Elucidating the Mode of Action of Marine Natural Products through an Immunoaffinity Fluorescent (IAF) Approach

Understanding the target and mode of action (MOA) of small molecules activity has become a critical feature in guiding the transition between drug discovery and clinical evaluation. While classically placed at the latter stages of a discovery program, we now describe how addressing the mode of action of natural products at the early stages of a program provides an important vehicle to inform the drug discovery process. In this review, we outline a streamlined cellular and molecular biological system and explore its utility through a series of four cases studies. We believe that this approach offers critical lessons to guide future drug discovery programs.

Keywords:
marine natural product; mode of action; drug discovery; target identification; confocal microscopy

Authorship

James J. La Clair ^**e-mail: jlaclair@ucsd.edu, wfenical@ucsd.edu, costalotufo@usp.br

Department of Chemistry and Biochemistry, University of California at San Diego, 92093 La Jolla-CA, USAUniversity of CaliforniaUSALa Jolla, CA, USADepartment of Chemistry and Biochemistry, University of California at San Diego, 92093 La Jolla-CA, USA

James J. La Clair (Jim) received his PhD degree in Chemistry from the State University at Buffalo under the tutelage of Peter T. Lansbury. His studies in organic chemistry continued through a postdoctoral fellowship with Gilbert Stork at Columbia University. He then undertook a collaborative position between the laboratories Richard Lerner at Scripps Research Institute and Manfred Eigen at the Max Planck Institute für Biophysikalische Chemie. In 1999, he went independent in order to focus his time on research applications and concepts. This has since included research scientist positions at UC San Diego, the Salk Institute among other collaborative endeavors. To date, his efforts have resulted in publications spanning a wide diversity of natural and physical sciences.

William Fenical ^**e-mail: jlaclair@ucsd.edu, wfenical@ucsd.edu, costalotufo@usp.br

Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California at San Diego, 92093-0204 La Jolla-CA, USAUniversity of CaliforniaUSALa Jolla, CA, USACenter for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California at San Diego, 92093-0204 La Jolla-CA, USA

William Fenical (Bill) is distinguished professor of oceanography and pharmaceutical science at the Scripps Institution of Oceanography, University of California at San Diego. His career in marine natural products has spanned more than 45 years ever since his early work on the pigments of abalone shells. Fenical has more than 450 publications in marine natural products and 200 of these have involved developing the field of marine microbial chemistry. He is the recipient of the Paul Scheuer Award in Marine Natural Products Chemistry (1996), the International Society of Chemical Ecology's Silver Medal Award (1997), the Ernest Guenther Award in Natural Products from the American Chemical Society (2006). And, in 2008 Bill was Elected as Fellow of the American Association of Science (AAAS).

Leticia V. Costa-Lotufo ^**e-mail: jlaclair@ucsd.edu, wfenical@ucsd.edu, costalotufo@usp.br

Departamento de Farmacologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-900 São Paulo-SP, BrazilUniversidade de São PauloBrazilSão Paulo, SP, BrazilDepartamento de Farmacologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-900 São Paulo-SP, Brazil

Leticia Costa-Lotufo (Leca) recently moved to University of São Paulo, where she holds an associate professor position. Previously, she was a professor at Federal University of Ceara, Fortaleza, Ceara, Brazil from 2002 to 2015. Her lab is focused on the discovery of new anticancer compounds from Brazilian marine biodiversity and studies on their mechanisms of action. Recently, she started a multidisciplinary project to access marine microorganisms diversity and biotechnological potential along the Brazilian coast and islands. PhD Costa-Lotufo's activities include the supervision of undergraduate and graduate students and post-doctoral fellows. She has published book chapters, review articles and more than 210 articles in peer-reviewed journals. On 2010, PhD Costa-Lotufo was nominated as a young scientist of the Brazilian Academy of Science.

^*e-mail: jlaclair@ucsd.edu, wfenical@ucsd.edu, costalotufo@usp.br

James J. La Clair (Jim) received his PhD degree in Chemistry from the State University at Buffalo under the tutelage of Peter T. Lansbury. His studies in organic chemistry continued through a postdoctoral fellowship with Gilbert Stork at Columbia University. He then undertook a collaborative position between the laboratories Richard Lerner at Scripps Research Institute and Manfred Eigen at the Max Planck Institute für Biophysikalische Chemie. In 1999, he went independent in order to focus his time on research applications and concepts. This has since included research scientist positions at UC San Diego, the Salk Institute among other collaborative endeavors. To date, his efforts have resulted in publications spanning a wide diversity of natural and physical sciences.

William Fenical (Bill) is distinguished professor of oceanography and pharmaceutical science at the Scripps Institution of Oceanography, University of California at San Diego. His career in marine natural products has spanned more than 45 years ever since his early work on the pigments of abalone shells. Fenical has more than 450 publications in marine natural products and 200 of these have involved developing the field of marine microbial chemistry. He is the recipient of the Paul Scheuer Award in Marine Natural Products Chemistry (1996), the International Society of Chemical Ecology's Silver Medal Award (1997), the Ernest Guenther Award in Natural Products from the American Chemical Society (2006). And, in 2008 Bill was Elected as Fellow of the American Association of Science (AAAS).

Leticia Costa-Lotufo (Leca) recently moved to University of São Paulo, where she holds an associate professor position. Previously, she was a professor at Federal University of Ceara, Fortaleza, Ceara, Brazil from 2002 to 2015. Her lab is focused on the discovery of new anticancer compounds from Brazilian marine biodiversity and studies on their mechanisms of action. Recently, she started a multidisciplinary project to access marine microorganisms diversity and biotechnological potential along the Brazilian coast and islands. PhD Costa-Lotufo's activities include the supervision of undergraduate and graduate students and post-doctoral fellows. She has published book chapters, review articles and more than 210 articles in peer-reviewed journals. On 2010, PhD Costa-Lotufo was nominated as a young scientist of the Brazilian Academy of Science.

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Figures

Figures (9)

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Figure 1
The IAF approach. A five-step procedure is used that begins with (Step 1) labeling a natural product with an IAF tag (shaded in light blue). Step 2: the uptake and cellular tracking of the probe is then explored using a combination of confocal and super-resolution microscopies. Once the subcellular localization and trafficking is understood, samples of cell lysates are prepared from these cells or their subcellular fractions. Step 3: the targets within these lysates are then isolated using an immunoprecipitation (IP) technique that applies monoclonal mAbs designed against the IAF tag as a tool to selectively isolate proteins or other biomolecules that bind to the natural product. Step 4: the identity of the immunoprecipitated biomolecules is then elucidated. For proteins, this is accomplished using a combination of SDS-PAGE purification and trypsin-digest liquid chromatography-mass spectrometry (LC-MS/MS) protein ID and MudPIT analyses. Step 5: if a protein target is found, it is cloned, expressed and purified and used to (Step 6) validate binding to the natural product. We find that nano-isothermal titration calorimetry (ITC) offers an ideal means to complete these studies. For low molecular weight proteins, we have also found that nuclear magnetic resonance (NMR) titration methods using either unlabeled or ¹⁵N-labeled protein also provide effective identification of binding events.

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Figure 2
Structures of four marine natural products examined using the IAF approach.

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Figure 3
Application of the IAF approach to elucidate the targeting of myosin by ammosamide B. (a) An image from 10⁶ HeLa cells incubated with 1 mL of 50 µM ammosamide B probe in Dulbecco's Modified Eagle's Medium (DMEM) after incubation at 37 °C for 12 h; (b-d) immunoprecipitation studies; (b) a 3-8 % tris-acetate SDS-PAGE gel stained with GelCode arising from the IP of lysate from 10⁸ HCT-116 cells treated with the ammosamide B probe and Affigel Hz resin containing 12.5 mg mL^-1 of XRI-TF35 mAb. After incubation for 12 h and multiple washings with PBS pH 7.2 at 4 °C, the bound protein was eluted from XRI-TF35-Affigel Hz resin with 0.1 M Tris-HCl pH 6.8 (L1), 5 mM ammosamide B probe in 0.1 M Tris-HCl pH 6.8 (L2), or 50 mM ammosamide B probe in 0.1 M Tris-HCl pH 6.8 (L3) at 23 °C; (c) HCT-116 lysate stained with GelCode blue; (d) a fluorescent image of the gel in (b) prior to GelCode blue staining. Box denotes protein bands identified through trypsin-digest LC-MS/MS as a member of the myosin family (adapted from reference 9).

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Figure 4
Application of the IAF approach to elucidate the targeting of actin by marinopyrrole A. (a) Confocal fluorescent images of HCT-116 cells depicting the red and blue fluorescence from the uptake of 10 µM marinopyrrole A IAF probe after 6 h. A set of confocal fluorescent images showing blue (447 nm), red (692 nm), and two-color mixed channel (mix) with the blue arising from the IAF tag and the red from the marinopyrrole motif; (b) a fluorescent SDS PAGE gel showing cell lysate obtained from HCT-116 cells (10⁸ cells) treated with 10 µM the marinopyrrole IAF probe in L1 and 10 µM of a related IAF probe¹² in L2 for 12 h; (c) a Silver Blue stained SDS-PAGE gel depicting proteins immunoprecipitated from the lysate in (b) using Affigel 10 resin containing 3.5 mg mL^-1 of an anti-IAF antibody XRI-TF35; (d) SDS-PAGE gel depicting the HCT-116 cell lysate used in (c). Proteins were identified by Trypsin-digest LC-MS-MS analysis as indicated by red and blue boxes (adapted from reference 12).

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Figure 5
Application of the IAF approach to elucidate the targeting of dermcidin (DCD) by seriniquinone. (a) Fluorescence from treatment of HCT-116 cells with seriniquinone for 1 h results in the appearance of green-red fluorescence from around the nucleus, as indicated by counterstaining with DAPI (blue). Counterstaining of the (b) endoplasmic reticulum (ER) and (c) autophagosomes (AP) was used to validate the subcellular localization events. Images depict staining of cells with ER stain at 1 and 6 h and AP stain at 6 and 24 h; (d) a silver-stained SDS PAGE gel depicting proteins immunoprecipitated from HCT-116 cell lysates treated with the seriniquinone IAF probe over 8 h at 4 °C. This response was dose dependent with an increasing return of immunoprecipitated protein at higher concentrations of the seriniquinone IAF probe (L2 versus L3), but was not apparent in experiments lacking the probe (L1). Trypsin-digest LC-MS/MS analysis of the protein in a 90-kDa band returned peptides corresponding to DCD (red) and Hsp70 (blue) (adapted from reference 15).

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Figure 6
Application of the IAF approach to elucidate the targeting of glycolytic enzymes by chlorizidine A. (a) Immunoprecipitation of HCT-116 cell lysate (1 mg mL^-1 total protein) in PBS (pH 7.2) containing 10 µm chlorizidine A IAF probe returned four distinct protein bands after staining with GelCode blue stain. Lanes L1-L3 depict HCT-116 cell lysate (L1), the IP fraction (L2) from the chlorizidine A IAF probe, and the IP fraction (L3) from an IAF control; (b) subcellular localization of the chlorizidine A IAF probe in HCT-116 cells. A confocal microscopy image depicting HCT-116 cells treated with 5 mm chlorizidine A IAF probe for 16 h and then co-stained with 2.5 mm LysoTracker Red DND-99 for 15 min before imaging. The appearance of violet in the combined image indicates the co-localization of blue from the chlorizidine A IAF probe and red from the LysoTracker Red DND-99. (adapted from reference 16).

Figure 1 The IAF approach. A five-step procedure is used that begins with (Step 1) labeling a natural product with an IAF tag (shaded in light blue). Step 2: the uptake and cellular tracking of the probe is then explored using a combination of confocal and super-resolution microscopies. Once the subcellular localization and trafficking is understood, samples of cell lysates are prepared from these cells or their subcellular fractions. Step 3: the targets within these lysates are then isolated using an immunoprecipitation (IP) technique that applies monoclonal mAbs designed against the IAF tag as a tool to selectively isolate proteins or other biomolecules that bind to the natural product. Step 4: the identity of the immunoprecipitated biomolecules is then elucidated. For proteins, this is accomplished using a combination of SDS-PAGE purification and trypsin-digest liquid chromatography-mass spectrometry (LC-MS/MS) protein ID and MudPIT analyses. Step 5: if a protein target is found, it is cloned, expressed and purified and used to (Step 6) validate binding to the natural product. We find that nano-isothermal titration calorimetry (ITC) offers an ideal means to complete these studies. For low molecular weight proteins, we have also found that nuclear magnetic resonance (NMR) titration methods using either unlabeled or ¹⁵N-labeled protein also provide effective identification of binding events.

Figure 2 Structures of four marine natural products examined using the IAF approach.

Figure 3 Application of the IAF approach to elucidate the targeting of myosin by ammosamide B. (a) An image from 10⁶ HeLa cells incubated with 1 mL of 50 µM ammosamide B probe in Dulbecco's Modified Eagle's Medium (DMEM) after incubation at 37 °C for 12 h; (b-d) immunoprecipitation studies; (b) a 3-8 % tris-acetate SDS-PAGE gel stained with GelCode arising from the IP of lysate from 10⁸ HCT-116 cells treated with the ammosamide B probe and Affigel Hz resin containing 12.5 mg mL^-1 of XRI-TF35 mAb. After incubation for 12 h and multiple washings with PBS pH 7.2 at 4 °C, the bound protein was eluted from XRI-TF35-Affigel Hz resin with 0.1 M Tris-HCl pH 6.8 (L1), 5 mM ammosamide B probe in 0.1 M Tris-HCl pH 6.8 (L2), or 50 mM ammosamide B probe in 0.1 M Tris-HCl pH 6.8 (L3) at 23 °C; (c) HCT-116 lysate stained with GelCode blue; (d) a fluorescent image of the gel in (b) prior to GelCode blue staining. Box denotes protein bands identified through trypsin-digest LC-MS/MS as a member of the myosin family (adapted from reference 9).

Figure 4 Application of the IAF approach to elucidate the targeting of actin by marinopyrrole A. (a) Confocal fluorescent images of HCT-116 cells depicting the red and blue fluorescence from the uptake of 10 µM marinopyrrole A IAF probe after 6 h. A set of confocal fluorescent images showing blue (447 nm), red (692 nm), and two-color mixed channel (mix) with the blue arising from the IAF tag and the red from the marinopyrrole motif; (b) a fluorescent SDS PAGE gel showing cell lysate obtained from HCT-116 cells (10⁸ cells) treated with 10 µM the marinopyrrole IAF probe in L1 and 10 µM of a related IAF probe¹² in L2 for 12 h; (c) a Silver Blue stained SDS-PAGE gel depicting proteins immunoprecipitated from the lysate in (b) using Affigel 10 resin containing 3.5 mg mL^-1 of an anti-IAF antibody XRI-TF35; (d) SDS-PAGE gel depicting the HCT-116 cell lysate used in (c). Proteins were identified by Trypsin-digest LC-MS-MS analysis as indicated by red and blue boxes (adapted from reference 12).

Figure 5 Application of the IAF approach to elucidate the targeting of dermcidin (DCD) by seriniquinone. (a) Fluorescence from treatment of HCT-116 cells with seriniquinone for 1 h results in the appearance of green-red fluorescence from around the nucleus, as indicated by counterstaining with DAPI (blue). Counterstaining of the (b) endoplasmic reticulum (ER) and (c) autophagosomes (AP) was used to validate the subcellular localization events. Images depict staining of cells with ER stain at 1 and 6 h and AP stain at 6 and 24 h; (d) a silver-stained SDS PAGE gel depicting proteins immunoprecipitated from HCT-116 cell lysates treated with the seriniquinone IAF probe over 8 h at 4 °C. This response was dose dependent with an increasing return of immunoprecipitated protein at higher concentrations of the seriniquinone IAF probe (L2 versus L3), but was not apparent in experiments lacking the probe (L1). Trypsin-digest LC-MS/MS analysis of the protein in a 90-kDa band returned peptides corresponding to DCD (red) and Hsp70 (blue) (adapted from reference 15).

Figure 6 Application of the IAF approach to elucidate the targeting of glycolytic enzymes by chlorizidine A. (a) Immunoprecipitation of HCT-116 cell lysate (1 mg mL^-1 total protein) in PBS (pH 7.2) containing 10 µm chlorizidine A IAF probe returned four distinct protein bands after staining with GelCode blue stain. Lanes L1-L3 depict HCT-116 cell lysate (L1), the IP fraction (L2) from the chlorizidine A IAF probe, and the IP fraction (L3) from an IAF control; (b) subcellular localization of the chlorizidine A IAF probe in HCT-116 cells. A confocal microscopy image depicting HCT-116 cells treated with 5 mm chlorizidine A IAF probe for 16 h and then co-stained with 2.5 mm LysoTracker Red DND-99 for 15 min before imaging. The appearance of violet in the combined image indicates the co-localization of blue from the chlorizidine A IAF probe and red from the LysoTracker Red DND-99. (adapted from reference 16).

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Elucidating the Mode of Action of Marine Natural Products through an Immunoaffinity Fluorescent (IAF) Approach

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[1] ^*e-mail: jlaclair@ucsd.edu, wfenical@ucsd.edu, costalotufo@usp.br