Phagocytosis increases an oxidative metabolic and immune suppressive signature in tumor macrophages.

Phagocytosis is a key macrophage function, but how phagocytosis shapes tumor-associated macrophage (TAM) phenotypes and heterogeneity in solid tumors remains unclear. Here, we utilized both syngeneic and novel autochthonous lung tumor models in which neoplastic cells express the fluorophore tdTomato (tdTom) to identify TAMs that have phagocytosed neoplastic cells in vivo. Phagocytic tdTompos TAMs upregulated antigen presentation and anti-inflammatory proteins, but downregulated classic proinflammatory effectors compared to tdTomneg TAMs. Single-cell transcriptomic profiling identified TAM subset-specific and common gene expression changes associated with phagocytosis. We uncover a phagocytic signature that is predominated by oxidative phosphorylation (OXPHOS), ribosomal, and metabolic genes, and this signature correlates with worse clinical outcome in human lung cancer. Expression of OXPHOS proteins, mitochondrial content, and functional utilization of OXPHOS were increased in tdTompos TAMs. tdTompos tumor dendritic cells also display similar metabolic changes. Our identification of phagocytic TAMs as a distinct myeloid cell state links phagocytosis of neoplastic cells in vivo with OXPHOS and tumor-promoting phenotypes.

Multiplexed screens identify RAS paralogues HRAS and NRAS as suppressors of KRAS-driven lung cancer growth.

Oncogenic KRAS mutations occur in approximately 30% of lung adenocarcinoma. Despite several decades of effort, oncogenic KRAS-driven lung cancer remains difficult to treat, and our understanding of the regulators of RAS signalling is incomplete. Here to uncover the impact of diverse KRAS-interacting proteins on lung cancer growth, we combined multiplexed somatic CRISPR/Cas9-based genome editing in genetically engineered mouse models with tumour barcoding and high-throughput barcode sequencing. Through a series of CRISPR/Cas9 screens in autochthonous lung cancer models, we show that HRAS and NRAS are suppressors of KRASG12D-driven tumour growth in vivo and confirm these effects in oncogenic KRAS-driven human lung cancer cell lines. Mechanistically, RAS paralogues interact with oncogenic KRAS, suppress KRAS-KRAS interactions, and reduce downstream ERK signalling. Furthermore, HRAS and NRAS mutations identified in oncogenic KRAS-driven human tumours partially abolished this effect. By comparing the tumour-suppressive effects of HRAS and NRAS in oncogenic KRAS- and oncogenic BRAF-driven lung cancer models, we confirm that RAS paralogues are specific suppressors of KRAS-driven lung cancer in vivo. Our study outlines a technological avenue to uncover positive and negative regulators of oncogenic KRAS-driven cancer in a multiplexed manner in vivo and highlights the role RAS paralogue imbalance in oncogenic KRAS-driven lung cancer.

A multiplexed in vivo approach to identify driver genes in small cell lung cancer.

Small cell lung cancer (SCLC) is a lethal form of lung cancer. Here, we develop a quantitative multiplexed approach on the basis of lentiviral barcoding with somatic CRISPR-Cas9-mediated genome editing to functionally investigate candidate regulators of tumor initiation and growth in genetically engineered mouse models of SCLC. We found that naphthalene pre-treatment enhances lentiviral vector-mediated SCLC initiation, enabling high multiplicity of tumor clones for analysis through high-throughput sequencing methods. Candidate drivers of SCLC identified from a meta-analysis across multiple human SCLC genomic datasets were tested using this approach, which defines both positive and detrimental impacts of inactivating 40 genes across candidate pathways on SCLC development. This analysis and subsequent validation in human SCLC cells establish TSC1 in the PI3K-AKT-mTOR pathway as a robust tumor suppressor in SCLC. This approach should illuminate drivers of SCLC, facilitate the development of precision therapies for defined SCLC genotypes, and identify therapeutic targets.

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.

Impact of Methotrexate Discontinuation, Interruption, or Persistence in US Patients with Rheumatoid Arthritis Initiating Tofacitinib + Oral Methotrexate Combination.

PURPOSE: Using data from real-world practice, this analysis compared outcomes in patients with rheumatoid arthritis (RA) initiating treatment with an oral Janus kinase inhibitor, tofacitinib, in combination with persistent, discontinued, or interrupted treatment with oral methotrexate (MTX). METHODS: This retrospective claims analysis (MarketScan® databases) included data from US patients with RA and at least one prescription claim for tofacitinib, dated between January 1, 2013, and April 30, 2017. Eligible patients were continuously enrolled for ≥12 months before and after treatment initiation, and initiated tofacitinib in combination with oral MTX, with at least two prescription claims for each. Patients were grouped according to treatment pattern (MTX-Persistent, MTX-Discontinued, or MTX-Interrupted). Tofacitinib treatment persistence, adherence, and effectiveness, as well as all-cause and RA-related health care costs, were assessed. FINDINGS: A total of 671 patients were eligible for inclusion; 504 (75.1%) were MTX-Persistent; 131 (19.5%), MTX-Discontinued; and 36 (5.4%), MTX-Interrupted. Rates of tofacitinib treatment persistence, adherence, and effectiveness at 12 months were similar between the MTX-Persistent and MTX-Discontinued cohorts. The percentage of patients switched from tofacitinib to another advanced disease-modifying antirheumatic drug within 12 months of tofacitinib initiation was greater in the MTX-Persistent cohort compared with that in the MTX-Discontinued cohort. RA-related health care costs at 12 months post-initiation were significantly greater in the MTX-Persistent cohort compared with those in the MTX-Discontinued cohort. IMPLICATIONS: The findings from this analysis of real-world data indicate that patients who initiate tofacitinib in combination with oral MTX may discontinue MTX and still experience outcomes similar to those in patients who persist with MTX, with lesser RA-related health care costs. These results support those from a previous clinical study on methotrexate withdrawal in patients with RA (NCT02831855).

Combinatorial Inactivation of Tumor Suppressors Efficiently Initiates Lung Adenocarcinoma with Therapeutic Vulnerabilities.

Lung cancer is the leading cause of cancer death worldwide, with lung adenocarcinoma being the most common subtype. Many oncogenes and tumor suppressor genes are altered in this cancer type, and the discovery of oncogene mutations has led to the development of targeted therapies that have improved clinical outcomes. However, a large fraction of lung adenocarcinomas lacks mutations in known oncogenes, and the genesis and treatment of these oncogene-negative tumors remain enigmatic. Here, we perform iterative in vivo functional screens using quantitative autochthonous mouse model systems to uncover the genetic and biochemical changes that enable efficient lung tumor initiation in the absence of oncogene alterations. Generation of hundreds of diverse combinations of tumor suppressor alterations demonstrates that inactivation of suppressors of the RAS and PI3K pathways drives the development of oncogene-negative lung adenocarcinoma. Human genomic data and histology identified RAS/MAPK and PI3K pathway activation as a common feature of an event in oncogene-negative human lung adenocarcinomas. These Onc-negativeRAS/PI3K tumors and related cell lines are vulnerable to pharmacologic inhibition of these signaling axes. These results transform our understanding of this prevalent yet understudied subtype of lung adenocarcinoma. SIGNIFICANCE: To address the large fraction of lung adenocarcinomas lacking mutations in proto-oncogenes for which targeted therapies are unavailable, this work uncovers driver pathways of oncogene-negative lung adenocarcinomas and demonstrates their therapeutic vulnerabilities.

LKB1 drives stasis and C/EBP-mediated reprogramming to an alveolar type II fate in lung cancer.

LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Inactivation of Lkb1 accelerates the growth and progression of oncogenic KRAS-driven lung tumors in mouse models. However, the molecular mechanisms by which LKB1 constrains lung tumorigenesis and whether the cancer state that stems from Lkb1 deficiency can be reverted remains unknown. To identify the processes governed by LKB1 in vivo, we generated an allele which enables Lkb1 inactivation at tumor initiation and subsequent Lkb1 restoration in established tumors. Restoration of Lkb1 in oncogenic KRAS-driven lung tumors suppressed proliferation and led to tumor stasis. Lkb1 restoration activated targets of C/EBP transcription factors and drove neoplastic cells from a progenitor-like state to a less proliferative alveolar type II cell-like state. We show that C/EBP transcription factors govern a subset of genes that are induced by LKB1 and depend upon NKX2-1. We also demonstrate that a defining factor of the alveolar type II lineage, C/EBPα, constrains oncogenic KRAS-driven lung tumor growth in vivo. Thus, this key tumor suppressor regulates lineage-specific transcription factors, thereby constraining lung tumor development through enforced differentiation.

Discovery of ASTX029, A Clinical Candidate Which Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the mitogen-activated protein kinase pathway frequently drives tumor growth, and the ERK1/2 kinases are positioned at a key node in this pathway, making them important targets for therapeutic intervention. Recently, a number of ERK1/2 inhibitors have been advanced to investigational clinical trials in patients with activating mutations in B-Raf proto-oncogene or Ras. Here, we describe the discovery of the clinical candidate ASTX029 (15) through structure-guided optimization of our previously published isoindolinone lead (7). The medicinal chemistry campaign focused on addressing CYP3A4-mediated metabolism and maintaining favorable physicochemical properties. These efforts led to the identification of ASTX029, which showed the desired pharmacological profile combining ERK1/2 inhibition with suppression of phospho-ERK1/2 (pERK) levels, and in addition, it possesses suitable preclinical pharmacokinetic properties predictive of once daily dosing in humans. ASTX029 is currently in a phase I-II clinical trial in patients with advanced solid tumors.

ASTX029, a Novel Dual-mechanism ERK Inhibitor, Modulates Both the Phosphorylation and Catalytic Activity of ERK.

The MAPK signaling pathway is commonly upregulated in human cancers. As the primary downstream effector of the MAPK pathway, ERK is an attractive therapeutic target for the treatment of MAPK-activated cancers and for overcoming resistance to upstream inhibition. ASTX029 is a highly potent and selective dual-mechanism ERK inhibitor, discovered using fragment-based drug design. Because of its distinctive ERK-binding mode, ASTX029 inhibits both ERK catalytic activity and the phosphorylation of ERK itself by MEK, despite not directly inhibiting MEK activity. This dual mechanism was demonstrated in cell-free systems, as well as cell lines and xenograft tumor tissue, where the phosphorylation of both ERK and its substrate, ribosomal S6 kinase (RSK), were modulated on treatment with ASTX029. Markers of sensitivity were highlighted in a large cell panel, where ASTX029 preferentially inhibited the proliferation of MAPK-activated cell lines, including those with BRAF or RAS mutations. In vivo, significant antitumor activity was observed in MAPK-activated tumor xenograft models following oral treatment. ASTX029 also demonstrated activity in both in vitro and in vivo models of acquired resistance to MAPK pathway inhibitors. Overall, these findings highlight the therapeutic potential of a dual-mechanism ERK inhibitor such as ASTX029 for the treatment of MAPK-activated cancers, including those which have acquired resistance to inhibitors of upstream components of the MAPK pathway. ASTX029 is currently being evaluated in a first in human phase I-II clinical trial in patients with advanced solid tumors (NCT03520075).

The AMBRA1 E3 ligase adaptor regulates the stability of cyclin D.

The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication1. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer2,3. However, the mechanisms that regulate levels of cyclin D are incompletely understood4,5. Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of  the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.

A Functional Taxonomy of Tumor Suppression in Oncogenic KRAS-Driven Lung Cancer.

Cancer genotyping has identified a large number of putative tumor suppressor genes. Carcinogenesis is a multistep process, but the importance and specific roles of many of these genes during tumor initiation, growth, and progression remain unknown. Here we use a multiplexed mouse model of oncogenic KRAS-driven lung cancer to quantify the impact of 48 known and putative tumor suppressor genes on diverse aspects of carcinogenesis at an unprecedented scale and resolution. We uncover many previously understudied functional tumor suppressors that constrain cancer in vivo. Inactivation of some genes substantially increased growth, whereas the inactivation of others increases tumor initiation and/or the emergence of exceptionally large tumors. These functional in vivo analyses revealed an unexpectedly complex landscape of tumor suppression that has implications for understanding cancer evolution, interpreting clinical cancer genome sequencing data, and directing approaches to limit tumor initiation and progression. SIGNIFICANCE: Our high-throughput and high-resolution analysis of tumor suppression uncovered novel genetic determinants of oncogenic KRAS-driven lung cancer initiation, overall growth, and exceptional growth. This taxonomy is consistent with changing constraints during the life history of cancer and highlights the value of quantitative in vivo genetic analyses in autochthonous cancer models.This article is highlighted in the In This Issue feature, p. 1601.

A versatile system to record cell-cell interactions.

Cell-cell interactions influence all aspects of development, homeostasis, and disease. In cancer, interactions between cancer cells and stromal cells play a major role in nearly every step of carcinogenesis. Thus, the ability to record cell-cell interactions would facilitate mechanistic delineation of the role of the cancer microenvironment. Here, we describe GFP-based Touching Nexus (G-baToN) which relies upon nanobody-directed fluorescent protein transfer to enable sensitive and specific labeling of cells after cell-cell interactions. G-baToN is a generalizable system that enables physical contact-based labeling between various human and mouse cell types, including endothelial cell-pericyte, neuron-astrocyte, and diverse cancer-stromal cell pairs. A suite of orthogonal baToN tools enables reciprocal cell-cell labeling, interaction-dependent cargo transfer, and the identification of higher order cell-cell interactions across a wide range of cell types. The ability to track physically interacting cells with these simple and sensitive systems will greatly accelerate our understanding of the outputs of cell-cell interactions in cancer as well as across many biological processes.

It takes the coordinated effort of more than 40 trillion cells to build and maintain a human body. This intricate process relies on cells being able to communicate across long distances, but also with their immediate neighbors. Interactions between cells in close contact are key in both health and disease, yet tracing these connections efficiently and accurately remains challenging. The surface of a cell is studded with proteins that interact with the environment, including with the proteins on neighboring cells. Using genetic engineering, it is possible to construct surface proteins that carry a fluorescent tag called green fluorescent protein (or GFP), which could help to track physical interactions between cells. Here, Tang et al. test this idea by developing a new technology named GFP-based Touching Nexus, or G-baToN for short. Sender cells carry a GFP protein tethered to their surface, while receiver cells present a synthetic element that recognizes that GFP. When the cells touch, the sender passes its GFP to the receiver, and these labelled receiver cells become 'green'. Using this system, Tang et al. recorded physical contacts between a variety of human and mouse cells. Interactions involving more than two cells could also be detected by using different colors of fluorescent tags. Furthermore, Tang et al. showed that, alongside GFP, G-baToN could pass molecular cargo such as proteins, DNA, and other chemicals to receiver cells. This new system could help to study interactions among many different cell types. Changes in cell-to-cell contacts are a feature of diverse human diseases, including cancer. Tracking these interactions therefore could unravel new information about how cancer cells interact with their environment.

Associations between Patient Global Assessment scores and pain, physical function, and fatigue in rheumatoid arthritis: a post hoc analysis of data from phase 3 trials of tofacitinib.

BACKGROUND: Tofacitinib is an oral Janus kinase inhibitor for the treatment of rheumatoid arthritis (RA). We examined the degree to which Patient Global Assessment of Disease Activity (PtGA) was driven by patient-reported assessments of pain (Pain), physical function, and fatigue in patients receiving tofacitinib 5 mg twice daily or placebo, each with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs). METHODS: This post hoc analysis used data pooled from three randomized controlled trials in csDMARD-inadequate responder (csDMARD-IR) patients (ORAL Scan: NCT00847613; ORAL Standard: NCT00853385; ORAL Sync: NCT00856544). Using subgroup analysis from 2 × 2 tables, associations between PtGA and Pain, Health Assessment Questionnaire-Disability Index (HAQ-DI), and Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) at month 3 were evaluated using Pearson's Phi correlation coefficients. To support the main analysis, associations between select patient-reported outcomes (PROs) were also evaluated in csDMARD-naïve (ORAL Start; NCT01039688) and biologic (b)DMARD-IR (ORAL Step; NCT00960440) patients. RESULTS: Across csDMARD-IR treatment groups, low disease activity (defined as PtGA ≤ 20 mm), and moderate (≥ 30%) and substantial (≥ 50%) improvements from baseline in PtGA were associated with mild Pain (Visual Analog Scale score ≤ 20 mm), and moderate (≥ 30%) and substantial (≥ 50%) improvements from baseline in Pain; lack of Pain improvement was associated with little/no improvement in PtGA. In contrast, large proportions of csDMARD-IR patients who reported PtGA improvements did not report HAQ-DI or FACIT-F scores ≥ normative values (≤ 0.25 and ≥ 43.5, respectively) or changes in HAQ-DI or FACIT-F scores ≥ minimum clinically important difference (≥ 0.22 and ≥ 4.0, respectively). Generally, PtGA and Pain outcomes were moderately-to-strongly correlated at month 3 in csDMARD-IR patients, with weaker correlations evident between PtGA and HAQ-DI/FACIT-F outcomes. Similar findings were generally evident in csDMARD-naïve and bDMARD-IR patients. CONCLUSIONS: This analysis supports the role of Pain as a key driver of PtGA in RA; physical function and fatigue play lesser roles in patients' perceptions of disease activity. These findings corroborate the importance of improved PROs and attainment of low symptom states for optimizing patient care. TRIAL REGISTRATION: Clinicaltrials.gov: NCT00847613 (registered: February 19, 2009); NCT00853385 (registered: March 2, 2009); NCT00856544 (registered: March 5, 2009); NCT01039688 (registered: December 25, 2009); NCT00960440 (registered: August 17, 2009).

Fragment-based drug discovery: opportunities for organic synthesis.

This Review describes the increasing demand for organic synthesis to facilitate fragment-based drug discovery (FBDD), focusing on polar, unprotected fragments. In FBDD, X-ray crystal structures are used to design target molecules for synthesis with new groups added onto a fragment via specific growth vectors. This requires challenging synthesis which slows down drug discovery, and some fragments are not progressed into optimisation due to synthetic intractability. We have evaluated the output from Astex's fragment screenings for a number of programs, including urokinase-type plasminogen activator, hematopoietic prostaglandin D2 synthase, and hepatitis C virus NS3 protease-helicase, and identified fragments that were not elaborated due, in part, to a lack of commercially available analogues and/or suitable synthetic methodology. This represents an opportunity for the development of new synthetic research to enable rapid access to novel chemical space and fragment optimisation.

An LKB1-SIK Axis Suppresses Lung Tumor Growth and Controls Differentiation.

The kinase LKB1 is a critical tumor suppressor in sporadic and familial human cancers, yet the mechanisms by which it suppresses tumor growth remain poorly understood. To investigate the tumor-suppressive capacity of four canonical families of LKB1 substrates in vivo, we used CRISPR/Cas9-mediated combinatorial genome editing in a mouse model of oncogenic KRAS-driven lung adenocarcinoma. We demonstrate that members of the SIK family are critical for constraining tumor development. Histologic and gene-expression similarities between LKB1- and SIK-deficient tumors suggest that SIKs and LKB1 operate within the same axis. Furthermore, a gene-expression signature reflecting SIK deficiency is enriched in LKB1-mutant human lung adenocarcinomas and is regulated by LKB1 in human cancer cell lines. Together, these findings reveal a key LKB1-SIK tumor-suppressive axis and underscore the need to redirect efforts to elucidate the mechanisms through which LKB1 mediates tumor suppression. SIGNIFICANCE: Uncovering the effectors of frequently altered tumor suppressor genes is critical for understanding the fundamental driving forces of cancer growth. Our identification of the SIK family of kinases as effectors of LKB1-mediated tumor suppression will refocus future mechanistic studies and may lead to new avenues for genotype-specific therapeutic interventions.This article is highlighted in the In This Issue feature, p. 1469.

Crystallographic screening using ultra-low-molecular-weight ligands to guide drug design.

We present a novel crystallographic screening methodology (MiniFrags) that employs high-concentration aqueous soaks with a chemically diverse and ultra-low-molecular-weight library (heavy atom count 5-7) to identify ligand-binding hot and warm spots on proteins. We propose that MiniFrag screening represents a highly effective method for guiding optimisation of fragment-derived lead compounds or chemical tools and that the high screening hit rates reflect enhanced sampling of chemical space.

Publisher Correction: Towards quantitative and multiplexed in vivo functional cancer genomics.

The originally published article failed to acknowledge the equal first authorship contribution of I. P. Winters and C. W. Murray. The article has now been corrected online. The editors apologize for this error.

Towards quantitative and multiplexed in vivo functional cancer genomics.

Large-scale sequencing of human tumours has uncovered a vast array of genomic alterations. Genetically engineered mouse models recapitulate many features of human cancer and have been instrumental in assigning biological meaning to specific cancer-associated alterations. However, their time, cost and labour-intensive nature limits their broad utility; thus, the functional importance of the majority of genomic aberrations in cancer remains unknown. Recent advances have accelerated the functional interrogation of cancer-associated alterations within in vivo models. Specifically, the past few years have seen the emergence of CRISPR-Cas9-based strategies to rapidly generate increasingly complex somatic alterations and the development of multiplexed and quantitative approaches to ascertain gene function in vivo.

Fragment-Based Discovery of a Potent, Orally Bioavailable Inhibitor That Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors.

The Fragment Network: A Chemistry Recommendation Engine Built Using a Graph Database.

The hit validation stage of a fragment-based drug discovery campaign involves probing the SAR around one or more fragment hits. This often requires a search for similar compounds in a corporate collection or from commercial suppliers. The Fragment Network is a graph database that allows a user to efficiently search chemical space around a compound of interest. The result set is chemically intuitive, naturally grouped by substitution pattern and meaningfully sorted according to the number of observations of each transformation in medicinal chemistry databases. This paper describes the algorithms used to construct and search the Fragment Network and provides examples of how it may be used in a drug discovery context.