Fragment Screening Research Articles

Covalent disruptor of YAP-TEAD association suppresses defective Hippo signaling.

The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.

Large-Scale Crystallographic Fragment Screening Expedites Compound Optimization and Identifies Putative Protein-Protein Interaction Sites.

The identification of starting points for compound development is one of the key steps in early-stage drug discovery. Information-rich techniques such as crystallographic fragment screening can potentially increase the efficiency of this step by providing the structural information of the binding mode of the ligands in addition to the mere binding information. Here, we present the crystallographic screening of our 1000-plus-compound F2X-Universal Library against the complex of the yeast spliceosomal Prp8 RNaseH-like domain and the snRNP assembly factor Aar2. The observed 269 hits are distributed over 10 distinct binding sites on the surface of the protein-protein complex. Our work shows that hit clusters from large-scale crystallographic fragment screening campaigns identify known interaction sites with other proteins and suggest putative additional interaction sites. Furthermore, the inherent binding pose validation within the hit clusters may accelerate downstream compound optimization.

Selective and Cell-Active PBRM1 Bromodomain Inhibitors Discovered through NMR Fragment Screening.

PBRM1 is a subunit of the PBAF chromatin remodeling complex that uniquely contains six bromodomains. PBRM1 can operate as a tumor suppressor or tumor promoter. PBRM1 is a tumor promoter in prostate cancer, contributing to migratory and immunosuppressive phenotypes. Selective chemical probes targeting PBRM1 bromodomains are desired to elucidate the association between aberrant PBRM1 chromatin binding and cancer pathogenesis and the contributions of PBRM1 to immunotherapy. Previous PBRM1 inhibitors unselectively bind SMARCA2 and SMARCA4 bromodomains with nanomolar potency. We used our protein-detected NMR screening pipeline to screen 1968 fragments against the second PBRM1 bromodomain, identifying 17 hits with Kd values from 45 μM to >2 mM. Structure-activity relationship studies on the tightest-binding hit resulted in nanomolar inhibitors with selectivity for PBRM1 over SMARCA2 and SMARCA4. These chemical probes inhibit the association of full-length PBRM1 to acetylated histone peptides and selectively inhibit growth of a PBRM1-dependent prostate cancer cell line.

Discovery and Structural Characterization of Small Molecule Binders of the Human CTLH E3 Ligase Subunit GID4.

Targeted protein degradation (TPD) strategies exploit bivalent small molecules to bridge substrate proteins to an E3 ubiquitin ligase to induce substrate degradation. Few E3s have been explored as degradation effectors due to a dearth of E3-binding small molecules. We show that genetically induced recruitment to the GID4 subunit of the CTLH E3 complex induces protein degradation. An NMR-based fragment screen followed by structure-guided analog elaboration identified two binders of GID4, 16 and 67, with Kd values of 110 and 17 μM in vitro. A parallel DNA-encoded library (DEL) screen identified five binders of GID4, the best of which, 88, had a Kd of 5.6 μM in vitro and an EC50 of 558 nM in cells with strong selectivity for GID4. X-ray co-structure determination revealed the basis for GID4–small molecule interactions. These results position GID4-CTLH as an E3 for TPD and provide candidate scaffolds for high-affinity moieties that bind GID4.

X-ray Screening of an Electrophilic Fragment Library and Application toward the Development of a Novel ERK 1/2 Covalent Inhibitor.

Fragment-based drug discovery (FBDD) has become an established method for the identification of efficient starting points for drug discovery programs. In recent years, electrophilic fragment screening has garnered increased attention from both academia and industry to identify novel covalent hits for tool compound or drug development against challenging drug targets. Herein, we describe the design and characterization of an acrylamide-focused electrophilic fragment library and screening campaign against extracellular signal-regulated kinase 2 (ERK2) using high-throughput protein crystallography as the primary hit-finding technology. Several fragments were found to have covalently modified the adenosine triphosphate (ATP) binding pocket Cys166 residue. From these hits, 22, a covalent ATP-competitive inhibitor with improved potency (ERK2 IC50 = 7.8 μM), was developed.

Fragment-Based and Structural Investigation for Discovery of JNK3 Inhibitors.

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family and are related to cell proliferation, gene expression, and cell death. JNK isoform 3 (JNK3) is an important therapeutic target in varieties of pathological conditions including cancers and neuronal death. There is no approved drug targeting JNKs. To discover chemical inhibitors of JNK3, virtual fragment screening, the saturation transfer difference (STD) NMR, in vitro kinase assay, and X-ray crystallography were employed. A total of 27 fragments from the virtually selected 494 compounds were identified as initial hits via STD NMR and some compounds showed the inhibition of the activity of JNK3 in vitro. The structures of JNK3 with a fragment and a potent inhibitor were determined by X-ray crystallography. The fragment and inhibitor shared a common JNK3-binding feature. The result shows that fragment screening by NMR spectroscopy is a very efficient method to screen JNK3 binders and the structure of JNK3-inhibitor complex can be used to design and develop more potent inhibitors.

Fragment-Based Discovery of a Novel, Brain Penetrant, Orally Active HDAC2 Inhibitor.

Fragment-based ligand discovery was successfully applied to histone deacetylase HDAC2. In addition to the anticipated hydroxamic acid- and benzamide-based fragment screening hits, a low affinity (∼1 mM) α-amino-amide zinc binding fragment was identified, as well as fragments binding to other regions of the catalytic site. This alternative zinc-binding fragment was further optimized, guided by the structural information from protein-ligand complex X-ray structures, into a sub-μM, brain penetrant, HDAC2 inhibitor (17) capable of modulating histone acetylation levels in vivo.

Demolition Waste Potential for Completely Cement-Free Binders.

Due to renovation and fighting in the world, a huge accumulation of construction and demolition waste is formed. These materials are effectively used as aggregates, but there is very little information about the use of scrap concrete to create cementless binders. The purpose of the work is to be a comprehensive study of the composition and properties of concrete wastes of various fractions with the aim of their rational use as cementless binders. The scientific novelty lies in the fact that the nature of the processes of structure formation of a cementless binder based on sandy fractions of the screening of fragments of destroyed buildings and structures, as a complex polyfunctional system, has been theoretically substantiated and experimentally confirmed. Different percentages of non-hydrated clinker minerals in concrete scrap were determined. In the smallest fraction (less than 0.16 mm), more than 20% of alite and belite are present. Waste of the old cement paste is more susceptible to crushing compared to the large aggregate embedded in it, therefore, particles of the old cement paste and fine aggregate predominate in the finer fractions of the waste. Comprehensive microstructural studies have been carried out on the possibility of using concrete scrap as a completely cementless binder using scanning electron microscopy, X-ray diffraction analysis, and differential thermal analysis. It has been established that for cementless samples prepared from the smallest fractions (less than 0.315 mm), the compressive strength is 1.5–2 times higher than for samples from larger fractions. This is due to the increased content of clinker minerals in their composition. The compressive strength of the cementless binder after 28 days (7.8 MPa), as well as the early compressive strength at the age of 1 day after steaming (5.9 MPa), make it possible to effectively use these materials for enclosing building structures.

Identification and structure-based drug design of cell-active inhibitors of interleukin 17A at a novel C-terminal site

Anti-IL17A therapies have proven effective for numerous inflammatory diseases including psoriasis, axial spondylitis and psoriatic arthritis. Modulating and/or antagonizing protein–protein interactions of IL17A cytokine binding to its cell surface receptors with oral therapies offers the promise to bring forward biologics-like efficacy in a pill to patients. We used an NMR-based fragment screen of recombinant IL17A to uncover starting points for small molecule IL17A antagonist discovery. By examining chemical shift perturbations in 2D [1H, 13C-HSQC] spectra of isotopically labeled IL17A, we discovered fragments binding the cytokine at a previously undescribed site near the IL17A C-terminal region, albeit with weak affinity (> 250 µM). Importantly this binding location was distinct from previously known chemical matter modulating cytokine responses. Subsequently through analog screening, we identified related compounds that bound symmetrically in this novel site with two copies. From this observation we employed a linking strategy via structure-based drug design and obtained compounds with increased binding affinity (< 50 nM) and showed functional inhibition of IL17A-induced cellular signaling (IC50~1 µM). We also describe a fluorescence-based probe molecule suitable to discern/screen for additional molecules binding in this C-terminal site.

From fragment screening to fragment growing – new methods for drug discovery

From fragment screening to fragment growing – new methods for drug discovery

Implementation of a crystallographic fragment screening pipeline at the Swiss Light Source

Implementation of a crystallographic fragment screening pipeline at the Swiss Light Source

2-Amino-1,3,4-thiadiazoles as Glutaminyl Cyclases Inhibitors Increase Phagocytosis through Modification of CD47-SIRPα Checkpoint.

Glutaminyl cyclases (QC, isoQC) convert N-terminal glutamine or glutamate into pyroglutamate (pGlu) on substrates. IsoQC has recently been demonstrated to promote pGlu formation on the N-terminus of CD47, the SIRPα binding site, contributing to the "don't eat me" cancer immune signaling of CD47-SIRPα. We developed new QC inhibitors by applying a structure-based optimization approach starting from fragments identified through library screening. Screening of metal binding fragments identified 5-(1H-benzimidazol-5-yl)-1,3,4-thiadiazol-2-amine (9) as a potent fragment, and further modification provided 5-(1-(3-methoxy-4-(3-(piperidin-1-yl)propoxy)benzyl)-1H-benzo[d]imidazol-5-yl)-1,3,4-thiadiazol-2-amine (22b) as a potent QC inhibitor. Treatment with 22b in A549 and H1975 lung cancer cells decreased the CD47/αhCD47-CC2C6 interaction, indicative of the CD47/SIRPα interaction, and enhanced the increased phagocytic activity of both THP-1 and U937 macrophages.

Phenotypic screening of low molecular weight compounds is rich ground for repurposed, on-target drugs.

A target-based drug discovery strategy has led to a bias away from low molecular weight (MWT) drug discovery. Analysis of the ACS chemistry registration system shows that most low MWT drugs were first made in the time era before target-based drug discovery. Therapeutic activity among most low MWT drugs was identified in the era of phenotypic drug discovery when drugs were selected based on their phenotypic effects and before in vitro screening, mechanism of action considerations and experiences with fragment screening became known. The common perception that drugs cannot be found among low MWT compounds is incorrect based on both drug discovery history and our own experience with MLR-1023. The greater proportion of low MWT compounds that are commercially available compared to higher MWT compounds is a factor that should facilitate biology study. We posit that low MWT compounds are more suited to identification of new therapeutic activity using phenotypic screens provided that the phenotypic screening method has enough screening capacity. On-target and off-target therapeutic activities are discussed from both a chemistry and biology perspective because of a concern that either phenotypic or low MWT drug discovery might bias towards promiscuous compounds that combine on-target and off-target effects. Among ideal drug repositioning candidates (late-stage pre-clinical or clinically-experience compounds), pleiotropic activity (multiple therapeutic actions) is far more likely due to on-target effects arising where a single target mediates multiple therapeutic benefits, a desirable outcome for drug development purposes compared to the off-target alternative. Our exemplar of a low MWT compound, MLR-1023, discovered by phenotypic screening and subsequently found to have a single mechanism of action would have been overlooked based on current era medicinal chemistry precedent. The diverse therapeutic activities described for this compound by us, and others arise from the same pleiotropic lyn kinase activation molecular target. MLR-1023 serves as a proof-of-principle that potent, on target, low MWT drugs can be discovered by phenotypic screening.

Life sciences discovery and technology highlights

Life sciences discovery and technology highlights

Immunopotentiation mechanism of a mannose-modified peptide fragment containing a dominant antigenic epitope of Micropterus salmoides rhabdovirus (MSRV) glycoprotein after coupling to nanocarrier

Micropterus salmoides rhabavirus (MSRV) is an important pathogen that threatens the aquaculture of bass farming worldwide, and the efficacy of vaccines developed against this pathogen still needs to be improved. In order to further enhance the immune efficacy of existing vaccines, a mannose-modified nanovector vaccine SWCNTs-M-G2 was constructed by screening for peptide fragments encoding highly immunogenic antigenic epitopes (G2) and mannose modifications. Mannose receptor (MR) is abundantly expressed on the surface of Antigen-presenting cells (APCs) and plays a role in recognizing pathogens and presenting antigens. In this study, the effect of mannosylated modification on the immunopotentiation of highly immunogenic peptide fragments was investigated by using the characteristics of MR-specific recognition of mannose and APCs surface. In vitro macrophage assays showed that SWCNTs-M-G2 was efficiently phagocytosed and taken up by largemouth black bass head kidney macrophages, and effectively stimulated macrophage activation and upregulated the secretion of macrophage cytokines IL-1β, IL-8 and IL-12. In vivo, SWCNTs-M-G2 was also shown to be better taken up by immune organs such as spleen and head kidney, and induced increased expression of IL-1β, IL-8 and IL-12 in the immune organs of Micropterus salmoides, and promoted the expression of TCR and CD4 molecules in specific immune cells. Meanwhile, in vivo fluorescence imaging revealed that SWCNTs-M-G2 could be in the immune organ of fish for a longer period of time, contributing to the efficient uptake of antigens. Finally, the challenge test showed that SWCNTs-M-G2 could mediate the production of specific antibodies at a higher level than the unmodified carrier vaccine SWCNTs-G2 and showed a higher relative immune protection rate.

Novel fragment-derived colchicine-site binders as microtubule-destabilizing agents

Microtubules (MTs) are dynamic filaments of the cytoskeleton, which are formed by the polymerization of their building block tubulin. Perturbation of MT dynamics by MT-targeting agents (MTAs) leads to cell cycle arrest or cell death, a strategy that is pursued in chemotherapy. We recently performed a combined computational and crystallographic fragment screening approach and identified several tubulin-binding fragments. Here, we sought to capitalize on this study with the aim to demonstrate that low affinity tubulin-binding fragments can indeed be used as valuable starting points for the development of active, lead-like antitubulin small molecules. To this end, we report on a new, rationally designed series of 2-aminobenzimidazole derivatives that destabilize MTs by binding tubulin at the colchicine-binding site (CBS). We applied a fragment growing strategy by combining X-ray crystallography and computer-aided drug design. Preliminary structure-activity-relationship studies afforded compound 18 that inhibits HeLa cell viability with a submicromolar activity (IC50 of 0.9 μM). X-ray crystallography confirmed the compound pose in the CBS, while immunostaining experiments suggested a molecular mechanism of action alike classical CBS ligands with antimitotic and antitumor activity associated with MTs destabilization. This promising outcome underpins that our previously performed combined computational and crystallographic fragment screening approach provides promising starting points for developing new MTAs binding to the CBS of tubulin and, eventually, to further tubulin pockets.

Paleoproteomics.

Paleoproteomics, the study of ancient proteins, is a rapidly growing field at the intersection of molecular biology, paleontology, archaeology, paleoecology, and history. Paleoproteomics research leverages the longevity and diversity of proteins to explore fundamental questions about the past. While its origins predate the characterization of DNA, it was only with the advent of soft ionization mass spectrometry that the study of ancient proteins became truly feasible. Technological gains over the past 20 years have allowed increasing opportunities to better understand preservation, degradation, and recovery of the rich bioarchive of ancient proteins found in the archaeological and paleontological records. Growing from a handful of studies in the 1990s on individual highly abundant ancient proteins, paleoproteomics today is an expanding field with diverse applications ranging from the taxonomic identification of highly fragmented bones and shells and the phylogenetic resolution of extinct species to the exploration of past cuisines from dental calculus and pottery food crusts and the characterization of past diseases. More broadly, these studies have opened new doors in understanding past human–animal interactions, the reconstruction of past environments and environmental changes, the expansion of the hominin fossil record through large scale screening of nondiagnostic bone fragments, and the phylogenetic resolution of the vertebrate fossil record. Even with these advances, much of the ancient proteomic record still remains unexplored. Here we provide an overview of the history of the field, a summary of the major methods and applications currently in use, and a critical evaluation of current challenges. We conclude by looking to the future, for which innovative solutions and emerging technology will play an important role in enabling us to access the still unexplored “dark” proteome, allowing for a fuller understanding of the role ancient proteins can play in the interpretation of the past.

Innovative Approach for a Classic Target: Fragment Screening on Trypanothione Reductase Reveals New Opportunities for Drug Design.

Trypanothione reductase (TR) is a key factor in the redox homeostasis of trypanosomatid parasites, critical for survival in the hostile oxidative environment generated by the host to fight infection. TR is considered an attractive target for the development of new trypanocidal agents as it is essential for parasite survival but has no close homolog in humans. However, the high efficiency and turnover of TR challenging targets since only potent inhibitors, with nanomolar IC50, can significantly affect parasite redox state and viability. To aid the design of effective compounds targeting TR, we performed a fragment-based crystal screening at the Diamond Light Source XChem facility using a library optimized for follow-up synthesis steps. The experiment, allowing for testing over 300 compounds, resulted in the identification of 12 new ligands binding five different sites. Interestingly, the screening revealed the existence of an allosteric pocket close to the NADPH binding site, named the “doorstop pocket” since ligands binding at this site interfere with TR activity by hampering the “opening movement” needed to allow cofactor binding. The second remarkable site, known as the Z-site, identified by the screening, is located within the large trypanothione cavity but corresponds to a region not yet exploited for inhibition. The fragments binding to this site are close to each other and have some remarkable features making them ideal for follow-up optimization as a piperazine moiety in three out of five fragments.

Structural Analysis and Development of Notum Fragment Screening Hits.

The Wnt signaling suppressor Notum is a promising target for osteoporosis, Alzheimer’s disease, and colorectal cancers. To develop novel Notum inhibitors, we used an X-ray crystallographic fragment screen with the Diamond-SGC Poised Library (DSPL) and identified 59 fragment hits from the analysis of 768 data sets. Fifty-eight of the hits were found bound at the enzyme catalytic pocket with potencies ranging from 0.5 to >1000 μM. Analysis of the fragments’ diverse binding modes, enzymatic inhibitory activities, and chemical properties led to the selection of six hits for optimization, and five of these resulted in improved Notum inhibitory potencies. One hit, 1-phenyl-1,2,3-triazole 7, and its related cluster members, have shown promising lead-like properties. These became the focus of our fragment development activities, resulting in compound 7d with IC50 0.0067 μM. The large number of Notum fragment structures and their initial optimization provided an important basis for further Notum inhibitor development.

Abstract 2779: A live tumor fragment (LTF) platform with real-time imaging for immune response assays

Abstract Immuno-Oncology (IO) therapies provide remarkable clinical benefits. However, too few patients respond, and there are no diagnostic tools that predict IO response with high accuracy. Moreover, as more IO drugs and combinations are approved, selecting the best IO-based regimen for each patient will become more complex. To facilitate this selection, we are developing an ex vivo live tumor fragment (LTF) screening platform that retains representation of a patient’s tumor microenvironment (TME) including immune cells, enabling development of deep neural networks to predict IO response and thereby individualize therapy. Here we present preliminary data from our proof-of-concept platform that performs tissue fragmentation/sorting, liquid handling, drug treatment, high-resolution dynamic imaging, and multiplex immuno-assays. Human tumor excisions were obtained from the Univ. of Wisconsin (IRB approved), and CT26 tumors were grown in mice subcutaneously. Live tumors were cut into 300 x 300 µm fragments of 100 - 300 µm thickness, sorted into multi-well plates, and cultured for 48 or 72 h in the presence or absence of anti-PD1 (nivolumab or mouse equivalent), anti-PD1 plus anti-CTLA4 (ipilimumab or mouse equivalent), or concanavalin A (ConA) as a positive control. Cell viability was measured by ATP luminescence assay or flow cytometry. Motility of CD8+ cells was tracked in human LTF using camelid VHH anti-hCD8a-AF594 and 3D multiphoton microscopy for 30 min. Supernatant cytokines were measured using bead immunoassay and T cell markers by flow cytometry. T cells were retained within LTFs, and the proportion of lymphocytes in LTFs was independent of fragment thickness (1.6%/2.1% for 300 µm, 1.4%/2.3% for 200 µm and 1.4%/2.8% for 100 µm thickness, for CD4+ and CD8+ T cells, respectively). Total cell viability and T cell viability exceeded 80% at 48h, and 3D LTF structure remained intact for at least 48 h. We treated LTFs with anti-PD1, anti-PD1 plus anti-CTLA4, or ConA and confirmed the presence of IFN-γ and 10 (mouse) or 16 (human) other cytokines associated with immune activation in both the ConA and anti-PD1 treated samples, but not the control. In human LTFs, cytokine panel upregulation was observed for anti-PD1 vs. control (p=1.1e-5) and anti-PD1 plus anti-CTLA4 vs. anti-PD1 (p=3.7e-9). Using multiphoton microscopy and CD8-binding nanobodies, we observed vigorous CD8+ T cell motility in human LTFs, with a speed of 10 µm/min, which is comparable to that reported in vivo. Our LTF platform has an immuno-competent TME in which we can detect cellular and secreted immune response markers, compare alternative treatments, and track the surveillance activity of infiltrating T cells. Future work will further advance the platform, enabling clinical trials for training and validating deep neural networks to predict response to checkpoint inhibitors and other IO drugs. Citation Format: Tomasz Zal, Leung Kau Tang, Laura Hrycyniak, Anura Shrestha, Dinesh Joshi, Pichet Adstamongkonkul, Kelsey Tweed, Wesley A. Cox-Muranami, Sean Caenepeel, Hinco J. Gierman, Kevin Eliceiri, Kristina A. Matkowskyj, Sam J. Lubner, John Rafter, Mike Szulczewski, Maneesh Arora, Jonathan Oliner. A live tumor fragment (LTF) platform with real-time imaging for immune response assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2779.