A data science roadmap for open science organizations engaged in early-stage drug discovery.

The Structural Genomics Consortium is an international open science research organization with a focus on accelerating early-stage drug discovery, namely hit discovery and optimization. We, as many others, believe that artificial intelligence (AI) is poised to be a main accelerator in the field. The question is then how to best benefit from recent advances in AI and how to generate, format and disseminate data to enable future breakthroughs in AI-guided drug discovery. We present here the recommendations of a working group composed of experts from both the public and private sectors. Robust data management requires precise ontologies and standardized vocabulary while a centralized database architecture across laboratories facilitates data integration into high-value datasets. Lab automation and opening electronic lab notebooks to data mining push the boundaries of data sharing and data modeling. Important considerations for building robust machine-learning models include transparent and reproducible data processing, choosing the most relevant data representation, defining the right training and test sets, and estimating prediction uncertainty. Beyond data-sharing, cloud-based computing can be harnessed to build and disseminate machine-learning models. Important vectors of acceleration for hit and chemical probe discovery will be (1) the real-time integration of experimental data generation and modeling workflows within design-make-test-analyze (DMTA) cycles openly, and at scale and (2) the adoption of a mindset where data scientists and experimentalists work as a unified team, and where data science is incorporated into the experimental design.

A chemical probe to modulate human GID4 Pro/N-degron interactions.

The C-terminal to LisH (CTLH) complex is a ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via its substrate receptor Glucose-Induced Degradation 4 (GID4), but its function and substrates in humans remain unclear. Here, we report PFI-7, a potent, selective and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation and quantitative proteomics enabled the identification of GID4 interactors and GID4-regulated proteins. GID4 interactors are enriched for nucleolar proteins, including the Pro/N-degron-containing RNA helicases DDX21 and DDX50. We also identified a distinct subset of proteins whose cellular levels are regulated by GID4 including HMGCS1, a Pro/N-degron-containing metabolic enzyme. These data reveal human GID4 Pro/N-degron targets regulated through a combination of degradative and nondegradative functions. Going forward, PFI-7 will be a valuable research tool for investigating CTLH complex biology and facilitating development of targeted protein degradation strategies that highjack CTLH E3 ligase activity.

A Second-Generation Oral SARS-CoV-2 Main Protease Inhibitor Clinical Candidate for the Treatment of COVID-19.

Despite the record-breaking discovery, development and approval of vaccines and antiviral therapeutics such as Paxlovid, coronavirus disease 2019 (COVID-19) remained the fourth leading cause of death in the world and third highest in the United States in 2022. Here, we report the discovery and characterization of PF-07817883, a second-generation, orally bioavailable, SARS-CoV-2 main protease inhibitor with improved metabolic stability versus nirmatrelvir, the antiviral component of the ritonavir-boosted therapy Paxlovid. We demonstrate the in vitro pan-human coronavirus antiviral activity and off-target selectivity profile of PF-07817883. PF-07817883 also demonstrated oral efficacy in a mouse-adapted SARS-CoV-2 model at plasma concentrations equivalent to nirmatrelvir. The preclinical in vivo pharmacokinetics and metabolism studies in human matrices are suggestive of improved oral pharmacokinetics for PF-07817883 in humans, relative to nirmatrelvir. In vitro inhibition/induction studies against major human drug metabolizing enzymes/transporters suggest a low potential for perpetrator drug-drug interactions upon single-agent use of PF-07817883.

Chemical tools for the Gid4 subunit of the human E3 ligase C-terminal to LisH (CTLH) degradation complex.

We have developed a novel chemical handle (PFI-E3H1) and a chemical probe (PFI-7) as ligands for the Gid4 subunit of the human E3 ligase CTLH degradation complex. Through an efficient initial hit-ID campaign, structure-based drug design (SBDD) and leveraging the sizeable Pfizer compound library, we identified a 500 nM ligand for this E3 ligase through file screening alone. Further exploration identified a vector that is tolerant to addition of a linker for future chimeric molecule design. The chemotype was subsequently optimized to sub-100 nM Gid4 binding affinity for a chemical probe. These novel tools, alongside the suitable negative control also identified, should enable the interrogation of this complex human E3 ligase macromolecular assembly.

Discovery of PFI-6, a small-molecule chemical probe for the YEATS domain of MLLT1 and MLLT3.

Epigenetic proteins containing YEATS domains (YD) are an emerging target class in drug discovery. Described herein are the discovery and characterization efforts associated with PFI-6, a new chemical probe for the YD of MLLT1 (ENL/YEATS1) and MLLT3 (AF9/YEATS3). For hit identification, fragment-like mimetics of endogenous YD ligands (crotonylated histone-containing proteins), were synthesized via parallel medicinal chemistry (PMC) and screened for MLLT1 binding. Subsequent SAR studies led to iterative MLLT1/3 binding and selectivity improvements, culminating in the discovery of PFI-6. PFI-6 demonstrates good affinity and selectivity for MLLT1/3 vs. other human YD proteins (YEATS2/4) and engages MLLT3 in cells. Small-molecule X-ray co-crystal structures of two molecules, including PFI-6, bound to the YD of MLLT1/3 are also described. PFI-6 may be a useful tool molecule to better understand the biological effects associated with modulation of MLLT1/3.

Target 2035 - an update on private sector contributions.

Target 2035, an international federation of biomedical scientists from the public and private sectors, is leveraging 'open' principles to develop a pharmacological tool for every human protein. These tools are important reagents for scientists studying human health and disease and will facilitate the development of new medicines. It is therefore not surprising that pharmaceutical companies are joining Target 2035, contributing both knowledge and reagents to study novel proteins. Here, we present a brief progress update on Target 2035 and highlight some of industry's contributions.

Discovery of High-Affinity Small-Molecule Binders of the Epigenetic Reader YEATS4.

A series of small-molecule YEATS4 binders have been discovered as part of an ongoing research effort to generate high-quality probe molecules for emerging and/or challenging epigenetic targets. Analogues such as 4d and 4e demonstrate excellent potency and selectivity for YEATS4 binding versus YEATS1,2,3 and exhibit good physical properties and in vitro safety profiles. A new X-ray crystal structure confirms direct binding of this chemical series to YEATS4 at the lysine acetylation recognition site of the YEATS domain. Multiple analogues engage YEATS4 with nanomolar potency in a whole-cell nanoluciferase bioluminescent resonance energy transfer assay. Rodent pharmacokinetic studies demonstrate the competency of several analogues as in vivo-capable binders.

Target 2035 - update on the quest for a probe for every protein.

Twenty years after the publication of the first draft of the human genome, our knowledge of the human proteome is still fragmented. The challenge of translating the wealth of new knowledge from genomics into new medicines is that proteins, and not genes, are the primary executers of biological function. Therefore, much of how biology works in health and disease must be understood through the lens of protein function. Accordingly, a subset of human proteins has been at the heart of research interests of scientists over the centuries, and we have accumulated varying degrees of knowledge about approximately 65% of the human proteome. Nevertheless, a large proportion of proteins in the human proteome (∼35%) remains uncharacterized, and less than 5% of the human proteome has been successfully targeted for drug discovery. This highlights the profound disconnect between our abilities to obtain genetic information and subsequent development of effective medicines. Target 2035 is an international federation of biomedical scientists from the public and private sectors, which aims to address this gap by developing and applying new technologies to create by year 2035 chemogenomic libraries, chemical probes, and/or biological probes for the entire human proteome.

An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.

Structure and Inhibitor Binding Characterization of Oncogenic MLLT1 Mutants.

Dysfunction of YEATS-domain-containing MLLT1, an acetyl/acyl-lysine dependent epigenetic reader domain, has been implicated in the development of aggressive cancers. Mutations in the YEATS domain have been recently reported as a cause of MLLT1 aberrant reader function. However, the structural basis for the reported alterations in affinity for acetylated/acylated histone has remained elusive. Here, we report the crystal structures of both insertion and substitution mutants present in cancer, revealing significant conformational changes of the YEATS-domain loop 8. Structural comparison demonstrates that not only did such alteration alter the binding interface for acetylated/acylated histones, but the sequence alterations in the loop in T1 mutant may enable dimeric assembly consistent with inducing self-association behavior. Nevertheless, we show that also the MLLT1 mutants can be targeted by developed acetyllysine mimetic inhibitors with affinities similarly to wild-type. Our report provides a structural basis for the altered behaviors and a potential strategy for targeting oncogenic MLLT1 mutants.

Investigating 3,3-diaryloxetanes as potential bioisosteres through matched molecular pair analysis.

Oxetanes have received increasing interest in medicinal chemistry as attractive polar and low molecular weight motifs. The application of oxetanes as replacements for methylene, methyl, gem-dimethyl and carbonyl groups has been demonstrated to often improve chemical properties of target molecules for drug discovery purposes. The investigation of the properties of 3,3-diaryloxetanes, particularly of interest as a benzophenone replacement, remains largely unexplored. With recent synthetic advances in accessing this motif we studied the effects of 3,3-diaryloxetanes on the physicochemical properties of 'drug-like' molecules. Here, we describe our efforts in the design and synthesis of a range of drug-like compounds for matched molecular pair analysis to investigate the viability of the 3,3-diaryloxetane motif as a replacement group in drug discovery. We conclude that the properties of the diaryloxetanes and ketones are similar, and generally superior to related alkyl linkers, and that diaryloxetanes provide a potentially useful new design element.

Discovery of Tyrosine Kinase 2 (TYK2) Inhibitor (PF-06826647) for the Treatment of Autoimmune Diseases.

Tyrosine kinase 2 (TYK2) is a member of the JAK kinase family that regulates signal transduction downstream of receptors for the IL-23/IL-12 pathways and type I interferon family, where it pairs with JAK2 or JAK1, respectively. On the basis of human genetic and emerging clinical data, a selective TYK2 inhibitor provides an opportunity to treat autoimmune diseases delivering a potentially differentiated clinical profile compared to currently approved JAK inhibitors. The discovery of an ATP-competitive pyrazolopyrazinyl series of TYK2 inhibitors was accomplished through computational and structurally enabled design starting from a known kinase hinge binding motif. With understanding of PK/PD relationships, a target profile balancing TYK2 potency and selectivity over off-target JAK2 was established. Lead optimization involved modulating potency, selectivity, and ADME properties which led to the identification of the clinical candidate PF-06826647 (22).

Selective, Small-Molecule Co-Factor Binding Site Inhibition of a Su(var)3-9, Enhancer of Zeste, Trithorax Domain Containing Lysine Methyltransferase.

The first chemical probe to primarily occupy the co-factor binding site of a Su(var)3-9, enhancer of a zeste, trithorax (SET) domain containing protein lysine methyltransferase (PKMT) is reported. Protein methyltransferases require S-adenosylmethionine (SAM) as a co-factor (methyl donor) for enzymatic activity. However, SAM itself represents a poor medicinal chemistry starting point for a selective, cell-active inhibitor given its extreme physicochemical properties and its role in multiple cellular processes. A previously untested medicinal chemistry strategy of deliberate file enrichment around molecules bearing the hallmarks of SAM, but with improved lead-like properties from the outset, yielded viable hits against SET and MYND domain-containing protein 2 (SMYD2) that were shown to bind in the co-factor site. These leads were optimized to identify a highly biochemically potent, PKMT-selective, and cell-active chemical probe. While substrate-based inhibitors of PKMTs are known, this represents a novel, co-factor-derived strategy for the inhibition of SMYD2 which may also prove applicable to lysine methyltransferase family members previously thought of as intractable.

Donated chemical probes for open science.

Potent, selective and broadly characterized small molecule modulators of protein function (chemical probes) are powerful research reagents. The pharmaceutical industry has generated many high-quality chemical probes and several of these have been made available to academia. However, probe-associated data and control compounds, such as inactive structurally related molecules and their associated data, are generally not accessible. The lack of data and guidance makes it difficult for researchers to decide which chemical tools to choose. Several pharmaceutical companies (AbbVie, Bayer, Boehringer Ingelheim, Janssen, MSD, Pfizer, and Takeda) have therefore entered into a pre-competitive collaboration to make available a large number of innovative high-quality probes, including all probe-associated data, control compounds and recommendations on use (https://openscienceprobes.sgc-frankfurt.de/). Here we describe the chemical tools and target-related knowledge that have been made available, and encourage others to join the project.

Selective Targeting of Bromodomains of the Bromodomain-PHD Fingers Family Impairs Osteoclast Differentiation.

Histone acetyltransferases of the MYST family are recruited to chromatin by BRPF scaffolding proteins. We explored functional consequences and the therapeutic potential of inhibitors targeting acetyl-lysine dependent protein interaction domains (bromodomains) present in BRPF1-3 in bone maintenance. We report three potent and selective inhibitors: one (PFI-4) with high selectivity for the BRPF1B isoform and two pan-BRPF bromodomain inhibitors (OF-1, NI-57). The developed inhibitors displaced BRPF bromodomains from chromatin and did not inhibit cell growth and proliferation. Intriguingly, the inhibitors impaired RANKL-induced differentiation of primary murine bone marrow cells and human primary monocytes into bone resorbing osteoclasts by specifically repressing transcriptional programs required for osteoclastogenesis. The data suggest a key role of BRPF in regulating gene expression during osteoclastogenesis, and the excellent druggability of these bromodomains may lead to new treatment strategies for patients suffering from bone loss or osteolytic malignant bone lesions.

Design of a Biased Potent Small Molecule Inhibitor of the Bromodomain and PHD Finger-Containing (BRPF) Proteins Suitable for Cellular and in Vivo Studies.

The BRPF (bromodomain and PHD finger-containing) family are scaffolding proteins important for the recruitment of histone acetyltransferases of the MYST family to chromatin. Evaluation of the BRPF family as a potential drug target is at an early stage although there is an emerging understanding of a role in acute myeloid leukemia (AML). We report the optimization of fragment hit 5b to 13-d as a biased, potent inhibitor of the BRD of the BRPFs with excellent selectivity over nonclass IV BRD proteins. Evaluation of 13-d in a panel of cancer cell lines showed a selective inhibition of proliferation of a subset of AML lines. Pharmacokinetic studies established that 13-d had properties compatible with oral dosing in mouse models of disease (Fpo 49%). We propose that NI-42 (13-d) is a new chemical probe for the BRPFs suitable for cellular and in vivo studies to explore the fundamental biology of these proteins.

Identification of a Chemical Probe for Family VIII Bromodomains through Optimization of a Fragment Hit.

The acetyl post-translational modification of chromatin at selected histone lysine residues is interpreted by an acetyl-lysine specific interaction with bromodomain reader modules. Here we report the discovery of the potent, acetyl-lysine-competitive, and cell active inhibitor PFI-3 that binds to certain family VIII bromodomains while displaying significant, broader bromodomain family selectivity. The high specificity of PFI-3 for family VIII was achieved through a novel bromodomain binding mode of a phenolic headgroup that led to the unusual displacement of water molecules that are generally retained by most other bromodomain inhibitors reported to date. The medicinal chemistry program that led to PFI-3 from an initial fragment screening hit is described in detail, and additional analogues with differing family VIII bromodomain selectivity profiles are also reported. We also describe the full pharmacological characterization of PFI-3 as a chemical probe, along with phenotypic data on adipocyte and myoblast cell differentiation assays.

SETD7 Controls Intestinal Regeneration and Tumorigenesis by Regulating Wnt/ß-Catenin and Hippo/YAP Signaling.

Intestinal tumorigenesis is a result of mutations in signaling pathways that control cellular proliferation, differentiation, and survival. Mutations in the Wnt/ß-catenin pathway are associated with the majority of intestinal cancers, while dysregulation of the Hippo/Yes-Associated Protein (YAP) pathway is an emerging regulator of intestinal tumorigenesis. In addition, these closely related pathways play a central role during intestinal regeneration. We have previously shown that methylation of the Hippo transducer YAP by the lysine methyltransferase SETD7 controls its subcellular localization and function. We now show that SETD7 is required for Wnt-driven intestinal tumorigenesis and regeneration. Mechanistically, SETD7 is part of a complex containing YAP, AXIN1, and ß-catenin, and SETD7-dependent methylation of YAP facilitates Wnt-induced nuclear accumulation of ß-catenin. Collectively, these results define a methyltransferase-dependent regulatory mechanism that links the Wnt/ß-catenin and Hippo/YAP pathways during intestinal regeneration and tumorigenesis.

Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance.

Mammalian SWI/SNF [also called Brg/Brahma-associated factors (BAFs)] are evolutionarily conserved chromatin-remodeling complexes regulating gene transcription programs during development and stem cell differentiation. BAF complexes contain an ATP (adenosine 5'-triphosphate)-driven remodeling enzyme (either BRG1 or BRM) and multiple protein interaction domains including bromodomains, an evolutionary conserved acetyl lysine-dependent protein interaction motif that recruits transcriptional regulators to acetylated chromatin. We report a potent and cell active protein interaction inhibitor, PFI-3, that selectively binds to essential BAF bromodomains. The high specificity of PFI-3 was achieved on the basis of a novel binding mode of a salicylic acid head group that led to the replacement of water molecules typically maintained in other bromodomain inhibitor complexes. We show that exposure of embryonic stem cells to PFI-3 led to deprivation of stemness and deregulated lineage specification. Furthermore, differentiation of trophoblast stem cells in the presence of PFI-3 was markedly enhanced. The data present a key function of BAF bromodomains in stem cell maintenance and differentiation, introducing a novel versatile chemical probe for studies on acetylation-dependent cellular processes controlled by BAF remodeling complexes.

Lysine Methyltransferase SETD7 (SET7/9) Regulates ROS Signaling through mitochondria and NFE2L2/ARE pathway.

Reactive oxygen species (ROS) homeostasis requires stringent regulation. ROS imbalance, especially ROS accumulation, has profound implications in various disease pathogenesis. Lysine methylation of histone and non-histone proteins has been implicated in various cellular responses. The main objective of this study is to investigate the role of SET domain containing lysine methyltransferase SETD7 (SET7/9) in the regulation of ROS-mediated signaling. Here we report that inhibition of SETD7 with siRNA or a SETD7 small molecule inhibitor in both macrophages and a human bronchial epithelial cell line (Beas-2B) were able to counter NF-ĸB-induced oxidative stress and pro-inflammatory cytokine production. Meanwhile, inhibition of SETD7 elevates mitochondria antioxidant functions via negative regulation of PPARGC1A and NFE2L2. Using a co-expression system and purified proteins, we detected direct interaction between SETD7 and NFE2L2. These results indicate that lysine methylation by SETD7 is important for the fine-tuning of ROS signaling through its regulation on pro-inflammatory responses, mitochondrial function and the NFE2L2/ARE pathway. Up-regulation of multiple antioxidant genes and improved ROS clearance by inhibition of SETD7 suggests the potential benefit of targeting SETD7 in treating ROS-associated diseases.