The OCTOPUS Project: Open Collaboration for Transformative Open Pedagogy to support Undergraduate Open Science Education

As part of the ORCA-funded OCTOPUS project, we are developing guidelines for student use of open lab notebooks.

Student Lab Notebooks and Open Science

Topic: 

Accessible and Open Electronic Lab Notebooks using Pressbooks and Existing H5P Resources

Why?

Documentation of lab procedures and results is critical for reproducible science. While a variety of electronic lab notebooks are commercially available, the cost may be prohibitive for institutions and an additional financial burden for students. Free options have limitations or are cumbersome to implement in a classroom. Importantly, this is an opportunity to integrate documentation training into an open science curriculum and leverage existing knowledge available on Pressbooks. 

How?

We will identify, adapt, and develop resources to help instructors explain to students the importance of lab documentation and its connections to open science. We will create an H5P activity that is modified to allow students to submit lab entries, as well as provide the necessary training for instructors and students to use and improve the system.

For relevant background please see relevant page: Assessing Ovastacin and Fetuin B as a Non-Hormonal Contraceptive Targets  (https://openlabnotebooks.org/assessing-ovastacin-an-fetuin-b-as-a-non-hormonal-contraceptive-targets/)

Donor Plasmid:

pFastBac-Dual vector was used for the dual expression of hOvastacin-twinStrep (driven by polyhedrin promoter) and hFetuin B-Fc-6xHis (driven by p10 promoter).

Recombinant bacmid DNA preparation:

• DH10Bac coli was used for the production of the recombinant bacmid (1ng donor plasmid to 100ul DH10Bac competent cells);
• The LB medium containing kanamycin, gentamicin, tetracycline, X-β-Gal, and IPTG for Blue-White screening was used to pre-select the coli colonies with transposed bacmid, and the white colonies were then confirmed by re-spreading on the fresh LB medium containing the above mentioned antibiotics, X-β-Gal, and IPTG;

50 ug/ml kanamycin

7 ug/ml gentamicin

10 ug/ml tetracycline

200 ug/ml X-β-Gal

40 ug/ml IPTG

• Double-check the pre-selected white colonies by performing colony PCR using pUC/M13 Forward and Reverse primers (the PCR amplified DNA band from the negative colonies should be around 350bp, while the DNA band from the positive colonies should be 350bp plus the size of the DNA inserted, which is around 2kb);

pUC/M13 Forward primer: 5′-CCCAGTCACGACGTTGTAAAACG-3′

pUC/M13 Reverse primer:  5′-AGCGGATAACAATTTCACACAGG-3′

• High-pure extraction of the recombinant bacmid DNA from the positive colony identified.

Baculovirus preparation:

• Pre-incubate the insect cell sf9 (in Insect-XPRESS protein-free cell medium with L-glutamine), make sure the cells are healthy with greater than 95% viability and are growing in the logarithmic phase with a density of 2×10^6 cells/ml before proceeding to transfection;
• Prepare 2ml of the insect cells (8×10^5 cells/ml) in a 6-well tissue culture plate, transfect the cells with 1ug of the purified recombinant bacmid DNA using Cellfectin® II Reagent;
• Incubate the transfected insect cells at 27°C for 72 hours, and harvest the supernatant to fresh 15 ml snap-cap tubes, centrifuge the cells at 800g x 5min to collect the supernatant (P1 virus stock, around 2ml). Store the P1 virus stock at 4°C, protect from light;
• Amplify the P1 viral stock in suspension culture at 2×10^6 cells/ml to get a higher titer of P2 viral stock (50ml medium with 2ml P1 virus stock in a 1L flask). Make sure that the cells used are healthy, and have >95% viability before proceeding to infection. Incubate the cells in a shaker with a setting of 27°C, 170rpm for 72h. Centrifuge the transfected cells at 800g x 5min to collect the supernatant (P2 viral stock). Store the virus stock at 4°C (protect from light) for recombinant protein production. Amplify the P2 virus to get a large volume of P3 virus if necessary.

Co-expression and purification of hFetuin B-Fc-6xHis and hOvastacin-twinStrep:

• To express the recombinant protein, infect 2L of sf9 cells with Baculovirus (each of the 500ml sf9 suspension cell cultures with 12ml P2 or P3 baculovirus). Make sure the cells are healthy with greater than 95% viability and are growing in the logarithmic phase with a density of 2×10^6 cells/ml. Incubate the infected cells in a shaker with a setting of 27°C, 170rpm for 72h;
• Centrifuge the cell culture at 1000g x 20min, RT, collect the supernatant (hFetuin B-Fc-6xHis and hOvastacin-twinStrep are secreted proteins) to a beaker, add solid ammonium sulfate slowly with gentle agitation (allow to dissolve before adding more solid, try to prevent foaming) to get a final 60% saturated buffer solution. Let the protein precipitate in ammonium sulfate buffer at 4°C overnight with gentle agitation;
• Centrifuge the overnight precipitated buffer at 17000g for 2h, 4°C, carefully discard the supernatant and leave the precipitation, dissolve the precipitation with 200ml (1/10 volume relative to the cell culture used) of loading buffer;
• Dialysis the dissolved precipitation buffer solution in 2L loading buffer at 4°C for 4h with gentle agitation. An extra centrifuge step (8000g for 20min at 4°C) is necessary if the solution is turbid, otherwise not necessary;

• Ni-IMAC purification of protein:

1. Rinse the Nickel (Ni) beads with loading buffer (pre-incubate the loading buffer at 4°C);
2. For each of the 40ml protein solution from the above step 4), add 1ml of Ni beads in a 50ml falcon tube, incubate at a shaker with gentle shake at 4°C overnight;
3. Transfer the protein solution with Ni beads to the IMAC column at 4°C, collect the flow through (FT);
4. Wash the beads with 200ml of wash buffer, collect the wash buffer for SDS-PAGE analysis;
5. Elute with 10ml of elution buffer, and concentrate the elute to around 5ml;
6. Take 2ug protein for mass spectrometry (MS) analysis to check the protein molecular weight. Run gel filtration (GF) of the 5ml elute (filtered with 0.22um filter before GF) with S75 in 500ml of chromatography buffer;
7. Run SDS-PAGE of FT, wash buffer, elute and the proteins with peak from the GF;
8. Collect the protein after GF, concentrate the protein, aliquots into small volume and flash freeze with liquid N2, store at -80°C.

Loading buffer used:

20mM Tris-HCl, 20mM imidazole, 500mM NaCl, 1mM TCEP, 10% glycerol

Wash buffer used:

20mM Tris-HCl, 30mM imidazole, 500mM NaCl, 1mM TCEP, 10% glycerol

Elution buffer used:

20mM Tris-HCl, 300mM imidazole, 100mM NaCl, 0.5mM TCEP, 10% glycerol

Chromatography buffer used:

20mM Tris-HCl, 100mM NaCl, 0.5mM TCEP, 10% glycerol

• Strep-Tactin®T 4Flow® high capacity FPLC column (2-5028-001) purification of protein:

1. Equilibrate the column (5ml bed volume) with 25ml (5 CVs) of wash buffer (pre-incubated at 4°C), the flow rate should be in the range of 1-3 ml/min;
2. Apply the protein solution from the above step 4) into the Strep-Tactin®T 4Flow® column, collect the flow through (FT);
3. Wash the column with 40ml (8 CVs) of wash buffer, collect the wash buffer;
4. Elute the protein with 25ml (5 CVs) elution buffer, collect the elute, concentrate the elute;
5. Run SDS-PAGE with FT, wash buffer, elute;
6. Regenerate the Strep-Tactin®T 4Flow® column with 75ml (15 CVs) of 3M MgCl2, wash with 25ml (5CVs) of wash buffer, store the column at 4°C;
7. Aliquots the concentrated protein into a small volume and flash freeze with liquid N2, store at -80°C.

Wash buffer used:

100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1mM TCEP, 10% glycerol

Elution buffer used:

100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 50 mM biotin, 0.5mM TCEP, 10% glycerol

Ni-IMAC purification of hFetuin B-Fc-6xHis and hOvastacin-twinStrep protein complex:

Strep-Tactin®T 4Flow® high capacity FPLC column purification of hFetuin B-Fc-6xHis and hOvastacin-twinStrep protein complex:

Western Blot to confirm hFetuin B-Fc-6xHis and hOvastacin-twinStrep:

This project is funded by the Bill and Melinda Gates Foundation. The Structural Genomics Consortium is a registered charity (no: 1097737) that receives funds from AbbVie, Bayer AG, Boehringer Ingelheim, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD in Canada and US), Pfizer, Takeda and the Wellcome Trust [106169/ZZ14/Z].

It’s been one year since the last update on the α/β hydrolase domain 2 (ABHD2) as a target for non-hormonal contraception (see my previous post here). The major challenges were the production of ABHD2 and development of a reliable activity assay to screen and quantify inhibitory compounds. At that point 14 different constructs had been tested in E. coli and 17 in BEVS, both displaying only mediocre amounts of soluble protein either in the initial screen or after scale-up that were not sufficient for the use in downstream applications. In the meantime, other ABHDs (ABHD10, 11, and 14B) were purified with the aim to develop a selectivity panel as well as an activity assay using p-nitrophenyls as general hydrolase substrates.

The goal of this project is two-fold: Characterization of ABHD2 to evaluate the protein as a target for non-hormonal contraception as well as the generation of a full target-enabling package (TEP) that offers molecular tools for further investigation of the enzyme. This includes protocols for production of soluble ABHD2, assays that test folding status and activity of the purified protein, investigation of the effect of progesterone as well as the published inhibitor (CBK600192) for a molecular characterization. On this knowledge will then be expanded to develop more potent inhibitory compounds towards a chemical probe. In parallel, crystal structures of the free protein and in complex with ligands as well as the development of a specific antibody are needed for a complete TEP.

The Results

Protein production

Production of ABHD2 was approach by multiple efforts in parallel. 120 different constructs with different truncations and terminal tags were cloned and tested in different E. coli strains (BL21(DE3)R3pRARE2, T7 express cells from NEB), 20 in BEVS, and 4 in mammalian cells at SGC Toronto and SGC Karolinska. A full list of tested constructs can be found in the Zenodo entry. In parallel, LiberumBio tested the cell-free expression of one construct with an N-terminal His-tag for residues L33-E425. Ca. 300 µg of ABHD2 with other contaminating proteins were acquired with 1 mM Tween-80 and reducing agent. Genscript was contracted with testing two different secreted constructs containing residues R31 to E425 of the protein with an N-terminal signal sequence and C-terminal His-tag in their TurboCHO system, in which the produced protein is secreted. SDS-PAGE revealed high molecular-weight aggregates of expressed protein, but no secreted protein was detected in the medium. The band of the monomeric protein could be recovered under reducing conditions, which indicated misfolding under oxidative conditions.

As an alternative approach, mutational screening using the PROSS tool was used to create three constructs with 9, 18, and 33 mutations inserted, based on multiple sequence alignment, to increase solubility and stability.¹ Only small amounts (less than 1 mg) of each construct could be extracted for the constructs carrying 18 and 33 mutations, that however displayed activity in a general hydrolase activity assay.

At the same time, the team at SGC Toronto found that addition of 1% Triton X-100 to the lysis buffer could substantially increase the amounts of soluble ABHD2, based on the observation from cell-free expression, where soluble ABHD2 could retained in solution by addition of a detergent. For a construct of L33-E425 carrying an N-terminal His-FLAG tag, as described in this post by Madison Edwards, several milligrams of protein were purified. This could be reproduced for other constructs with an N-terminal His-tag as well as a His-FLAG-tag after which a TEV cutting site was inserted for tag-removal. The identity of the purified protein was confirmed by mass spectrometry.

Figure 1: A) Exemplary SEC profile of ABHD2-c049 (N-His-FLAG-ABHD2(L33-E425)) on a HiLoad 16/60 Superdex 200 pg. Fractions of green and orange boxes were pooled and concentrated. B) SDS-PAGE and Coomassie stain of final samples in diluted and concentrated form are displayed, too. Theoretical molecular weight of purified construct is indicated by an black arrow.

Subcellular localization

Based on results from Genscript, which showed no secretion of the protein but only high molecular-weight aggregates under oxidative conditions, as well as the lack of disulfide bonds in the structure prediction by AlphaFold and behavior of the protein in buffer (stable in the presence of reducing agent), the extracellular localization of ABHD2 was put into question.

At SGC Frankfurt and SGC Toronto, this was investigated by transient transfection of HEK293T and U2OS cells with different full-length constructs of ABHD2, one carrying either a C-terminal MYC- or FLAG-tag, that was subsequently detected by immunofluorescence, after permeabilizing the cells. The second construct carried a C-terminal eGFP for detection of eGFP-fluorescence without immunostaining.

Figure 2: Confocal images of U2OS cells transiently transfected with full-length ABHD2 constructs, carrying either a C-terminal FLAG-tag (A) or C-terminal eGFP (B). A) Immunofluorescence of permeabilized cells using an anti-FLAG is shown, while in B) the intrinsic fluorescence of eGFP from untreated cells is displayed. For neither, fluorescence at the plasma membrane could be observed.

Both, immune- and eGFP-fluorescence were only detected in the cytosol, while no fluorescence was observed at the membrane, indicating no membrane-localization of ABHD2 for the transfected cells.

Folding and interactions

Folding of recombinantly purified N-FLAG-ABHD2 (L33-E425) was tested by differential scanning fluorimetry (DSF) using tryptophan fluorescence on a Prometheus^TM NT.48 (NanoTemper Technologies). For this, 4 µM ABHD2 (L33-E425) were prepared in 50 mM HEPES pH 8, 500 mM NaCl, 10% glycerol, 1 mM TCEP, with and without 1% DMSO and after 5 min of centrifugation at 15,000xg transferred to capillaries Prometheus^TM NT.48 Capillaries (NanoTemper Technologies). Triplicate samples were heated with 1 °C/min from 25 to 95 °C, while intrinsic fluorescence at 330 and 350 nm as well as the backscattering was recorded. A melting curve was observed from which an infliction point at 44.2 °C could be derived, corresponding to the melting temperature, indicating structural integrity of the purified protein. No significant difference with or without the presence of 1% DMSO could be observed.

Furthermore, DSF was used to investigate interactions between ABHD2 (L33-E425) and progesterone or CBK600192, which were described previously.^2,3 For this, the protein was prepared as described with the addition of 40 µM of the respective compound and incubated on ice for 30 min prior to centrifugation. Presence of progesterone in solution was monitored by measuring absorbance at 248 nm for different concentrations in sample buffer and the sample.⁴ For progesterone no significant shift in melting temperature was observed, indicating no stabilization and thereby interaction between progesterone and ABHD2 (L33-E425). Presence of CBK600192 lead to stabilization of the protein by 4.9 °C, which was also observed after incubation with both progesterone and CBK600192 (each 40 µM), indicating binding of CBK600192 which was not disturbed by progesterone.

Figure 3: Melting curves of His-FLAG-ABHD2(L33-E425) are displayed in the presence of progesterone, CBK600192, or both in comparison to a DMSO control. 350 nm/330 nm is shown in dependence of temperature [°C].

Activity assay

The more general activity assay for hydrolases that was used for ABHDs 10, 11, and 14B (described here) was adapted to ABHD2. For this, a p-nitrophenyl analogue is incubated with the enzyme, which leads to hydrolysis of the substrate to p-nitrophenol and the respective acid. p-nitrophenol can then be detected due to its absorbance at 405 nm. After initial screens, 50 nM of ABHD2 were incubated with 500 µM p-nitrophenyl octanoate (p-NPO). In contrast to p-nitrophenyl butyrate, which was the substrate used for the other ABHDs, p-NPO was chosen for ABHD2. Not all ABHDs displayed activity towards p-nitrophenyl substrates with longer aliphatic chains, leading to a higher specificity. Additionally, p-NPO displayed higher stability in aqueous solution than analogues with shorter aliphatic chains. Usually, 40 µl of protein solution was pre-incubated with a specific compound on ice for 30 min, transferred to a 96-well plate and then placed in a plate reader with a thermostat function at 37 °C for 2 min. In parallel, buffer was incubated at 37 °C for 30 min, then p-NPO was added to the buffer, mixed, and 160 µl of substrate solution added to each well containing 40 µl protein solution, thereby starting the reaction. This resulted in final assay conditions of 20 mM HEPES, 500 mM NaCl, 10% glycerol, pH 8, 1 mM TCEP, 2% DMSO, 1.44% Methanol, 500 µM p-NPO. Absorbance at 405 nm was typically monitored for 10 min at 37 °C.

Alternatively, a second, fluorescence based, activity assay was developed for ABHD2, using 7-Hydroxycoumarinyl arachidonate (7-HCA) as substrate. As a derivative of arachidonic acid, 7-HCA is structurally very close to ABHD2’s proposed natural substrate 2-arachidonoyl glycerol (2-AG), resulting in a more specific enzyme-substrate interaction.² 7-HCA is hydrolyzed by ABHD2, producing arachidonic acid and umbelliferone, the latter of which can be detected via fluorescence. Also, greater sensitivity due to fluorescence of the product and therefore use of lower concentrations of protein enables determination of lower IC₅₀ values as well as less consumption of protein, which is better suited for high-throughput screens. Solutions were typically prepared as described above, with final concentrations for ABHD2 and 7-HCA of 10 nM and 5 µM, respectively. Solutions were incubated at 37 °C for 10 min and changes in fluorescence monitored (λ_ex = 335 nm, λem = 450 nm).

Figure 4: Principle of substrate consumption and mode of detection for both developed activity assays for ABHD2.

Based on the observation that the purified ABHD2 (L33-E425) showed activity in both assays, the effects of progesterone and CBK600192 on the enzymatic activity were investigated. For progesterone, two different concentrations of the ABHD2 were used to be able to observe an increase in activity, however no effect on the enzymatic activity was observed. Contrarily, pre-incubation with CBK600192 lead to a decrease in activity of 87.4% after 10 min of incubation at 37 °C.

Figure 5: Effect of progesterone (left) and CBK600192 (right) on enzymatic activity of ABHD2. No effect on activity could be observed in the presence of 10 µM progesterone at two different concentrations of ABHD2. Pre-incubation with CBK600192 lead to decrease of activity after 10 min of 87.4%.

Compound screens

Because of the initial results ABHD2 of inhibition by CBK600192, a library consisting of 53 synthesized or commercially available derivatives was collated and screened against at 10 µM in triplicates. The full library can be found in the Zenodo deposition. IC₅₀ values for the eight compounds displaying greatest inhibitory effect relative to the DMSO control.

Figure 6: Summary of screening derivatives of published ABHD2 inhibitor CBK600192.3 Three compounds, CBK600218, 191, and 209 showed ~10-fold more potent inhibition in activity assay.

Compounds CBK600218, CBK600191, and CBK600209 displayed a 10-fold greater potency than the published inhibitor found by Baggelaar et al. These were verified by DSF and lead to thermal shifts of 5.8, 13, and 14.4 °C, respectively. The most potent inhibitor, compound 218, also known as KT-109, is a known inhibitor of ABHD6 and proposed to covalently modify the catalytic serine.⁵ This was investigated using mass spectrometry. Pre-incubation of ABHD2 (L33-E425) with compound 192 did not lead to an increase in mass, however for 218 an increase of 201 g/mol was observed, which is consistent with a covalent modification of the catalytic serine by the mechanism shown below.

Figure 7: A) CBK600218, 191, and 209 stabilize ABHD2 in DSF. B) Change in molecular weight suggests covalent modification of ABHD2 by CBK600218, which was not observed for CBK600192. C) Proposed mechanism of modification by CBK600218.

Structure

For a complete TEP as well as SAR to develop a more potent and specific inhibitor for ABHD2, a structure of the protein bound or unbound to a small compound is needed. For this, several buffer screens were tested for a construct carrying an N-terminal His-FLAG tag for ABHD2(L33-E425) with or without CBK600192. Several conditions yielded cubic crystals that contained protein according to UV imaging, however none of these crystals diffracted.

Figure 8: Cubic crystals of ABHD2 incubated with and without the published inhibitor CBK600192. Illumination in UV imaging suggests presence of protein, however none of the crystals diffracted.

Because crystallization of ABHD2 turned out to be challenging, hydrogen-deuterium exchange mass spectrometry (HDXMS) was done in Derek Wilson’s lab at York university to get an initial idea of compound interaction. For this, the protein is incubated with an excess of compound before placed in D₂O, proteolytic digestion, and analysis of the peptides by MS, which is then compared to a control without compound. Interaction with a small molecule and/or accompanying structural rearrangements will lead to changes in exchange between amide protons or other polar protons and deuterons of the solvent because of the formation of new hydrogen bonds. These changes in exchange and subsequently mass can be detected and localized by proteolytic digestion and following MS.

For CBK600192, changes in exchange were observed for helices X and Y, which are directly next to the active site, suggesting binding of the inhibitory compound to that region. Still, for effective SAR a better resolution of the binding site (i.e., crystal structure) is needed.

Figure 9: HDXMS data of ABHD2 in presence of CBK600192 revealed changes in H-D exchange close to the active site, suggesting interaction of the inhibitor close by.

Antibodies

At SGC-Montreal currently commercially available antibodies against ABHD2 were tested with lysates from RKO, U-87, and OCUM-1 cell. ABHD2 expression was verified before via RNA sequencing. From these antibodies, none resulted to be specific, producing multiple bands on Western blots, while the most dominant band detected by 14039-1-AP from proteintech correlates to the molecular weight of ABHD2.

In the meantime, ThermoFisher Scientific was contracted to produce specific antibody against ABHD2, based on the recombinantly purified ABHD2 (L33-E425) construct, which is still ongoing.

Figure 10: Western blots of different commercially available polyclonal antibodies with lysates of RKO, U-87, and OCUM-1 cells, for which endogenous expression of ABHD2 was verified on RNA level previously.

The Verdict

With all this data, where are we now? We made good progress towards the TEP goal – protocols for protein expression and purification as well as two activity assays are established and tested, also, compounds that are more potent than the published inhibitor CBK600192 have been identified. Antibody generation and efforts towards crystal structures are ongoing, so is the development of a chemical probe. However, the characterization of ABHD2 as a target for non-hormonal contraception led to questions regarding the proposed mechanism. In HEK293T and U2OS cells, no membrane localization of ABHD2 was observed using immunofluorescence or intrinsic eGFP-fluorescence. Moreover, no effect of progesterone on protein stability or activity could be observed, indicating no interaction between progesterone and ABHD2. *Insert passage about hyperactivation assay if Nuvisan is OK with it*

Based on these results, ABHD2 was de-prioritized in a joint meeting between people from SGC, Nuvisan, and the Gates foundation.

The Outlook

What is still missing? After developing a specific antibody against ABHD2, this needs to be validated against a single-knock-out cell line, which is generated at the moment at SGC-Montreal. Afterwards, this antibody can be used to investigate ABHD2 localization in cells with endogenous expression as well as sperm cells. While the results presented here do not support a membrane-bound extracellular domain, overexpression of the protein might lead to non-representative results. Moreover, target engagement of the inhibitory compounds needs to be considered, if ABHD2 is localized inside the cell. Although the compounds are able to inhibit ABHD2, they might not penetrate the plasma membrane. This is explored at SGC Toronto using cellular thermal shaft assays (CETSA) coupled to HiBit as a detection method. Crystal structures of ABHD2 in presence of different ligand will aid the development of more specific compounds. Testing different constructs as well as seeding approaches will hopefully yield diffracting crystals. Finally, the specificity of the identified compounds needs to be validated. For this, a selectivity panel consisting of more ABHDs needs to be developed. Alternatively, CETSA followed by proteomic analysis can be utilized as a more wholistic approach using a cell-line with endogenous ABHD2 expression. The chemical space of the identified compounds will be explored, and a small collection has already been selected. In this context, we are currently starting a collaboration with Recursion. They are specialized in generating compounds libraries guided by machine learning and will provide more compounds to be tested if the current hits are not well suited as chemical probes.

Acknowledgements

All the work presented here is based on the amazing effort of multiple people across the SGC as well as partners from the industry. I would like to thank Aled Edwards, Claudia Tredup, Opher Gileadi, and Daniel Goldberg for scientific guidance and discussions. Madison Edwards, Alma Seitova, Peter Loppnau, and everyone else at SGC Toronto that worked diligently on the production of ABHD2, as well as facilitating experiments conducted at SGC Toronto. Furthermore, I’m grateful for the help of Dalia Barsyte-Lovejoy and her lab at SGC Toronto, especially Michelle Cao and Magdalena Szewczyk, with immunofluorescence and CETSA-HiBit experiments. Likewise, I thank Susanne Müller-Knapp, Yufeng Pan, and Amelie Tjaden at SGC Frankfurt, for their help on cell-based assays, too. On the chemistry side, I appreciate the input and efforts of Matthew Todd and Eve Carter at SGC London, as well as Evert Homan, Martin Haraldsson, and Pauline Ribera at Science for Life Laboratory in Stockholm, likewise the Chemical Biology Consortium Sweden for collating the library and synthesizing compounds. I would like to thank Carl Laflamme, Vincent Francis, and Riham Ayoubi at SGC Montreal for their work on antibody characterization and generation of KO cell lines. Thank you to Martin Moche at the Protein Science Facility of the Karolinksa Institute for his help on crystallization efforts as well as Derek Wilson and Vimanda Chow for their expertise in HDXMS.

This project is funded by the Bill and Melinda Gates Foundation. The Structural Genomics Consortium is a registered charity (no: 1097737) that receives funds from AbbVie, Bayer AG, Boehringer Ingelheim, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD in Canada and US), Pfizer, Takeda and the Wellcome Trust [106169/ZZ14/Z].

References

1. Goldenzweig, A. et al. Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability. Mol. Cell 63, 337–346 (2016).
2. Miller, M. R. et al. Unconventional endocannabinoid signaling governs sperm activation via the sex hormone progesterone. Science (80-. ). 352, 555–559 (2016).
3. Baggelaar, M. P. et al. ABHD2 Inhibitor Identified by Activity-Based Protein Profiling Reduces Acrosome Reaction. ACS Chem. Biol. 14, 2295–2304 (2019).
4. Haskins Jr., A. L. Solubility of Progesterone in Water and in Saline. Proc. Soc. Exp. Biol. Med. 70, 228–229 (1949).
5. Hsu, K. L. et al. DAGLβ inhibition perturbs a lipid network involved in macrophage inflammatory responses. Nat. Chem. Biol. 8, 999–1007 (2012).

Scheme 1. Re-synthesis identifies cyclic analog 4.

The formation of the cyclic analog 4 occurred via an intramolecular Michael reaction (Scheme 2) where the acyclic analog 3 cyclizes to form the cyclic compound 4.

Scheme 2. Intramolecular Michael reaction favors cyclization.

NMR-based characterization of acyclic and cyclic analogs:

The structures of the acyclic (3) and cyclic analogs (4) were confirmed by 2D NMR spectroscopic data including COSY, HSQC, and HMBC spectra. ¹H NMR analysis of compound 3 showed characteristic olefinic protons at δ 6.69 ppm (dt, J = 15.3, 1.8 Hz, 1H) and δ 6.82 ppm (dt, J = 15.3, 4.4 Hz, 1H) respectively (Figure 1a). The disappearance of the olefinic protons and appearance of a multiplet at δ 5.07 ppm (m, J = 8.9, 4.5 Hz, H-9) was a key point of difference for the formation of compound 4 (Figure 1b). Another key difference was the disappearance of H-12 at δ 4.11 ppm (ddd, J = 6.2, 4.2, 1.8 Hz, 2H) in the cyclic analog 4. After cyclization, H-8 appeared at δ 3.70 ppm (td, J = 6.3, 3.5 Hz, 1H) and δ 3.93 ppm (ddd, J = 13.3, 4.4, 2.3 Hz, 1H) respectively in analog 4 (Figure 1b). H-11 in analog 4 appeared as distinct protons at δ 3.76-3.72 ppm (m, 1H) and δ 3.98 ppm (dd, J = 14.4, 3.9 Hz, 1H) respectively (Figure 1b). Important to note that the H-12 protons in compound 3 appeared together at δ 4.11 ppm (ddd, J = 6.2, 4.2, 1.8 Hz, 2H) (Figure 1a). However, in the cyclic compound 4, the corresponding protons denoted as H-8 are non-equivalent (showing different signals in the ¹H NMR spectra) (Figure 1b). These protons were initially equivalent in the open form (denoted by H-13 and H-14 in Figure 1a).

Figure 1. ¹H NMR spectra of compounds 3 and 4 depicting the key distinguishable peaks between the open and cyclic analogs.

¹H-¹³C HMBC analysis of cyclic analog 4 revealed that H-9 illustrates a three-bond correlation with atom C-4 (Figure 2). This correlation is the key evidence for formation of (E)-vinyl sulfone 4.

Figure 2. HMBC spectrum of 4.

Vinyl sulfones have (E)-configuration:

It is important to note that the vinyl sulfone warhead 2 was synthesized as (E)-isomer (Figure 3a), which yielded the corresponding acyclic analog 3 in (E)-configuration during the coupling reaction (Figure 3b). The (E)-isomer is confirmed in both cases from the coupling constant (J = 15 Hz) values of the olefinic protons in vinyl sulfones 2 and 3.

Figure 3. ¹H NMR spectra of (E)-vinyl sulfones 2 and 3 depicting the olefinic protons with corresponding J values.

Although we were able to characterize the acyclic and the cyclic sulfones, full biological evaluation of the screening hit and the synthesis of analogs required the development of robust protocols to access the pure acyclic and cyclic pyrazoles.

This encouraged us to solve three major issues associated with the chemistry:

• Develop a robust analytical method to identify and separate the acyclic and cyclic analogs
• Develop a robust synthetic protocol to access the acyclic analog exclusively in high yields
• Develop a synthetic method to completely convert acyclic to cyclic analog

1. Development of Analytical Method

 Initial LCMS run time of 3 min and 6 min did not separate out the acyclic and cyclic analogs and they overlapped with one another in the LCMS spectra. Even on preparative (8 min) and analytical HPLC (6 min) runs, compounds 3 and 4 did not show up as distinct separable peaks. This encouraged us to develop an in-house analytical method to monitor the reactions, separate the acyclic and cyclic analogs, and identify the products. After several trials, it was observed that a run time of 12 min on LCMS (Figures 4a and 4b) and 26 min on HPLC (Figure 4c) could separate out the acyclic and cyclic analogs as two distinct peaks (LCMS column: C18 2.7 mm (Agilent); HPLC column: Luna 5 mm phenyl-hexyl (Phenomenex). Thus, the acyclic and cyclic forms were separable only with extended HPLC runs.

Figure 4. LCMS (4a–b) and HPLC (4c) profiles of acyclic (3) and (4) cyclic analogs.

The acyclic (3) and cyclic (4) vinyl sulfones were isolated with high purity from the HPLC purification (Figure 5).

Figure 5. LCMS profiles of pure acyclic and cyclic analogs after preparative-HPLC purification.

2. Synthesis of Acyclic Analog and Methods for Cyclization

a) Optimization of Reaction Conditions for Amide Coupling:

As the amide coupling conditions were prone to form both the acyclic and cyclic analogs, it was important to reduce the propensity to form the cyclic analog. In this regard, we tried different conditions for performing the acid-amine coupling. Using pyrazole carboxylic acid 1 (1.0 equiv.), amine 2 (1.2 equiv.), HATU (1.5 equiv.), DIPEA (3.0 equiv.) in DMF at 25°C for 2h afforded the acyclic (3) and the cyclic (4) analogs in 60:40 ratio (Condition 1, Table 1). Similar ratio of formation of 3 and 4 were observed when treated with T₃P (1.5 equiv.) and TEA (3.0 equiv.) in DMF at 25°C for 2h (Condition 2, Table 1). When the coupling was carried out using EDC.HCl (1.5 equiv.), HOBt (1.5 equiv.) in DMF at 25°C for 2h, ratio of acyclic (3) and cyclic (4) analogs was 70:30 (Condition 3, Table 1). Using PyBOP (1.0 equiv.), DIPEA (2.0 equiv.) in DMF at 25°C for 16h (Condition 4, Table 1) or DIC (1.2 equiv.), DMAP (2.0 equiv.) in DMF at 25°C for 16h (Condition 5, Table 1) did not improve the fate of the reaction. However, with TBTU (1.5 equiv.) and pyridine (0.2M), exclusive formation of pyrazole (E)-vinyl sulfone 3 was observed in 70% yield (Condition 6, Table 1). This optimized reaction condition will be used for coupling the substituted pyrazole carboxylic acids with the cysteine-capturing warheads during SAR exploration.

Scheme 3. Amide coupling leads to both acyclic and cyclic analogs.

Table 1. Optimization of Reaction Conditions for Amide Coupling^a

 ^aReaction conditions: 1 (1.0 eq.), 2 (1.2 eq.), solvent (0.2 M). ^bBased on LCMS and ¹H NMR analyses.

b) Alternative Route to Access Acyclic Analogs by Protection of Pyrazole NH:

An alternate regioselective protocol using MOM-protection of pyrazole 1 was developed to enable synthesis of pyrazole analogs that were more prone to cyclization (Scheme 4). MOM (methoxymethyl) protection at the N-1 position of pyrazole 1 was achieved using 3.0 eq. of CH₃OCH₂Cl (MOMCl), 1.2 eq. of K₂CO₃ in DMSO at 25°C for 2h (Scheme 4, Step 1). TBTU-pyridine mediated amide coupling (Scheme 4, Step 2), followed by MOM deprotection yielded acyclic (E)-vinyl sulfone 3 (Scheme 4, Step 3). This strategy will be useful to access pyrazole-containing acyclic covalent inhibitors in good yields, avoiding intramolecular cyclization, and minimizing tedious purification steps. The high regioselectivity of the N-1 substituted pyrazole is illustrated by previous reports from Almena et al.² and Huang et al^3a, Norman et al^3b.

Scheme 4. K₂CO₃-mediated MOM-protection of pyrazole NH (Step 1), amide coupling (Step 2), and subsequent deprotection (Step 3) ^a,b

^aReaction conditions: Step-1: 1 (1.0 eq.), MOMCl (3.0 eq.), K₂CO₃ (1.2 eq.), DMSO (0.3 M), 25°C, 2h. Step-2: 1a (1.0 eq.), 2 (1.2 eq.), TBTU (1.5 eq.), pyridine (0.2 M), 25°C, 2h. Step-3: 2a (1.0 eq.), 4N HCl in dioxane (10.0 eq.), 25°C, 0.5h. ^bYields are isolated yields.

c) Optimization of Reaction Conditions for Intramolecular Cyclization:

The optimal reaction conditions for conversion to cyclic form by intramolecular aza-Michael reaction were also identified.⁴ Stability studies demonstrated that acyclic pyrazole 3 is converted cyclic pyrazole 4 in mild basic media, but 4 is stable under standard laboratory conditions. Intramolecular aza-Michael reaction was performed under basic conditions with polar protic and aprotic solvents. Different bases viz. K₂CO₃, NaHCO₃, TEA, DIPEA, DBU were used at room temperature to favor the cyclization (Table 2). Interestingly, two reaction conditions:  K₂CO₃ (3.5 equiv.), EtOH, 25°C, 12h (Condition 1) and Na₂CO₃ (3.0 equiv.), dioxane:H₂O (1:1), 25°C, 12h (Condition 5) afforded the cyclic analog 4 with 100% conversion. Important to mention that other bases like NaHCO₃ (53% in H₂O and 88% in MeOH), TEA (49% in MeOH), DIPEA (96% in DMF), DBU (92% in ACN) also favored the cyclization with good conversion rates (Table 2). However, due to the difficulty in separation of the acyclic and cyclic analogs, we favored the conditions with 100% conversion. This will avoid the need to perform difficult HPLC purifications of all the analogs for SAR studies.

Table 2. Optimization of Reaction Conditions for Intramolecular Cyclization

^aBased on LCMS analyses of crude reaction mixtures.

We are trying to understand what factors (electronics, sterics, basicity) might affect the propensity for the formation of acyclic and cyclic analogs. Accordingly, suitable modifications will be made on the pyrazole ring and the warhead to understand the relative stability between the formation of acyclic and cyclic vinyl sulfones.

References:

1. https://readdi-ac.org/
2. Almena et al. 1998, 35, 1263-1268.
3. (a) Huang et al. J Org. Chem. 2017, 82, 8864-8872. (b) Norman et al. J Org. Chem. 2022, 87, 10018-10025.
4. (a) McDowell et al. J Chem. Soc. B: Phys. Org. 1967, 0, 343-348; (b) McDowell et al. J Chem. Soc. B: Phys. Org. 1967, 0, 348-350.

1. Physicochemical Filters

Physicochemical filters are based on the molecular properties of the compounds, such as molecular weight, lipophilicity, polarity, and hydrogen bonding. These properties may affect the drug-likeness and pharmacokinetics of the compounds. One of the most widely used physicochemical filters is the Lipinski’s rule of five (Lipinski et al., 2001), which states that most orally active drugs have no more than five violations of the following criteria: molecular weight ≤ 500 Da, logP ≤ 5, number of hydrogen bond donors ≤ 5, and number of hydrogen bond acceptors ≤ 10. Other physicochemical filters include the Veber rule (Veber et al., 2002), which limits the number of rotatable bonds ≤ 10 and the polar surface area ≤ 140 Å, and the Ghose filter (Ghose et al., 1999), which defines ranges for molecular weight (160-480 Da), logP (-0.4 to 5.6), number of atoms (20-70), and molar refractivity (40-130).

The advantages of physicochemical filters are that they are based on empirical data from known drugs and are easy to calculate computationally. They can help to reduce the size of the compound library and eliminate hit compounds that may be difficult to optimize into drug leads. However, a key disadvantage of physicochemical filters is that they are not specific for the HTS target and may exclude some novel chemotypes that can be optimized for activity on viral enzymes.

2. Pan Assay Interference Filters

Pan Assay Interference (PAINS) filters are based on the structural features of the compounds that are known to cause promiscuous off-target activity across multiple HTS (Baell et al., 2010). These features include reactive groups (such as aldehydes, thiols, or Michael acceptors), protein aggregators (such as aromatic amines or sulfonamides)(Irwin et al., 2015), fluorescent compounds (such as coumarins or rhodamines), or metal chelators (such as catechols or hydroxamic acids). These types of compounds can interfere with the assay by covalently modifying the target or other proteins, forming aggregates that sequester the target or other molecules, quenching or enhancing the signal of the assay readout, or binding to metal ions that are essential for the enzymatic activity of the target.

The advantage of PAINS filters is that they can be applied computationally to rapidly eliminate potential false positives arising from non-specific interactions or assay artifacts. However, a major disadvantage of PAINS filters is that they may exclude compounds that have genuine activity against viral targets, such as covalent inhibition or metal chelation. These filters will fail to flag those interfering chemotypes that have not been widely reported in the literature.

3. Data Interpretation Filters

Data interpretation filters are based on the statistical analysis of the primary HTS data to eliminate outliers and false positives (Moffat et al., 2017). These filters include methods such as Z’-score normalization, selectivity index, dose-response curve fitting, hillslope, and cluster analysis. These methods can help to correct for systematic errors, such as plate effects or edge effects, as well as to flag compounds with poor solubility or protein aggregators to help distinguish between true hits and false positives or negatives.

The advantages of data interpretation filters are that they can be used to triage the primary HTS screening data to remove many of the false positives and to prioritize the most promising hits for further validation. However, the disadvantages of data interpretation filters are that they require careful selection and optimization of parameters and algorithms, and they may depend on the quality and quantity of the HTS data. They may also introduce biases or artifacts if not applied properly.

Figure 1: Hit Confirmation Process in AViDD

4. Hit filtering criteria and hit confirmation

In the READDI AViDD Center, all primary hits from virtual and high-throughput screening are filtered through the set of physicochemical, experimental, and data interpretation filters depicted in Figure 1. The accepted parameter for each step is presented in Table 1 for hit confirmation and progression.

Table: READDI AViDD Center Hit Selection Parameters Limit

Conclusion

Compound filtering from HTS is a crucial step in hit identification for small molecule drug discovery. For the viral enzyme targets in the READDI AViDD Center it has been used to eliminate primary hits that have undesirable phyiochemical properties or potetial for assay interference, and to select compounds that have the best potential for on target activity. However, no single filter is perfect or universal. Therefore, it is important to apply multiple filters in a rational and balanced way, taking into account the specific characteristics of the target protein and the assay format. By doing so, one can increase the chances of finding progessible hits for optimization into effective antiviral drugs.

To know more:

Lipinski Rules: AdvancedDrugDeliveryReviews, 46 (2001) 3–26

To access STOPLIGHT: Click here; Ref. Holli et al., 2023

Aggregators: J Med Chem. 2015 Sep 10; 58(17): 7076–7087.

Solubility models: https://practicalcheminformatics.blogspot.com/2023/06/getting-real-with-molecular-property.html?m=1

PLCζ1 background

One promising protein target for NHCs is phospholipase C zeta 1 (PLCζ1). PLCζ1 is found within the sperm and is known to be a sperm oocyte activation factor (SOAF). PLCζ1 catalyses the hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP₂) into inositol-1,4,5-triphosphate (IP₃) and diacylglycerol (DAG).^1,2 Once released, IP₃ is then able to bind to its receptors located in specialised compartments of the endoplasmic reticulum (ER) membrane and release intracellular calcium (Ca²⁺) from the ER. These Ca²⁺ oscillations are vital for successful fertilisation.^1,3

Scheme 1: Hydrolysis of PIP₂ into IP₃ and DAG.

Protein Production

Madison Edwards has purified MBP-tagged human PLCζ1 from E. coli in a low yield, similar to previously reported expressions.⁴ However, the protein appears to be in soluble aggregates and contains a chaperone protein contaminant, GroEL, which was not removed through size exclusion chromatography or by washing under recommended conditions.

Expression of human PLCζ1 constructs from mammalian cells was not successful. Dalia Barsyte-Lovejoy and Peter Loppnau expressed codon-optimised human PLCζ1 in mammalian cells, which was visualised by FLAG-tag immunoblot. Expression was low yielding, and the protein was found to have solubility issues. Opher Gileadi tried to improve upon these results by using a different expression vector and exchanging the tag from flag to strep, but expression was still low yielding.

Madison attempted to express PLCζ1 from six animals (chicken, mouse, macaque, cow, pig and horse) from E. coli, but only insoluble protein was obtained. Expression of chicken, mouse and macaque PLCζ1 in a baculovirus/SF9 system was successful, with macfa and mouse found to be active through an IP-one assay⁵. Chicken PLCζ1 is the only construct that reliably provided high yields of monomeric protein. Chicken PLCζ1 has 62% identity to human PLCζ1 and 68% in the X and Y catalytic domains. Work is ongoing to assess whether chicken PLCζ1 is similar enough to human PLCζ1 for this to be a relevant target to screen for small molecule inhibitors.

Biochemical Assays

Eve Carter is working to establish a biochemical assay with Aldol 518 as a substrate for hydrolysis and Aldol 355 as a fluorescence enhancer, as previously reported for PLCγ1.⁵ This would provide a cheap and straightforward assay to measure PLCζ1 activity. Two other assays, IP-one⁵ and XY-69⁶, may also be utilised.

Scheme 2: Hydrolysis of Aldol 518 by PLC can be measured by fluorescence intensity.

Future work

HiBiT Cellular Thermal Shift Assay (CETSA): Dalia Barsyte-Lovejoy, Peter Loppnau and Magdalena Szewczyk are working on setting up a CETSA assay for human and chicken PLCζ1. This would provide an orthogonal assay to confirm any proposed binders/inhibitors to the protein.

DNA Encoded Library (DEL) screen: If we confirm that chicken PLCζ1 is a suitable orthologue to investigate, we will send this for a DEL screen. This will provide a starting point for the identification of chemical probes to investigate PLCζ1.

Crystallography: We will attempt to crystalise PLCζ1 to further the understanding of this protein and assist with the discovery of small molecule binders. So far, the only PLC crystal structure is that of PLCδ1.⁷

Antibodies: In collaboration with YCharOS and Thermo Fisher Scientific, we are working towards generating a PLCζ1 antibody.

References

(1)        Thanassoulas, A.; Swann, K.; Lai, F. A.; Nomikos, M. The Structure and Function Relationship of Sperm PLCZ1. Reproduction 2022, 164 (1), F1–F8. https://doi.org/10.1530/REP-21-0477.

(2)        Nomikos, M.; Kashir, J.; Lai, F. A. The Role and Mechanism of Action of Sperm PLC-Zeta in Mammalian Fertilisation. Biochem. J. 2017, 474 (21), 3659–3673. https://doi.org/10.1042/BCJ20160521.

(3)        Saleh, A.; Kashir, J.; Thanassoulas, A.; Safieh-Garabedian, B.; Lai, F. A.; Nomikos, M. Essential Role of Sperm-Specific PLC-Zeta in Egg Activation and Male Factor Infertility: An Update. Front. Cell Dev. Biol. 2020, 8 (28), 1–9. https://doi.org/10.3389/fcell.2020.00028.

(4)        Nomikos, M.; Stamatiadis, P.; Sanders, J. R.; Beck, K.; Calver, B. L.; Buntwal, L.; Lofty, M.; Sideratou, Z.; Swann, K.; Lai, F. A. Male Infertility-Linked Point Mutation Reveals a Vital Binding Role for the C2 Domain of Sperm PLC ζ. Biochem. J. 2017, 474, 1003–1016. https://doi.org/10.1042/BCJ20161057.

(5)        Le Huray, K. I. P.; Bunney, T. D.; Pinotsis, N.; Kalli, A. C.; Katan, M. Characterization of the Membrane Interactions of Phospholipase Cγ Reveals Key Features of the Active Enzyme. Sci. Adv. 2022, 8 (25), 1–16. https://doi.org/10.1126/sciadv.abp9688.

(6)        Huang, W.; Wang, X.; Endo-Streeter, S.; Barrett, M.; Waybright, J.; Wohlfeld, C.; Hajicek, N.; Harden, T. K.; Sondek, J.; Zhang, Q. A Membrane-Associated , Fluorogenic Reporter for Mammalian Phospholipase C Isozymes. J. Biol. Chem. 2018, 293 (5), 1728–1735. https://doi.org/10.1074/jbc.RA117.000926.

(7)        Essen, L.-O.; Perisic, O.; Cheungt, R.; Katant, M.; Williams, R. L. Crystal Structure of a Mammalian Phosphoinositide-Specific Phospholipase C Delta. Nature 1996, 380, 595–602. https://doi.org/10.1038/380595a0.

A promising protein target for NHCs is Protein Associated with Topoisomerase II Homolog 2 (PATL2), a highly conserved, oocyte-specific mRNA-binding protein that represses translation¹. For consanguineous families, the inheritance pattern for PATL2 is recessive; thus, infertility is often caused by homozygous mutations in the gene². Although some mutations in PATL2 reduce expression levels, the molecular mechanism through which mutations lead to oocyte maturation arrest is not fully understood^2–4. During oocyte growth, mRNA that is essential to maturation accumulates in the oocyte, in which about 30% of mRNA must be translationally repressed by proteins such as PATL2 until after fertilization⁵. PATL2 mRNA is expressed in immature oocytes with significant translation occurring during the primary follicle stage⁵. PATL2 concentration peaks during the secondary follicle stage, but the total amount of PATL2 increases as oocytes mature (Figure 1)⁵. In the absence of PATL2, genes involved in oocyte maturation and embryonic development are deregulated, demonstrating that PATL2’s function is critical to these processes^5,6.

Figure 1: Diagram depicting PATL2 expression during oocyte maturation. PATL2 is expressed in immature oocytes, with significant translation starting at the primary oocyte stage. As oocytes mature, the total amount of PATL2 increases, but the PATL2 concentration peaks during the secondary follicle stage. Interestingly, PATL2 has a unique expression profile relative to other known RNA-binding proteins in oocytes. Both overexpression and the absence of PATL2 expression can cause oocyte maturation arrest. Figure made in bioRender.

PATL2 is a homolog of PATL1, which also contains a C-terminal PAT1 domain; the two proteins share 38% identity. PAT proteins contain a conserved N-terminal sequence, a Mid domain, a proline-rich region, and a C-terminal domain^1,7. Within the C-terminus of PATL2, a PAT1 domain (amino acids 252-491) mediates PATL2’s mRNA-binding capabilities (Figure 2)². In contrast to the C-terminus of PATL2, the N-terminal half (approximately 53% of the 543 amino acids) is predicted to be disordered (Figure 2). PATL1 has been shown to couple mRNA decapping and deadenylation in the 5’-3’ decay pathway through its interactions with the Lsm 1-7 complex^8,9. PATL2 associates with some of the same proteins as PATL1, such as Lsm1 and Lsm4, of the Lsm1-7 complex, as well as Xrn1⁹. Since PATL1 and PATL2 share sequence identity in the region of PATL1 that allows it to bind RNA, PATL2 likely retains similar RNA-binding properties^1,10.

Figure 2: Schematic and predicted structure of human PATL2. A) Schematic depicting the relevant domains of PATL2. B) Predicted structure of full-length human PATL2 from AlphaFold2. The N and C-termini are labeled in black, along with amino acid G288, which serves as the start site for one of our PATL2 constructs. The disordered N-termini of the protein (aa 1-290) is colored magenta, while the ordered C-termini containing the PAT1 domain is colored blue (aa 291-543).

Proposed research:

The overarching goal of the proposed research is to enable drug discovery studies on PATL2. To this end, we will (1) structurally and biophysically characterize PATL2 interactions with RNA and protein binding partners (as this information is missing in the literature), (2) develop a biophysical assay (Protein-protein or protein-RNA interaction assays) suitable for compound screening and (3) identify small molecule binders that can be progressed to chemical probes to assess PATL2’s role in reproductive biology. To achieve these ends, PATL2, and various constructs of this protein, will be produced recombinantly and purified (Aim 1). Purified PATL2 will be used to characterize its RNA-binding abilities, protein-protein interactions, translation repression, and other biophysical properties (Aim 2). Several of the planned constructs are also suitable for X-ray crystallography, allowing us to structurally elucidate the PATL2 interaction with its binding partners (Aim 3). Finally, we will combine the knowledge gained from Aims 1-2 to screen for small molecules capable of inhibiting the PATL2’s RNA-binding or protein-binding functions (Aim 4).

Aim 1: Recombinant Expression and Purification of PATL2 Constructs

Several PATL2 constructs for recombinant expression and purification were designed using the AlphaFold2 program to analyze the predicted structure of PATL2 and design potential domain boundaries for protein expression constructs. Expression constructs were first tested on a small scale to determine which constructs could generate milligram quantities of soluble protein. From the constructs tested, we were able to successfully purify several PATL2 constructs. We first purified an N-terminally tagged hexahistidine protein construct consisting of amino acids G288-Y543 from the SF9 insect cell/baculovirus system, however the yield was low (0.04 mg/liter of cells). When purifying the same construct from E. coli, our yield was tenfold higher. Both constructs were purified using nickel affinity chromatography, followed by reverse nickel affinity, and finally, gel filtration (Figure 3).

Figure 3: Purified PATL2 G288-Y543. The SDS-PAGE gel shows the purified protein. Protein identity was confirmed by mass spectrometry. Protein elutes as a monomer on gel filtration column.

Aim 2: Biophysical Assays to Explore PATL2’s Function

PATL2 regulates specific genes implicated in oocyte maturation and early embryonic development as opposed to globally repressing translation, suggesting that PATL2 has sequence or structure-specific binding properties⁵. This hypothesis is supported by the observation that both human PATL1 and Xenopus PATL2 were found to bind GQs, as well as poly(G) and poly(U) homopolymers,   in vitro¹⁰. Based on these findings, we purchased the synthetic RNA GQ from the 5’ UTR of the NRAS proto-oncogene used in this publication. We also created a mutant version that would be incapable of forming GQs. Using fluorescence anisotropy, we observed only very weak binding by our PATL2_G288-Y543 construct to either RNA. Our findings suggest that these RNAs may not be the target of Human PATL2, or perhaps the N-terminus of PATL2 is needed for binding to GQs. Moving forward, we will (1) attempt to purify a construct of PATL2 that contains the N-terminus, (2) test if PATL2 interacts with RNA as a part of a complex with other proteins (such as those mentioned below), and (3) we may perform RNA pull-down assays to identify RNA targets specifically of Human PATL2.

Likewise, it is a priority to examine PATL2 interaction partners and verify if PATL2 directly binds these proteins in vitro. Using coimmunoprecipitation (co-IP) experiments, previous studies have identified Lsm1, Lsm4, and Xrn1 as PATL1 and PATL2 interactors.^9,11. LSM1 and LSM4 have been successfully purified for Biolayer Interferometry assays to determine if PATL2 directly binds either of these proteins in vitro. The aforementioned co-IP experiments were performed with HEK293 cells as opposed to oocytes, where PATL2 is highly expressed. Therefore, we plan to ship our purified PATL2 for antibody generation, as antibodies will be needed for our co-IP assays in oocytes. In the meantime, we are currently in the process of testing if the LSM1-7 complex will co-precipitate with full-length PATL2, or with the PAT1 domain from SF9 cells. Additionally, Mario Bengtson’s lab is cloning PATL2 into a mammalian expression vector to identify additional PATL2 interaction partners.

Aim 3: Structural Characterization of PATL2 through X-ray Crystallography

To date, the experimental structure for human PATL2 has not been reported. As the PAT1 domain of PATL1 has been crystallized, there is a high probability that we will can crystallize this domain from PATL2 in order to support structure-guided drug discovery⁷. We set up initial crystal screens with the PAT1 domain of PATL2 yet were unsuccessful in generating crystals. We are currently testing if proteolytic digestion of the PAT1 domain will provide us with a smaller domain that is more amenable to crystallization. As proteins are often stabilized by their interaction partners, we will attempt to co-crystallize a complex of PATL2 with its binding partners, which would contribute greatly to the field’s knowledge of this protein. Moreover, this structure would enable us to design evidence-based molecules capable of disrupting PATL2’s interactions that promote reproduction.

Aim 4: Test the Ability of Small Molecules to Inhibit PATL2 Function

Once we have developed a fuller understanding of the functions of PATL2 from Aims 2 and 3, we will design assays to screen for compounds that inhibit the protein’s RNA-binding or protein-binding function. As alluded to earlier, we will benefit from knowing which regions of PATL2 contribute to its various functions. This will allow us to pinpoint regions of the protein to target for protein degradation or small molecule inhibitors. Another approach to determine how to target PATL2 will be analyzing the PATL2 mutants that result in infertility. Several PATL2 mutations have been identified, and work in previous aims will explore how these mutations alter PATL2’s ability to bind to RNA and to its protein interaction partners. This information will inform our decision on small molecules to test, and suitable assays to perform.

Acknowledgements

I would like to acknowledge Alma Seitova, Ashley Hutchinson, Maria Kutera, and Peter Loppnau for their contributions to cloning and expressing PATL2 protein constructs. I would like to thank Hong Zeng for her work to purify some of the PATL2 constructs, and Mario Bengtson for his lab’s collaboration. I would also like to acknowledge Levon Halabelian and Aled Edwards for their input and supervision of this work.

This project is funded by the Bill and Melinda Gates Foundation. The Structural Genomics Consortium is a registered charity (no: 1097737) that receives funds from AbbVie, Bayer AG, Boehringer Ingelheim, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD in Canada and US), Pfizer, Takeda and the Wellcome Trust [106169/ZZ14/Z].

References

(1)        Maddirevula, S.; Coskun, S.; Alhassan, S.; Elnour, A.; Alsaif, H. S.; Ibrahim, N.; Abdulwahab, F.; Arold, S. T.; Alkuraya, F. S. Female Infertility Caused by Mutations in the Oocyte-Specific Translational Repressor PATL2. Am. J. Hum. Genet. 2017, 101 (4), 603–608. https://doi.org/10.1016/j.ajhg.2017.08.009.

(2)        Liu, Z.; Zhu, L.; Wang, J.; Luo, G.; Xi, Q.; Zhou, X.; Li, Z.; Yang, X.; Duan, J.; Jin, L.; Zhang, X. Novel Homozygous Mutations in PATL2 Lead to Female Infertility with Oocyte Maturation Arrest. J. Assist. Reprod. Genet. 2020, 37 (4), 841–847. https://doi.org/10.1007/s10815-020-01698-6.

(3)        Huang, L.; Tong, X.; Wang, F.; Luo, L.; Jin, R.; Fu, Y.; Zhou, G.; Li, D.; Song, G.; Liu, Y.; Zhu, F. Novel Mutations in PATL2 Cause Female Infertility with Oocyte Germinal Vesicle Arrest. Hum. Reprod. 2018, 33 (6), 1183–1190. https://doi.org/10.1093/humrep/dey100.

(4)        Wu, L.; Chen, H.; Li, D.; Song, D.; Chen, B.; Yan, Z.; Lyu, Q.; Wang, L.; Kuang, Y.; Li, B.; Sang, Q. Novel Mutations in PATL2: Expanding the Mutational Spectrum and Corresponding Phenotypic Variability Associated with Female Infertility. J. Hum. Genet. 2019, 64 (5), 379–385. https://doi.org/10.1038/s10038-019-0568-6.

(5)        PATL2 Is a Key Actor of Oocyte Maturation Whose Invalidation Causes Infertility in Women and Mice. EMBO Mol. Med. 2018, 10 (5), e8515. https://doi.org/10.15252/emmm.201708515.

(6)        Chen, B.; Zhang, Z.; Sun, X.; Kuang, Y.; Mao, X.; Wang, X.; Yan, Z.; Li, B.; Xu, Y.; Yu, M.; Fu, J.; Mu, J.; Zhou, Z.; Li, Q.; Jin, L.; He, L.; Sang, Q.; Wang, L. Biallelic Mutations in PATL2 Cause Female Infertility Characterized by Oocyte Maturation Arrest. Am. J. Hum. Genet. 2017, 101 (4), 609–615. https://doi.org/10.1016/j.ajhg.2017.08.018.

(7)        Braun, J. E.; Tritschler, F.; Haas, G.; Igreja, C.; Truffault, V.; Weichenrieder, O.; Izaurralde, E. The C-Terminal Alpha-Alpha Superhelix of Pat Is Required for MRNA Decapping in Metazoa. EMBO J. 2010, 29 (14), 2368–2380. https://doi.org/10.1038/emboj.2010.124.

(8)        Sharif, H.; Conti, E. Architecture of the Lsm1-7-Pat1 Complex: A Conserved Assembly in Eukaryotic MRNA Turnover. Cell Rep. 2013, 5 (2), 283–291. https://doi.org/10.1016/j.celrep.2013.10.004.

(9)        Ozgur, S.; Chekulaeva, M.; Stoecklin, G. Human Pat1b Connects Deadenylation with MRNA Decapping and Controls the Assembly of Processing Bodies. Mol. Cell. Biol. 2010, 30 (17), 4308–4323. https://doi.org/10.1128/MCB.00429-10.

(10)      Marnef, A.; Maldonado, M.; Bugaut, A.; Balasubramanian, S.; Kress, M.; Weil, D.; Standart, N. Distinct Functions of Maternal and Somatic Pat1 Protein Paralogs. RNA 2010, 16 (11), 2094–2107. https://doi.org/10.1261/rna.2295410.

(11)      Szklarczyk, D.; Gable, A. L.; Nastou, K. C.; Lyon, D.; Kirsch, R.; Pyysalo, S.; Doncheva, N. T.; Legeay, M.; Fang, T.; Bork, P.; Jensen, L. J.; von Mering, C. The STRING Database in 2021: Customizable Protein-Protein Networks, and Functional Characterization of User-Uploaded Gene/Measurement Sets. Nucleic Acids Res. 2021, 49 (D1), D605–D612. https://doi.org/10.1093/nar/gkaa1074.