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Live Biotherapeutics-Derived Short-Chain Fatty Acids As Potent Class I HDAC Inhibitors

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Live Biotherapeutics-Derived Short-Chain Fatty Acids As Potent Class I HDAC Inhibitors Live Biotherapeutics-derived Short-Chain Fatty Acids as Potent Class I HDAC Inhibitors Samantha Yuille, Andrei Bolocan, Parthena Fotiadou, Nicole Reichardt, Alessandro Busetti, Imke Mulder 4D pharma Research Ltd, Life Sciences Innovation Building, Cornhill Road, AB25 2SZ, Aberdeen, UK 4D pharma is a pharmaceutical company focussed on developing Live Biotherapeutic products (LBPs) from the human gut microbiome. LBPs represent a new class of drugs that contain live organisms for the prevention, treatment or cure of disease. 4D pharma has clinical stage programmes in cancer, asthma, IBS and IBD, and a strong pipeline of pre-clinical programmes in oncology, CNS disease, autoimmunity and inflammation. We have developed a multi-disciplinary functional screening platform, MicroRx®, which enables us to target specific biological functions and select bacterial candidates from our large proprietary culture collection of commensal bacterial isolates from the gut microbiome of healthy human donors. ACETYLATION Screening for histone deacetylase inhibitors AC AC AC AC Posttranslational modifications of histones play a pivotal role in the development and progression of various diseases by HDI of Gut Bacteria & SCFAs? modulating gene expression, chromatin remodelling, and nuclear architecture. Overexpression of histone deacetylase (HDAC) AC AC AC AC Butyric acid isoforms has been implicated in a variety of pathologies, such as oncological and inflammatory conditions as well as cardiovascular HATHAT HDAC INHIBITORS Valeric acid and neurodegenerative diseases. As such, HDAC inhibitors have been identified as therapeutic targets. HDACHDAC The gut microbiota is known to modulate host physiology, particularly immunology, and microbial metabolites have been Caproic acid associated with host epigenetic mechanisms. HDAC inhibition (HDI) by gut commensals has been attributed to the short chain fatty Cancer Intestinal fibrosis acid (SCFA) butyrate. However, the potent and diverse metabolic reservoir provided by the gut microbiota and its role in host Metabolic disorders Immune dysfunction disease physiology warrants further investigation in a variety of diseases. Neurodegenerative disease DEACETYLATION Inflammatory diseases Results Screening gut isolates for HDAC activity analyses SCFAs quantification of bacterial supernatants The initial screening of the cell-free supernatants (CFS) of 79 bacterial strains for total HDAC inhibitory effects on Acetate Propionate Butyrate Valeric Acid Hexanoic acid HT-29 whole cells resulted in the identification of potential HDAC inhibiting bacterial strains (Fig 1). MRX1342 200 All three bacterial strains, MRx0029, MRx0071 and MRx1342 produced butyrate (Fig 3). MRx0071 150 The most potent butyrate producer MRx0071 (25.6 mM) closely MRx0029 matches MRx0029 (butyrate 16.7 mM, valeric acid 4.4 mM) with 100 regard to HDI. This suggests a cumulative effect of butyrate and % Activity % molecules other than SCFA produced by MRx0029 being 50 0 -40 -20 20 40 involved in the observed HDI. (Fig 2) mM 0 Fig 3. SCFAs profile of three candidate strains sp sp sp sp sp sp sp sp sp sp sp sp sp sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. TSA YCFA YCFA Dorea Dorea Untreated Blautia Blautia Blautia SCFAs MRx0029 Alistipes Roseburia Prevotella Prevotella Prevotella Prevotella Prevotella Prevotella Prevotella Prevotella Prevotella Prevotella Prevotella standards MRx0029 (Fig 4), the Bariatricus Clostridium Clostridium Emergencia Emergencia Emergencia Bacteroides Bacteroides Bacteroides Bacteroides Bacteroides Bacteroides Bacteroides Bacteroides Bacteroides Bacteroides Bacteroides Bacteroides Bacteroides Bacteroides Bacteroides Bacteroides Bacteroides Bacteroides Bacteroides Bacteroides Odoribacter Longicatena Longicatena Longicatena Eubacterium Eubacterium Eubacterium Eubacterium Eubacterium Eubacterium Lactobacillus Lactobacillus Lactobacillus Lactobacillus Enterococcus Enterococcus Faecalicatena Megasphaera Megasphaera Bifidobacterium Bifidobacterium Bifidobacterium Bifidobacterium Bifidobacterium Bifidobacterium Bifidobacterium Bifidobacterium Bifidobacterium Bifidobacterium Bifidobacterium Bifidobacterium Bifidobacterium strain whose supernatant Bifidobacterium Parabacteroides Parabacteroides Parabacteroides Acidaminococcus Acidaminococcus Acetic acid Fig 1. Screening of cell free supernatants (CFS) from 79 bacterial strains for total HDAC inhibition on whole HT-29 cells. Trichostatin A (TSA) is a positive control showed the strongest Pentanoicacid HDI, was the only strain 150 Propionic acid Hexanoic acid which produced valeric HT-29 HTwhole-29 whole cells cells HT-29 cellHT- 29lysate cell lysate Butyric acid Three strains with strong HDAC acid and hexanoic acid inhibitory effect were identified as (1.0 mM) (Fig 5). Megasphaera massiliensis MRx0029, 100 Roseburia intestinalis MRx0071, and Fig 4. Gram staining of M. massiliensis MRx0029 Fig 5. HPLC SCFAs profile of M. massiliensis MRx0029 Bariatricus massiliensis MRx1342. *** ** CFS of these selected strains were 150 ** *** *** HT-29 whole HTcells-29 whole cells *** tested again to confirm their HDAC % Activity % To investigate what SCFAs were the responsible for the 50 inhibition in HT-29 whole cells and on total HDAC inhibition, different concentrations of sodium HT-29 cell lysate to confirm that the 100 butyrate, valeric acid and hexanoic acid were tested for HDAC inhibition was not a result of ** ** * HDI on whole HT-29 cells. the treatment of the cells prior to % Activity% 50 The results (Fig 6) show a significant (P<0.05) inhibition of 0 nuclear protein extraction. HDAC activity by sodium butyrate on HT-29 whole cells, TSA TSA YCFA YCFA The results in Fig 2 show a similar while hexanoic acid did not have a significant effect with 0 any of the tested concentrations. Valeric acid MRx0029 MRx0071 MRx0029 MRx0071 HDAC inhibition of the supernatants MRX1342 MRX1342 Untreated Untreated TSA 2mM 4 mM 2 mM 4 mM 2 mM concentrations above 2 mM were toxic to the HT-29 cells, YCFA 10 mM on HT-29 cell lysates as compared to 10 mM Fig 2. CFS of three selected bacterial strains tested for HDAC inhibition on HT-29 whole cell and HT-29 whole cells. Untreated thus HDI could not be measured. HT-29 cell lysates. Trichostatin A (TSA) is used as a positive control. Significances tested against Butyrate Valeric acid Hexanoic acid YCFA ** (p<0.005) *** (P<0.001). Fig 6. HPLC SCFAs profile of M. massiliensis MRx0029 HDAC inhibition by M. massiliensis MRx0029 is Correlation of SCFAs profile with specific HDI activity transferable to a microbiota model system (SimMi) 150 A) Specific inhibition of HDAC 1 MRx0029 shifted the SCFA profile of a consortium of The results from the HDAC activity assay demonstrate that 100 17 bacteria (SimMi ) towards higher butyrate the SimMi consortium + M. massiliensis MRx0029 exhibited a % Activity % Positive control **** 50 * concentrations (18.5 and 13.2 mM), and lower acetate more potent total HDAC inhibition than the standard TSA * YCFA **** (47 and 55 mM) and propionate (5.6 and 7 mM) consortium on whole HT-29 cells (p<0.001) and on HT-29 cell MRx0029 MRx0071 0 MRx1342 concentrations than SimMi without MRx0029 (Fig 7). lysate (p<0.05) (Fig 8). This demonstrates the physiologically SImMi (+MRx0029) TSA YCFA SImMi C) SimMi HDAC2 MRX0029 MRX0071 MRX1342 Butyrate 10 mM 150 relevant potential of M. massiliensis MRx0029, as a butyrate Butyrate 4mM Butyrate 2mM Butyrate 1mM Butyrate 10mM Butyrate 4 mM Positive control B) Specific inhibition of HDAC 4mM Acid Valeric 2mM Acid Valeric 2 1mM Acid Valeric Valeric Acid 10mM Acid Valeric Acetate Butyrate 2 mM 4mM Acid Hexanoic 2mM Acid Hexanoic 1mM Acid Hexanoic Hexanoic Acid 10mM Acid Hexanoic YCFA and valeric acid producing bacteria, to stimulate HDAC Butyrate 1 mM (withSimMi MRX0029) Propionate Valeric acid 10 mM **** 100 **** **** **** **** inhibition within an established bacterial community. Valeric acid 4 mM **** Butyrate **** **** SimMi Day 12 Valeric acid 2 mM **** **** **** Valeric Acid Valeric acid 1mM **** % Activity % Hexanoicacid 10mM 50 **** SimMi (+MRx0029) Day 12 Hexanoic acid HT-29 whole cells HT-29 cell lysate Hexanoic acid 4 mM Hexanoic acid 2 mM Hexanoic acid 1 mM SimMi Day 11 0 TSA YCFA D) SimMi HDAC3 MRX0029 MRX0071 MRX1342 Butyrate 4mM Butyrate 2mM Butyrate 1mM SimMi (+MRx0029) Day 11 150 Butyrate 10mM Fig 10. Inhibition of different isoforms of Class I and Class II HDACs by CFS of Positive control Valeric Acid 4mM Acid Valeric 2mM Acid Valeric 1mM Acid Valeric C) Specific inhibition of 10mM Acid Valeric HDAC 3 Hexanoic Acid 4mM Acid Hexanoic 2mM Acid Hexanoic 1mM Acid Hexanoic Hexanoic Acid 10mM Acid Hexanoic MRx0029, MRx0071, MRx1342, SimMi (+/- M. massiliensis MRx0029) and (withSimMi MRX0029) 0 50 dilutions of SCFAs butyrate, valeric acid and MCFA hexanoic acid. 100 -50 100 150 *** ** ** * **** Millimolar (mM) **** % Activity % 50 Fig 7. SCFA and MCFA concentrations of SimMi consortia (+/- MRx0029) on day 11 and 12 of continuous culture The Class I isoforms HDAC2 and HDAC3 were inhibited by 0 TSA 4 mM 1 mM YCFA 4 mM 2 mM 1 mM 4 mM 2 mM 1 mM 2 mM SimMi 10 mM 10 mM the CFS and SCFAs tested (Fig 10). 10 mM MRx0029 MRx0071 MRX1342 Untreated Within the Class I HDACs the strongest effects were Butyrate Valeric acid Hexanoic acid measured for the HDAC2 isoform, where CFS from all three SimMi (+ MRx0029)
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