependent and immune independent Preclinical findings support previously reported Phase 1 clinical results for TPST-1495 showing tumor shrinkage and prolonged disease control in both monotherapy and in combination with pembrolizumab BRISBANE, Calif., July 19, 2023 (GLOBE NEWSWIRE) -- Tempest Therapeutics, Inc. (Nasdaq: TPST), a clinical-stage oncology company developing first-in-classi therapeutics that combine both targeted and immune-mediated mechanisms, announced today that in vivo and in vitro data on the unique mechanism of TPST-1495, the company’s novel dual receptor inhibitor of prostaglandin E2 (PGE2) signaling, were published in Cancer Research Communications, a journal of the American Association for Cancer Research. “While the biology of PGE2 in promoting tumor growth and immune suppression is well established, there are still no approved drugs for cancer that effectively block the prostaglandin pathway,” said Tom Dubensky, Ph.D., president of Tempest. “Our innovation with TPST-1495 shows for the first time the effect of blocking PGE2 signaling through the EP2 and EP4 pro-tumor receptors while maintaining the important anti-tumor signaling of PGE2 through the EP1 and EP3 receptors, which could be an important advance to inhibiting PGE2. Additionally, these results further support what we believe is an innovative and robust pipeline at Tempest that includes TPST-1120, a novel PPAR⍺ antagonist, which has shown early positive data from an ongoing global randomized study in first-line HCC patients.” About TPST-1495 Described in the Cancer Research Communications publication, TPST-1495 is an orally-available and potent small molecule designed to block the receptors EP2 and EP4 in the prostaglandin pathway. PGE2 both promotes tumor cell growth and has strong immune-suppressive signaling through these receptors. Several malignancies are thought to be prostaglandin driven through expression of high levels of COX-2, the cellular enzyme that produces PGE2, including endometrial, bladder, breast, colorectal, and cervical cancers. Tempest has conducted multiple studies with peripheral blood mononuclear cells (PBMCs) from healthy adult donors and in several mouse tumor models that demonstrate a significant increase in immune activation and anti-tumor potency by inhibiting both EP2 and EP4, when compared to EP4-only targeted molecules and non-steroidal anti-inflammatory drugs (NSAIDS), such as celecoxib. About Tempest Therapeutics Tempest Therapeutics is a clinical-stage oncology company advancing small molecules that combine both tumor-targeted and immune-mediated mechanisms with the potential to treat a wide range of tumors. The company has a diverse portfolio of novel programs ranging from early research to investigation in a randomized global study in first-line cancer patients. The company’s two clinical programs, TPST-1120 and TPST-1495, target PPARα and EP2/EP4, respectively, and are advancing through trials designed to study the agents as monotherapies and in combination with approved agents. TPST-1495 is currently being evaluated in combination with pembrolizumab in a Phase 1b expansion cohort in patients with advanced endometrial cancer. TPST-1120 is being evaluated in first line hepatocellular carcinoma (HCC) in combination with atezolizumab and bevacizumab, the standard of care for first-line HCC, in a Phase 1b/2 randomized global study where an early data cut demonstrated positive results in multiple categories; the company expects to receive the first full data set in the second half of 2023. Tempest is also developing an orally available inhibitor of TREX1, a target that controls activation of the cGAS/STING pathway. Tempest is headquartered in Brisbane, California. More information about Tempest can be found on the company’s website at www.tempesttx.com. Forward-Looking Statements This press release contains forward-looking statements (including within the meaning of Section 21E of the Securities Exchange Act of 1934, as amended, and Section 27A of the Securities Act of 1933, as amended (the “Securities Act”)) concerning Tempest Therapeutics, Inc. These statements may discuss goals, intentions, and expectations as to future plans, trends, events, results of operations or financial condition, or otherwise, based on current beliefs of the management of Tempest Therapeutics, as well as assumptions made by, and information currently available to, management of Tempest Therapeutics. Forward-looking statements generally include statements that are predictive in nature and depend upon or refer to future events or conditions, and include words such as “may,” “will,” “should,” “would,” “could”, “expect,” “anticipate,” “plan,” “likely,” “believe,” “estimate,” “project,” “intend,” and other similar expressions. All statements that are not historical facts are forward-looking statements, including any statements regarding: the design, initiation, progress, timing, scope and results of clinical trials; anticipated therapeutic benefit and regulatory development of Tempest Therapeutic’s product candidates; the Company’s ability to deliver on potential value-creating milestones; the Company’s guidance regarding cash runway, as well as our operational plans. Forward-looking statements are based on information available to Tempest Therapeutics as of the date hereof and are not guarantees of future performance. Any factors may cause differences between current expectations and actual results, including: unexpected safety or efficacy data observed during preclinical or clinical trials; clinical trial site activation or enrollment rates that are lower than expected; changes in expected or existing competition; changes in the regulatory environment; and unexpected litigation or other disputes. Other factors that may cause actual results to differ from those expressed or implied are discussed in greater detail in the “Risk Factors” section of the Company’s Quarterly Report on Form 10-Q for the quarter ended March 31, 2023 and other documents filed by the Company from time to time with the Securities and Exchange Commission. Except as required by applicable law, Tempest Therapeutics undertakes no obligation to revise or update any forward-looking statement, or to make any other forward-looking statements, whether as a result of new information, future events or otherwise. These forward-looking statements should not be relied upon as representing Tempest Therapeutics’ views as of any date subsequent to the date of this press release and should not be relied upon as prediction of future events. In light of the foregoing, investors are urged not to rely on any forward-looking statement in reaching any conclusion or making any investment decision about any securities of Tempest Therapeutics. Investor Contacts: Sylvia WheelerWheelhouse Life Science Advisorsswheeler@wheelhouselsa.com Aljanae Reynolds Wheelhouse Life Science Advisorsareynolds@wheelhouselsa.com ———————————————i If approved by the FDA

Researchers have identified key neurons that regulate body temperature at 37°C in mammals. This finding could pave the way for developing a technology that artificially adjusts body temperature to help treat heat stroke, hypothermia, and even obesity. A research group at Nagoya University in Japan has reported that a group of neurons, called EP3 neurons, in the preoptic area of the brain play a key role in regulating body temperature in mammals. The finding could pave the way for the development of a technology that artificially adjusts body temperature to help treat heat stroke, hypothermia, and even obesity. The new study was published in the journal Science Advances. Body temperature in humans and many other mammals is regulated at around 37°C (98.6°F), which optimizes all regulatory functions. When their body temperature noticeably deviates from the normal range, the functions are impaired, which could lead to heat stroke, hypothermia, and, in the worst case, death. However, these conditions might be treated if body temperature can be artificially adjusted to the normal range. The brain's temperature regulation center resides in the preoptic area, a part of the hypothalamus that controls the body's vital functions. For example, when the preoptic area receives signals from a mediator called prostaglandin E (PGE2) that is produced in response to infections, this area releases a command to raise body temperature to fight against viruses, bacteria, and other disease-causing organisms. However, it is still unclear exactly which neurons in the preoptic area release commands to increase or decrease body temperature. To identify such neurons, Professor Kazuhiro Nakamura, Lecturer Yoshiko Nakamura, and their colleagues at Nagoya University, in collaboration with Professor Hiroyuki Hioki at Juntendo University, conducted a study using rats. They focused on EP3 neurons in the preoptic area, which express EP3 receptors of PGE2, and investigated the function for regulating body temperature. Professor Nakamura and his colleagues first investigated how the activity of EP3 neurons in the preoptic area varies in response to changes in ambient temperature. A comfortable environmental temperature for rats is around 28°C. For two hours, the researchers exposed the rats to cold (4°C), room (24°C) and hot (36°C) temperatures. Results showed that exposure to 36°C activated EP3 neurons, while exposure to 4°C and 24°C did not. The group then observed nerve fibers of EP3 neurons in the preoptic area to identify where the signals from EP3 neurons are transmitted. The observation revealed that nerve fibers are distributed to various brain regions, particularly to the dorsomedial hypothalamus (DMH), which activates the sympathetic nervous system. Their analysis also showed that the substance that EP3 neurons use for the signal transmission to DMH is gamma-aminobutyric acid (GABA), a major inhibitor of neuronal excitation. To further investigate the role of EP3 neurons in temperature regulation, researchers artificially manipulated their activity using a chemogenetic approach. They found that activating the neurons led to a decrease in body temperature, whereas suppressing their activity led to their increase. Taken together, this study showed that EP3 neurons in the preoptic area play a key role in regulating body temperature by releasing GABA to send inhibitory signals to DMH neurons to control sympathetic responses. "Probably, EP3 neurons in the preoptic area can precisely regulate the signal strength to fine-tune body temperature," said Prof. Nakamura, the lead author of the study. "For example, in a hot environment, signals are augmented to suppress sympathetic outputs, resulting in increased blood flows in the skin to facilitate the radiation of the body's heat to prevent heat stroke. However, in a cold environment, signals are reduced to activate sympathetic outputs, which promote heat production in brown adipose tissue and other organs to prevent hypothermia. Furthermore, at the time of infection, PGE2 acts on EP3 neurons to suppress their activity, resulting in activation of sympathetic outputs to develop fever." This study's findings could pave the way for the development of a technology that artificially adjusts body temperature, which can be applied to a wide range of medical fields. Interestingly, this technology may be helpful in the treatment of obesity, by keeping body temperature slightly higher than normal to promote fat burning. "On top of that, this technology could lead to new strategies for survival of people in hotter global environments, which are becoming a serious worldwide problem," said Prof. Nakamura.