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Jun O. Liu
Department Affiliation: Primary: Pharmacology and Molecular Sciences; Secondary: Oncology
Degree: Ph.D., Massachusetts Institute of Technology
Telephone Number: 410-955-4619
Fax Number: 410-955-4520
E-mail address: email@example.com
School of Medicine Address: Room 516 Hunterian Building, 725 N. Wolfe Street, Baltimore, MD 21205
Chemical biology, molecular and cellular biology, and translational medicine
Our primary research interest lies at the interface between chemistry, biology and medicine. We employ high-throughput screening to identify modulators of various cellular processes and pathways that have been implicated in human diseases, ranging from cancer to autoimmunity. Once biologically active compounds are identified, they serve as both chemical probes of the biological processes of interest and leads for the development of new drugs for treating human diseases.
Biological processes of interest include cancer cell growth, metastasis, apoptosis, angiogenesis, calcium-dependent signaling pathways, eukaryotic transcription, and translation. Some ongoing projects are highlighted below:
• Exploration of the existing drug space for novel pharmacological activities with translational potential.
Drug discovery and development is a time-consuming and costly process. To accelerate the process, we have assembled a library of existing drugs, known as the Johns Hopkins Drug Library (JHDL). We have screened the JHDL in both target- and cell-based assays for novel pharmacological activities.
To date, we and our collaborators have identified a number of known drugs that exhibited previously unknown activity. The most interesting hits discovered in our lab alone include:
Itraconazole, a widely used antifungal drug, was found to possess potent anti-angiogenic activity and anti-hedgehog signaling activity. We and others have demonstrated that itraconazole inhibits angiogenesis and tumor xenograft growth in animal models, and now itraconazole has entered multiple Phase 2 human clinical studies. To date, itraconazole in combination with pemetrexed has shown efficacy in treating non-small cell lung cancer, metastatic and castration-resistant prostate cancer, and basal cell carcinoma.
Nitroxoline, a urinary tract antibiotic, was found to inhibit angiogenesis through dual inhibition of the type 2 methionine aminopeptidase and SIRT1 and 2, which culminate in differentiation of endothelial cells. As nitroxoline is uniquely concentrated in the urinary tract, it has potential in the treatment of bladder cancer. Upon confirming its effect in a mouse orthotopic bladder cancer model, it has entered into a Phase 3 human trial for treating bladder cancer.
Nelfinavir, an HIV protease inhibitor, was found to selectively inhibit HER2+ breast cancer cells. Follow-up studies revealed that nelfinavir is a HSP90 inhibitor with a novel mode of action, interacting with HSP90 at a site distinct from the binding sites of previously known HSP90 inhibitors. Nelfinavir and improved analogs have potential as new treatments for HER2+ breast cancer.
Clofazimine, an important component of the multidrug regimen for treating leprosy since the 1960s, was found to be a novel inhibitor of the Kv1.3 channel, thereby blocking the activation of effector memory T cells implicated in a multitude of autoimmune diseases.
In addition to the aforementioned hits, novel inhibitors of HIF-1, the hedgehog signaling pathway and the Hippo signaling pathway have also been identified from JHDL by our collaborators.
Among the leads, we have carried out mechanistic and translational studies on itraconazole in the greatest detail. We have found that itraconazole operates through a novel dual-targeted mechanism of action for its anti-angiogenic activity. We have identified and validated two molecular targets for itraconazole, the voltage-dependent anion channel (VDAC)1 and Niemann Pick Disease type C (NPC)1 (Fig. 1A). While inhibition of VDAC leads to a decrease in cellular ATP levels, leading to an increase in cellular AMP/ATP ratio and activation of the AMPK signaling pathway, inhibition of NPC1 causes accumulation of cholesterol in the endo-lysosome and consequently leads to defects in lysosomal calcium signaling. These effects converge to cause synergistic inhibition of mTOR (Fig. 1A). Itraconazole contains three stereocenters; the marketed drug is a mixture of four stereoisomers. We have shown that the four stereoisomers vary in both potency and toxicity. We
found that one of the four stereoisomers (2S, 4R, 2’S) is most potent for inhibition of angiogenesis and least toxic for the liver, a major side effect of the clinically used itraconazole mixture, rendering the specific stereoisomer a new lead for development (Fig. 1B). As a drug, itraconazole has a few drawbacks, including low solubility and potent inhibition of the drug metabolizing enzyme CYP3A4, preventing its use in combination with other drugs to treat cancer and other diseases. We have developed next-generation analogs, one of which was named tetrapyrizole, which is largely free of CYP3A4 inhibition and has significantly higher aqueous solubility and equally potent anti-angiogenic activity, making it an exciting lead to develop novel anticancer and anti-angiogenesis drugs (Fig. 1C).
• Learning from Nature--Natural products as probes of eukaryotic transcription and translation processes.
Natural products are an invaluable source of both molecular probes and drug leads, particularly those with anticancer and anti-infective activities. Triptolide is a natural product isolated from the Thunder God Vine, whose extracts have been used in traditional Chinese medicine as immunosuppressive and anti-inflammatory remedies for centuries. It displays strong inhibition of all cancer cell lines tested to date, with a mean IC50 value in the low nanomolar range. Its molecular mechanism of action remained elusive for decades. Using a top-down approach,
we identified XPB, a subunit of the general transcription factor TFIIH, as a molecular target of triptolide. We found that triptolide binds to XPB through covalent modification of an active site cysteine with one of its epoxide groups.
We and others have shown that binding of XPB by triptolide leads to degradation of the catalytic RPB subunit of RNA polymerase II. As RPB is not part of the TFIIH complex to which XPB belongs, this poses an intriguing mechanistic question of how binding of triptolide to one protein selectively induces degradation of another seemingly unrelated protein. We are currently attempting to elucidate the underlying molecular mechanism of this effect of triptolide.
On the translational front, various analogs of triptolide have been developed as leads for developing anticancer drugs with very limited success. Two of the limitations for triptolide as a drug lead are its general toxicity and low solubility. To address these problems, we have designed glucose-triptolide conjugates named glutriptolides. This modification both increases solubility and allows for selecting targeting of triptolide to cancer cells that overexpress glucose transporters (Fig. 2). We confirmed that the glucose-triptolide conjugate is more cytotoxic in cancer cells than normal cells. Moreover, it showed sustained anticancer activity in animal models. Glutriptolide is undergoing preclinical development and has the potential to become a new weapon with greater precision in the war against cancer.
• Imitating Nature--Generation of natural product-inspired macrocyclic combinatorial libraries for the discovery of novel inhibitors of protein-protein interactions and membrane proteins.
Nature has taught us many important lessons on how to harness the power of small molecules to manipulate the functions of proteins. An illuminating example is the duo of macrocyclic natural products rapamycin and FK506 that have become powerful immunosuppressive and/or anticancer drugs in their natural forms (Fig. 3). Rapamycin and FK506 share an extraordinary mode of action;
they act by recruiting an abundant and ubiquitously expressed cellular protein, the prolyl cis-trans isomerase FKBP, and the resultant binary complexes, FKBP-rapamycin and FKBP-FK506, subsequently bind to and allosterically inhibit their target proteins mTOR and calcineurin, respectively. Structurally, FK506 and rapamycin share a common FKBP-binding domain (FKBD) but differ in their effector domains. The presence of FKBD in FK506 and rapamycin confer a number of advantages, including stability, higher intracellular accumulation, larger size and superior in vivo pharmacological activity. Taking the cue from nature, we asked whether we could leverage this highly privileged scaffold to replace the effector domain of rapamycin with yet another structural scaffold to confer novel protein target specificity. We named our new hybrid macrocycles rapafucins.
We designed and generated a library of over 45,000 novel rapafucins (Fig. 3). To date, we have conducted multiple cell- and target-based screens of the rapafucin library and identified a number of potent inhibitors of undruggable or difficult-to-drug targets, including membrane protein targets as well as protein-protein interactions involving transcription factors. The representative hits include:
• Rapadocin, an isoform-specific inhibitor of the Equilibrative Nucleoside Transporter (ENT)-1, which upregulates adenosine signaling with implications in treating kidney reperfusion injury and organ transplantation. [Collaborators: Ville Paavilainen/University of Helsinki, Finland; Zhaoli Sun and Chung-Ming Tse/Johns Hopkins; Imogen Coe/Ryerson University, Canada; Cordelia Schiene-Fischer and Gunter Fischer/Martin Luther University, Germany; Seok-Yong Lee/Duke University]
• Rapaglutin A, a potent inhibitor of Class I glucose transporters (GLUT) with promising antitumor activity. [Collaborators: Sara Sukumar and Heng Zhu/Johns Hopkins; Jason Locasale, Duke University; Cordelia Schiene-Fischer and Gunter Fischer/Martin Luther University, Germany]
• Rapaglutin E, a potent inhibitor of GLUT1 and CD4+ T cell activation and promising lead to develop novel immunosuppressants. [Collaborators: Jason Locasale/Duke University]
• Rapayapin, an inhibitor of the protein-protein interaction between YAP and TEAD, downstream signal transducers of the Hippo signaling pathway and promising targets for immunotherapy due to the involvement of YAP in the regulation of Treg function. [Collaborators: Fan Pan and Drew Pardoll/Johns Hopkins; Duojia Pan/UT Southwestern]
• Rapayapon, an unexpected activator of YAP with potential in regenerative medicine through modulation of stem cell function. [Collaborators: Peter Devreotes and Andrew Ewald/Johns Hopkins; Jennifer Lippincott-Schwartz and Inhee Chung/HHMI GW Medical School]
• Rapupron, an activator of the unfolded protein response, particularly the IRE1-XBP1 signaling axis in the UPR pathway with implication in neurodegeneration and other indications. [Collaborators: Ville Paavilainen/University of Helsinki, Finland; Luke Wiseman/Scripps Research Institute]
• Rapaprotin, a novel latent (pro-drug) inhibitor of the human 26S proteasome that has a distinct binding site from all existing proteasome inhibitors. It showed cancer cell selectivity with low toxicity to normal cells. It exhibited strong synergy with bortezomib and other known proteasome inhibitors and overcomes bortezomib resistance. [Collaborators: Christian Goche/Johns Hopkins; Bill Matsui/UT Austin Dell Medical School; Jack Mao/Peking University, China]
• Rapashipin, an inhibitor of the protein phosphatase SHIP-2 with implications in immunotherapy. [Collaborator: Zhong-Yin Zhang/Purdue University]
To facilitate screening of rapafucin libraries, we developed a 3D rapafucin microarray (Raparray) by covalently immobilizing rapafucins onto 3-dimensional surface using carbene-based crosslinking chemistry. Raparray allows for the screening of the entire 45,000-compound
rapafucin library on a single chip (Fig. 4). Moreover, the Raparray chips can be used to screen any protein target of interest in less than 200 µL of cell lysate so long as a specific antibody is available for the endogenous protein, or a protein target of interest can be expressed with an epitope tag. This new Raparray platform will greatly facilitate the interrogation of the rapafucin library for new binding ligands for any protein of interest regardless of its intrinsic enzymatic/biological activity.
It is worth noting that several of the optimized hits have shown desirable pharmacological activity in animal disease models, confirming that the presence of FKBD in rapafucins affords favorable pharmacokinetic properties in vivo. Importantly, with a wide variety of commercially available non-natural amino acid building blocks, we can access a rapafucin library with a tetrapeptide effector domain that has a size approaching 3 billion compounds. One of the questions we will be attempting to address is how many proteins in the human proteome (ca. 22,000) can be targeted using a library of rapafucins of such a structural diversity (109). In rapadocin, we already observed exquisite isoform specificity for one of two closely related isoforms of hENTs. A related question is whether we can leverage the rapafucin platform to identify specific modulators of many, if not most, of cancer-driving mutations that have emerged from the extensive sequencing and annotation of human cancer genomes over the past decade. The discovery of mutant-specific inhibitors of cancer-driving mutant targets may give rise to novel targeted therapeutic drugs and the combination thereof for the treatment of cancer patients with much lower side effects than existing regimens. Given the target diversity we have already seen to date with the limited chemical space we have sampled using the 45,000-compound rapafucin library, we are optimistic that more rapafucin ligands will be identified for other protein targets and rapafucins are becoming an important new source of both novel chemical probes and drug leads.
• Translation from bench to bedside
The ultimate goal of our work is to leverage novel tools in chemistry and to exploit new knowledge in biology to discover and develop new therapeutic drugs for the betterment of human health. To date, many of the lead compounds we have discovered and developed have entered preclinical and human clinical studies, leading to a growing pipeline of drug candidates (Fig. 5).
Chong, C.R., Chen, X., Shi, L., Liu, J.O., and Sullivan, D.J. A clinical drug library screen identifies astemizole as an antimalarial agent. Nat Chem Biol, 2, 415-416, 2006. PubMed Reference
Chong, C.R., Xu, J., Lu, J., Bhat, S., Sullivan, D.J., Jr., Liu, J.O. Inhibition of angiogenesis by the antifungal drug itraconazole. ACS Chem Biol, 2, 263-70, 2007. PubMed Reference
Kim, J., Tang, J.Y., Gong, R., Kim, J., Lee, J.J., Clemons, K.V., Chong, C.R., Chang, K.S., Fereshteh, M., Gardner, D., Reya, T., Liu, J.O., Epstein, E. H., Stevens, D. A., Beachy, P. A. Itraconazole, a commonly used antifungal that inhibits Hedgehog pathway activity and cancer growth. Cancer Cell, 17, 388-399, 2010. PubMed Reference
Xu, J., Dang, Y., Ren, Y.R., Liu, J.O. Cholesterol trafficking is required for mTOR activation in endothelial cells. Proc Natl Acad Sci USA, 107, 4764-9, 2010. PubMed Reference
Shim, J.S., Matsui, Y., Bhat, S., Nacev, B.A., Xu, J., Bhang, H.E., Dhara, S., Han, K.C., Chong, C.R., Pomper, M.G., So, A., Liu, J.O. Effect of nitroxolibne on angiogenesis and growth of human bladder cancer. J Natl Cancer Inst, 102, 1855-1873, 2010. PubMed Reference
Head, S.A., Shi, W., Zhao, L., Gorshkov, K., Pasunooti, K., Chen, Y., Deng, Z., Li, R.J., Shim, J.S., Tan, W., Hartung, T., Zhang, J., Zhao, Y., Colombini, M., Liu, J.O. Antifungal drug itraconazole targets VDAC1 to modulate the AMPK/mTOR signaling axis in endothelial cells. Proc Natl Acad Sci USA, 112, E7276-7285, 2015. PubMed Reference
Shim, J.S., Li, R.J., Bumpus, N.N., Head, S.A., Kumar Pasunooti, K., Yang, E.J., Lv, J., Shi, W., and Liu, J.O. Divergence of Antiangiogenic Activity and Hepatotoxicity of Different Stereoisomers of Itraconazole. Clin Cancer Res, 22, 2709-2720, 2016. PubMed Reference
Head, S.A., Shi, W. Q, Yang, E.J., Nacev, B.A., Hong, S.Y., Pasunooti, K.K., Li, R.J., Shim, J.S., and Liu, J.O. Simultaneous Targeting of NPC1 and VDAC1 by Itraconazole Leads to Synergistic Inhibition of mTOR Signaling and Angiogenesis. ACS Chem Biol, 12, 174-182, 2017. PubMed Reference
Chemical biology of natural products
Low, W.-K., Dang, Y., Schneider-Poetsch, T., Shi, Z., Choi, N.S., Merrick, W.C., Romo, D., Liu, J.O. Inhibition of eukaryotic translation initiation by the marine natural product pateamine A. Mol Cell, 20, 709-722, 2005. PubMed Reference
Schneider-Poetsch, T., Ju, J., Eyler, D.E., Dang, Y., Bhat, S., Merrick, W.C., Green, R., Liu, J.O. Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nat Chem Biol, 6, 209-217, 2010. PubMed Reference
Titov, D.V., Gilman, B., He, Q.L., Bhat, S., Low, W.K., Dang, Y., Smeaton, M., Demain, A.L., Miller, P.S., Kugel, J.F., Goodrich, J.A., Liu, J.O. XPB, a subunit of TFIIH, is a target of the natural product triptolide. Nat Chem Biol, 7, 182-188. 2011. PubMed Reference
McClary, B., Zinshteyn, B., Meyer, M., Jouanneau, M., Pellegrino, S., Yusupova, G., Schuller, A., Reyes, J.C.P., Lu, J., Guo, Z., Ayinde, S., Luo, C., Dang, Y., Romo, D., Yusupov, M., Green, R., Liu, J.O. Inhibition of Eukaryotic Translation by the Antitumor Natural Product Agelastatin A. Cell Chem Biol, 24, 605-613, 2017. PubMed Reference
He, Q.L., Titov, D.V., Li, J., Tan, M., Ye, Z., Zhao, Y., Romo, D., Liu, J.O. Covalent modification of a cysteine residue in the XPB subunit of the general transcription factor TFIIH through single epoxide cleavage of the transcription inhibitor triptolide. Angew Chem Int Ed, 54, 1859-1863, 2014. PubMed Reference
He, Q.L., Minn, I., Wang, Q., Xu, P., Head, S.A., Datan, E., Yu, B., Pomper, M.G., Liu, J.O. Targeted Delivery and Sustained Antitumor Activity of Triptolide through Glucose Conjugation. Angew Chem Int Ed, 55, 12035-12039, 2016. PubMed Reference
Peiffer, B.J., Qi, L., Ahmadi, A.R., Wang, Y., Guo, Z., Peng, H., Sun, Z., Liu, J.O. Activation of BMP Signaling by FKBP12 Ligands Synergizes with Inhibition of CXCR4 to Accelerate Wound Healing. Cell Chem Biol, 26, 652-661, 2019. PubMed Reference
Youn, H.-D., Sun, L., Prywes, R., Liu, J.O. Apoptosis of T cells mediated by Ca2+-induced release of the transcription factor MEF2. Science, 286, 790-793, 1999. PubMed Reference
Han, A., Pan, F., Stroud, J.C., Youn, H.–D., Liu, J.O., Chen, L. Structural basis of sequence-specific recruitment of transcription corepressor Cabin1 by Myocyte Enhancer Factor-2. Nature, 422, 730-734, 2003. PubMed Reference
Pan, F., Sun, L., Dardian, D.B., Whartenby, K.A., Pardoll, D.M., Liu, J.O. Feedback inhibition of calcineurin and Ras by a dual inhibitory protein Carabin. Nature, 445, 433-436, 2007. PubMed Reference
Li, R.J., Xu, J., Fu, C., Zhang, J., Zheng, Y.G., Jia, H., Liu, J.O. Regulation of mTORC1 by lysosomal calcium and calmodulin. Elife, 5, e19360, 2016. PubMed Reference
Li, W., Bhat, S., Liu, J.O. (2011) A simple and efficient route to the FKBP-binding domain from rapamycin. Tetrehedron Lett, 52, 5070-5072, 2011. PubMed Reference
Guo, Z., Hong, S.Y., Wang, J., Liu, W., Peng, H., Das, M., Li, W., Rehan,S., Bhat, S., Peiffer, B., Tse, C.-M., Tarmakova, Z., Schiene-Fischer, C., Fischer, G., Coe, I., Paavilainen, V.O., Sun, Z., Liu, J.O. Rapafucins, rapamycin-inspired macrocycles with new target specificity. Nat Chem, 11, 254-263, 2019. PubMed Reference
Guo, Z., Cheng, Z., Wang, J., Liu, W., Peng, H., Wang, Y., Rao, A.V.S., Li, R.J., Ying, X., Korangath, P., Liberti, M.V., Li, Y., Xie, Y., Hong, S.Y., Schiene-Fischer, C., Fischer, G., Locasale, J.W., Sukumar, S., Zhu, H., Liu, J.O. Discovery of a Potent GLUT Inhibitor from a Library of Rapafucins by Using 3D Microarrays. Angew Chem Int Ed, 58, 17158-17162, 2019. PubMed Reference
Other graduate programs in which Dr. Liu participates: