Feature Review Activity-Based Diagnostics: An Emerging .

TRMOME 1539 No. of Pages 19Trends in Molecular MedicineFeature ReviewActivity-Based Diagnostics: An EmergingParadigm for Disease Detection and MonitoringAva P. Soleimany1,2,3 and Sangeeta N. Bhatia1,2,4,5,6,7,8,*Diagnostics to accurately detect disease and monitor therapeutic response areessential for effective clinical management. Bioengineering, chemical biology,molecular biology, and computer science tools are converging to guide thedesign of diagnostics that leverage enzymatic activity to measure or producebiomarkers of disease. We review recent advances in the development ofthese 'activity-based diagnostics' (ABDx) and their application in infectiousand noncommunicable diseases. We highlight efforts towards both molecularprobes that respond to disease-specific catalytic activity to produce a diagnosticreadout, as well as diagnostics that use enzymes as an engineered componentof their sense-and-respond cascade. These technologies exemplify howintegrating techniques from multiple disciplines with preclinical validation hasenabled ABDx that may realize the goals of precision medicine.HighlightsTools from an array of disciplines, suchas bio-orthogonal chemistry, responsivenanomaterials, machine learning, synthetic gene circuits, and CRISPR/Cassystems, have enabled the developmentof engineered activity‐based diagnostics(ABDx).ABDx leverage enzymatic activity tomeasure or produce biomarkers of disease. Because of the substrate recognition and catalytic signal amplificationproperties of enzymes, ABDx afford thepotential for highly specific, sensitive,and programmable diagnostics.Noninvasive Diagnostics: Methods, Challenges, and PossibilitiesAccurate detection and diagnosis of disease are essential for effective clinical management andtreatment. Rapid diagnostics for highly infectious diseases such as Ebola can enable early casedetection and intervention, thereby informing impactful public health interventions [1]. Similarly,sensitive and specific diagnostics for noncommunicable diseases such as cancer offer the promise of improved therapeutic outcomes through early diagnosis of localized disease and accurateclassification of high-risk patients [2–4]. Parallelizing therapeutic development with diagnosticsthat can measure treatment response could facilitate personalized treatment regimens tailoredspecifically to the disease state of each patient. Effective diagnostics improve patient outcomesby providing actionable information on the presence, prognosis, or progress of disease.Traditional noninvasive diagnostic strategies rely on a combination of imaging tests and assaysfor endogenous biomarkers. Imaging tests, such as computed tomography for lung cancerand magnetic resonance imaging (MRI) for brain scanning, remain the clinical standard fornoninvasive diagnostics and enable detection and localization of disease. However, they oftensuffer from poor specificity [5,6] and require investment in costly infrastructure and specializedpersonnel to interpret the findings. Recent advances in molecular diagnostics have yielded promising assays for endogenous disease biomarkers, such as nucleic acids for viral infections [7],stool-based tests for colon cancer screening [8], and circulating tumor DNA (ctDNA) [9–12] forcancer, that can be used in conjunction with or as an alternative to imaging.An ideal molecular test for infectious diseases would be simple, rapid, inexpensive, and accurate;such a test would enable specific disease detection and isolation directly at the point of care andwould have tremendous implications for global health [13]. In oncology, ctDNA has emerged as apromising tool for noninvasive disease detection and evaluation of treatment response in patientswith advanced cancers whose tumors shed ample cell-free DNA (cfDNA) into the bloodstream[12]. In this context, ctDNA has enabled comprehensive reconstruction of patient-specific mutation profiles via whole-exome sequencing [14], and can be used for longitudinal monitoring ofTrends in Molecular Medicine, Month 2020, Vol. xx, No. xxABDx have shown promise in preclinicalsettings for the detection of bothnoncommunicable (e.g., cancer) and infectious diseases, and have been applied for molecular sensing in vivo andfor in vitro diagnostics run on ex vivobiospecimens.1Koch Institute for Integrative CancerResearch, Massachusetts Institute ofTechnology, Cambridge, MA, USA2Harvard–MIT Division of Health Sciencesand Technology, Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA3Harvard Graduate Program in Biophysics,Harvard University, Boston, MA, USA4Department of Electrical Engineering andComputer Science, Massachusetts Institute of Technology, Cambridge, MA, USA5Department of Medicine, Brigham andWomen’s Hospital, Harvard MedicalSchool, Boston, MA, USA6Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA7Wyss Institute at Harvard, Cambridge,MA, USA8Howard Hughes Medical Institute, Cambridge, MA, USA*Correspondence:sbhatia@mit.edu (S.N. 13 2020 Elsevier Ltd. 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Trends in Molecular Medicinetreatment efficacy or disease relapse because ctDNA levels are thought to scale with tumorburden [12]. However, this correlation with tumor burden presents fundamental sensitivity limitsfor early-stage, localized disease [12,15]. Multi-analyte blood tests that measure both ctDNAand protein biomarkers, such as CancerSEEK [10], have recently been developed as a meansto improve detection rates. The median sensitivity of CancerSEEK was 70% for the eight common cancers tested, but the median sensitivity for stage I cancers was only 43%. Althoughmulti-analyte tests can improve specificity and resolution, strategies that detect endogenous biomarkers in circulation face intrinsic sensitivity limitations for minimal residual or early-stage diseaseowing to low analyte concentration, high background signal, and biomarker clearance [12]. As acomplementary strategy, engineered diagnostics that are selectively activated in disease states togenerate amplified readouts may complement existing tests to help address these challengesand realize more accurate and accessible diagnostics.Convergent efforts from bioengineering, chemical biology, and molecular biology have inspired anew class of smart diagnostics that leverage enzymatic activity to measure or produce biomarkers of disease. These engineered activity-based diagnostics (ABDx; see Glossary) offerthe potential to overcome the limitations faced by current standard tests because they harnessthe specialized substrate recognition and signal amplification properties of enzymes to achievespecific and sensitive disease detection (Figure 1). This Review broadly organizes ABDx intotwo classes, and focuses on recent efforts towards their preclinical development. First, we discuss molecular and chemical probes that monitor dysregulated enzyme activity as a functionalbiomarker of disease. Second, we highlight molecular and biological tools that use enzymatic activity as a means of sensing, measuring, or reporting on disease state. Throughout, we highlightthe enabling technologies that have catalyzed the emergence of these diagnostics, the performance advantages afforded by ABDx, as well as strategies for enhancing ABDx specificity(Box 1) and supporting clinical translation through dialogue with regulatory agencies. Continuedefforts to engineer and validate ABDx will encourage their use as next-generation tests forprecision medicine.Enzyme Activity as a Functional Biomarker of DiseaseBiomarkers such as proteins or nucleic acids are biological indicators of disease progression ortherapeutic response [16]. Because enzymes such as proteases play crucial roles in severalbiological processes that contribute to disease progression, many ABDx measure enzyme activity as a functional biomarker of disease [17] (Figure 1A). Furthermore, by leveraging the catalyticnature of enzymes for signal amplification, activity measurements may offer sensitivity advantagesrelative to endogenous blood biomarkers. Advances in chemistry and nanotechnology haveenabled new molecular probes that are selectively activated by disease-associated enzymaticactivity to generate a measurable diagnostic readout (Figure 1A).Substrate and Activity-Based Probes for Molecular Imaging In VivoSeveral ABDx function by converting the activity of enzymes involved in disease progression intoan imaging readout (Figure 2) [17,18]. For cancer in particular, accurate in vivo visualization ofmalignant tissues can aid in early diagnosis, surgical planning and resection, and monitoring oftreatment response. Because proteases play direct functional roles in all cancer hallmarks [17],imaging the proteases involved in cancer progression has emerged as a promising detectionstrategy.Advances in chemistry and nanotechnology have spawned both substrate cleavage- and bindingbased probes for molecular imaging of enzymes involved in cancer (Figure 2). Substrate-basedprobes that fluoresce in the near-IR upon proteolytic cleavage have been extensively used for2Trends in Molecular Medicine, Month 2020, Vol. xx, No. xxGlossaryActivity-based diagnostics (ABDx):engineered diagnostics that leverageenzymatic activity to measure orproduce biomarkers of disease.Activity-based probes (ABPs):chemical probes that interact withcatalytically active enzymatic targets andremain covalently bound to the activesite.Activity-based nanosensors (ABNs):enzyme-responsive nanoparticles thatrespond to dysregulated proteaseactivity in vivo to generate urinaryreporters of disease. ABNs havedemonstrated utility in preclinical modelsof several disease conditions includingcancer, bacterial infection, andthrombosis.Area under the curve (AUC): aperformance metric for a binary classifierthat is established by constructingreceiver operating characteristic (ROC)curves and quantifying the area underthe curve (AUC). AUC is used as a metricto characterize the predictive power ofthe candidate diagnostic in the testedcondition, where a baseline AUC of 0.5is equivalent to the predictive power of arandom binary test.Boolean logic operation: Booleanlogic describes an algebra governed bythe truth values 'True' (1) and 'False' (0).Boolean logic is dictated by the Booleanoperators 'AND', 'OR', and 'NOT'.Cell-penetrating peptide: shortpeptides, usually positively charged, thatfacilitate the cellular uptake of associatedcargo. The cargo can be covalentlylinked to the cell-penetrating peptide orassociated with it through non-covalentinteractions.Chemical warhead: a reactivechemical group that can covalently bindto a target site on a biological target. Inthe case of activity-based probes, thiswarhead is usually an electrophile thatcan covalently link to a nucleophilicresidue in the enzyme active site.CRISPR-associated (Cas) effector:an enzyme that uses CRISPR guides torecognize and cleave nucleic acids.DNA recombinase: an enzyme thatcatalyzes site-specific rearrangement(excision, inversion, insertion, ortranslocation) of DNA.Multi-compartment pharmacokineticmodel: a mathematical model thatdescribes the absorption, distribution,metabolism, and excretion of anadministered molecule or particle interms of transmission between