Faculty Bibliography

By providing broad resistance to environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations. A fundamental understanding of substrate selectivity by SMR transporters is needed to identify the types of selective pressures that contribute to this process. Using solid-supported membrane electrophysiology, we find that promiscuous transport of hydrophobic substituted cations is a general feature of SMR transporters. To understand the molecular basis for promiscuity, we solved X-ray crystal structures of a SMR transporter Gdx-Clo in complex with substrates to a maximum resolution of 2.3 Å. These structures confirm the family's extremely rare dual topology architecture and reveal a cleft between two helices that provides accommodation in the membrane for the hydrophobic substituents of transported drug-like cations.

The COVID-19 pandemic remains a global threat, and host immunity remains the main mechanism of protection against the disease. The spike protein on the surface of SARS-CoV-2 is a major antigen and its engagement with human ACE2 receptor plays an essential role in viral entry into host cells. Consequently, antibodies targeting the ACE2-interacting surface (ACE2IS) located in the receptor-binding domain (RBD) of the spike protein can neutralize the virus. However, the understanding of immune responses to SARS-CoV-2 is still limited, and it is unclear how the virus protects this surface from recognition by antibodies. Here, we designed an RBD mutant that disrupts the ACE2IS and used it to characterize the prevalence of antibodies directed to the ACE2IS from convalescent sera of 94 COVID19-positive patients. We found that only a small fraction of RBD-binding antibodies targeted the ACE2IS. To assess the immunogenicity of different parts of the spike protein, we performed in vitro antibody selection for the spike and the RBD proteins using both unbiased and biased selection strategies. Intriguingly, unbiased selection yielded antibodies that predominantly targeted regions outside the ACE2IS, whereas ACE2IS-binding antibodies were readily identified from biased selection designed to enrich such antibodies. Furthermore, antibodies from an unbiased selection using the RBD preferentially bound to the surfaces that are inaccessible in the context of whole spike protein. These results suggest that the ACE2IS has evolved less immunogenic than the other regions of the spike protein, which has important implications in the development of vaccines against SARS-CoV-2.

Citrate, α-ketoglutarate and succinate are TCA cycle intermediates that also play essential roles in metabolic signaling and cellular regulation. These di- and tricarboxylates are imported into the cell by the divalent anion sodium symporter (DASS) family of plasma membrane transporters, which contains both cotransporters and exchangers. While DASS proteins transport substrates via an elevator mechanism, to date structures are only available for a single DASS cotransporter protein in a substrate-bound, inward-facing state. We report multiple cryo-EM and X-ray structures in four different states, including three hitherto unseen states, along with molecular dynamics simulations, of both a cotransporter and an exchanger. Comparison of these outward- and inward-facing structures reveal how the transport domain translates and rotates within the framework of the scaffold domain through the transport cycle. Additionally, we propose that DASS transporters ensure substrate coupling by a charge-compensation mechanism, and by structural changes upon substrate release.

The transcription factor STAT3 is frequently activated in human solid and hematological malignancies and remains a challenging therapeutic target with no approved drugs to date. Here, we develop synthetic antibody mimetics, termed monobodies, to interfere with STAT3 signaling. These monobodies are highly selective for STAT3 and bind with nanomolar affinity to the N-terminal and coiled-coil domains. Interactome analysis detects no significant binding to other STATs or additional off-target proteins, confirming their exquisite specificity. Intracellular expression of monobodies fused to VHL, an E3 ubiquitin ligase substrate receptor, results in degradation of endogenous STAT3. The crystal structure of STAT3 in complex with monobody MS3-6 reveals bending of the coiled-coil domain, resulting in diminished DNA binding and nuclear translocation. MS3-6 expression strongly inhibits STAT3-dependent transcriptional activation and disrupts STAT3 interaction with the IL-22 receptor. Therefore, our study establishes innovative tools to interfere with STAT3 signaling by different molecular mechanisms.

The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) "killer" domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the "necrosome" and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites.

Despite being the subject of intense effort and scrutiny, kinases have proven to be consistently challenging targets in inhibitor drug design. A key obstacle has been promiscuity and consequent adverse effects of drugs targeting the ATP binding site. Here we introduce an approach to controlling kinase activity by using monobodies that bind to the highly specific regulatory allosteric pocket of the oncoprotein Aurora A (AurA) kinase, thereby offering the potential for more specific kinase modulators. Strikingly, we identify a series of highly specific monobodies acting either as strong kinase inhibitors or activators via differential recognition of structural motifs in the allosteric pocket. X-ray crystal structures comparing AurA bound to activating vs inhibiting monobodies reveal the atomistic mechanism underlying allosteric modulation. The results reveal 3 major advantages of targeting allosteric vs orthosteric sites: extreme selectivity, ability to inhibit as well as activate, and avoidance of competing with ATP that is present at high concentrations in the cells. We envision that exploiting allosteric networks for inhibition or activation will provide a general, powerful pathway toward rational drug design.

Background Current immunotherapies have shown promise in treating multiple cancer types but success remains elusive for cancers like pancreatic, the majority of colorectal cancers as well as cholangiocarcinoma. Poor prognosis in these tumor types is invariably linked to immune dysfunction. Galectin-9, which is a fundamental immune modulator and global immune suppressor, was previously identified as a molecule that plays a significant role in orchestrating and maintaining a tumor-permissive immune environment. In light of this, we have studied the relevance of galectin-9 as a biomarker and have developed LYT-200, a therapeutic, fully human IgG4 antibody to target galectin-9. Methods A synthetic, human antibody library was screened via phage display using purified carbohydrate binding domains (CRDs) of human and mouse galectin-9. Blockade of galectin-9-receptor binding and signaling by antibodies was assayed in both biochemical and cell-based formats. Antibody efficacy was assessed in orthotopic pancreatic cancer and subcutaneous melanoma mouse models as single agent and in combination with anti-PD1 and chemotherapy. Patient-derived samples (FFPE tissues, organotypic tumor spheroids (PDOTs)), which recapitulate complex tumor architecture, and patient blood samples were analyzed by immunohistochemistry, flow cytometry and ELISA to measure galectin-9 expression and compare with healthy controls across tumor types. Additionally, PDOTs were treated with clinical anti-galectin-9 antibodies and the resulting immune profile was analyzed. Results Significantly higher levels of galectin-9 were detected across patient blood/tissue samples (n >100 patients) compared with healthy controls. Analyses of PDOTs showed high levels of galectin-9 on tumor, myeloid and T cells. Its high expression, for example in pancreatic adenocarcinoma, suggest that galectin-9 is a promising therapeutic target and a biomarker that correlates with disease stage. From a therapeutic perspective, LYT-200 was selected out of numerous antibody candidates as the lead clinical clone based on its high affinity (dissociation constant < 1 nM) to galectin-9 across species, high specificity, stability, and blocking galectin-9 interactions with its ligands in biochemical and cellbased assays. Treatment of tumor bearing mice with LYT-200 significantly reduced in tumor size (p Conclusions LYT-200, a first-in-class human antibody that inhibits tumorpermitting activity of Galectin-9 was developed. The data collectively reveal novel galectin-9 biology and demonstrate therapeutic efficacy of LYT-200. Blockade of galectin-9 using LYT-200 is an innovative and promising strategy for treating aggressive solid tumors

Background Targeting and engineering gammadelta T cells has recently emerged as an orthogonal therapeutic approach in oncology with capacity to modulate both innate and adaptive immune properties. In solid tumors such as pancreatic ductal adenocarcinoma (PDA), melanoma, glioblastoma, ovarian, and breast cancer, gammadelta1 T cells express immunosuppression-related molecules and possess a protumorigenic capacity. We have shown that intra-tumoral gammadelta T cells from PDA, colorectal cancer (CRC) and hepatocellular carcinoma (HCC) potently suppress patients' alphabeta T cells. To harness the therapeutic potential of gammadelta1 T cell blockade, we developed highly specific, fully human antibodies against delta1-subset of gammadeltaT cell receptor (gammadelta1- TCR). Methods We determined the amino acid sequences of tumor specific delta1-TCR chains from primary PDA, CRC and gastric cancer samples. Multiple gammadelta-TCR proteins were produced and used to screen a proprietary synthetic, human antibody library using phage display. Surface plasmon resonance and bead-based assays were used to measure binding affinity and specificity. Affinity maturation was performed to improve cross-reactivity to monkey gammadelta1-TCR. Cell based assays were used to evaluate antibody-dependent cell cytotoxicity and phagocytosis (ADCC and ADCP). The levels of gammadelta1-T cell infiltration was measured in patient tumors. Efficacy in reversing immunesuppression was assayed using patient-derived organotypic tumor spheroids (PDOTS, n = 32), which recapitulate complex tumor architecture. Results Because the tumor-derived delta1 chains showed diverse CDR3 sequences, we developed antibodies that bind diverse delta1 TCRs. Our first-in-class anti-delta1 antibodies had low nanomolar affinity to delta1 TCRs and showed no binding to delta2 TCRs. Our lead clinical candidate showed no preference for the gamma chains of the TCR, which enables it to target diverse set of gammadelta1-TCRs. It had equivalent affinity for the human and cynomolgus monkey gammadelta1-TCRs and was potent in mediating cell based ADCC and ADCP. We showed that patient tumors can have a high delta1 T cell infiltration (up to 40% of the total T cell infiltrate). Our lead candidate achieved reproducible and robust efficacy in the up-regulation of pro-inflammatory T cell activation markers in PDOTS from a diverse set of gastrointestinal tumors. Furthermore, gammadelta knockout mice had an improved response to checkpoint inhibitors, anti-PD1 and anti-CTLA4, in melanoma and lung cancer models. Conclusions We have defined a novel therapeutic immuno-oncology strategy and translated it to develop a lead clinical candidate anti-delta1 monoclonal antibody. Our efficacious, novel immunotherapy has the potential to be transformative for the treatment of cancers where gammadelta1 T cells drive a pro-tumorigenic, immunosuppressive environment

Rapidly determining the biological effect of perturbing a site within a potential drug target could guide drug discovery efforts, but it remains challenging. Here, we describe a facile target validation approach that exploits monobodies, small synthetic binding proteins that can be fully functionally expressed in cells. We developed a potent and selective monobody to WDR5, a core component of the mixed lineage leukemia (MLL) methyltransferase complex. The monobody bound to the MLL interaction site of WDR5, the same binding site for small-molecule inhibitors whose efficacy has been demonstrated in cells but not in animals. As a genetically encoded reagent, the monobody inhibited proliferation of an MLL-AF9 cell line in vitro, suppressed its leukemogenesis and conferred a survival benefit in an in vivo mouse leukemia model. The capacity of this approach to readily bridge biochemical, structural, cellular characterization and tests in animal models may accelerate discovery and validation of druggable sites.