Activity-Based Diagnostics: An Emerging Paradigm for Disease Detection and Monitoring

Abstract

Diagnostics to accurately detect disease and monitor therapeutic response are essential for effective clinical management. Bioengineering, chemical biology, molecular biology, and computer science tools are converging to guide the design of diagnostics that leverage enzymatic activity to measure or produce biomarkers of disease. We review recent advances in the development of these 'activity-based diagnostics' (ABDx) and their application in infectious and noncommunicable diseases. We highlight efforts towards both molecular probes that respond to disease-specific catalytic activity to produce a diagnostic readout, as well as diagnostics that use enzymes as an engineered component of their sense-and-respond cascade. These technologies exemplify how integrating techniques from multiple disciplines with preclinical validation has enabled ABDx that may realize the goals of precision medicine.

Keywords: CRISPR/Cas; activity-based probes; diagnostics; enzymes; protease activity; synthetic biology; synthetic biomarkers.

Figure 1.. Principles of activity-based diagnostics (ABDx).

ABDx leverage enzymatic activity to measure or produce biomarkers of disease. (A) Molecular and chemical probes can be used to measure dysregulated enzyme (orange pacman) activity, either in vivo or ex vivo, as a functional biomarker of disease. Prior to catalysis, probes remain off (grey star). Enzyme-specific probe activation generates a measurable output as a diagnostic readout (green star). (B) Biological sensors, such as engineered bacteria or mammalian cells, carry enzyme-driven genetic circuits that enable them to sense and report on disease state. Synthetic reporter enzymes produced following sensor activation can generate amplified diagnostic readouts as output. (C) Guided, programmable nucleases, such as CRISPR-associated (Cas) effector enzymes, can be exploited for sequence-specific nucleic acid detection. Select nucleases can cleave synthetic reporter probes upon target binding to produce a signal-amplified readout of specific nucleic acid detection.

Figure 2.. Activatable probes for molecular imaging of disease-associated enzymes.

(A) Approaches that convert enzyme activity to an imaging readout include probes that assess substrate catalysis (top) or bind to the enzyme active site (bottom). Probes carry a dye or contrast agent and remain “OFF” prior to enzyme activation (grey star). Substrate-based probes carry an enzyme substrate and recognition motif (blue ellipse) that may be processed by active enzymes, while binding-based probes carry a chemical warhead (blue triangle) that can covalently bind to active enzymes. These probes turn “ON” to produce an imaging readout (green star) upon processing by enzymes such as proteases. (B) These probes can be used in vivo for diagnostic or intraoperative imaging (top), or ex vivo for visualization of tumor margins or for pathogen detection in clinical specimens (bottom).

Figure 3.. Activity-based nanosensors (ABNs).

(A) Protease-cleavable peptide substrates appended with a reporter molecule, such as a mass-encoded barcode, are conjugated to a nanocarrier, such as a (poly)ethylene-glycol (PEG) scaffold. (B) The protease-sensitive ABNs are administered intravenously, subcutaneously, or intratracheally, and designed to specifically disassemble upon engagement with dysregulated proteases at the site of disease. After protease cleavage, liberated reporters, whose size is below the renal filtration limit of ~5 nm, are filtered into the urine. (C) Reporters can be detected in the cleared urine via a corresponding analytical method, such as liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). Disease state is detected noninvasively based on urinary reporter signatures.

Figure 4.. Enzyme-driven biological sensors.

(A) Bacteria modified with sense-and-respond systems use disease-specific biomarkers (red hexagon) to sense a disease state and, in turn, produce a diagnostic readout. Synthetic reporter enzymes such as β-lactamase (orange pacman) can be expressed as a way to generate an activity-based readout of disease state. The engineered probiotic diagnostics can then be administered in vivo, for example orally for diagnostic applications in the gastrointestinal tract. (B) Quorum signaling through cholera autoinducer 1 (CAI-1, red hexagon) in Vibrio cholerae has been used to induce expression of the reporter enzyme β-lactamase in Lactococcus lactis engineered as a cholera diagnostic [100]. The engineered V. cholerae-sensing circuit consists of a hybrid two-component system, comprised of the sensory histidine kinase CqsS linked to the NisR signal transduction domain, and a TetR/P_tet reporter module. Upon CAI-1 binding to CqsS (i), phosphorylation through the two-component system is halted, preventing TetR expression and Ptet repression (ii). Without TetR, the β-lactamase reporter is freely expressed and secreted (iii). (C) An activity assay for the reporter enzyme can be used to produce a diagnostic readout, for example directly in collected fecal samples [100].

Figure 5.. Guided, programmable nucleases for ABDx.

(A) Nucleic acid detection with CRISPR-Cas augmented toehold sensing. The presence of a specific protospacer adjacent motif (PAM; orange) in a DNA input results in cleavage by Cas9 with a PAM-specific guide RNA. Following transcription, this differential cleavage leads to either truncated or full-length trigger RNAs, which can be designed to differentially activate an engineered toehold sensor. Since only full-length triggers can activate the toehold sensor, a downstream signal can be used to discriminate the two inputs [111]. (B) SHERLOCK diagnostic test. Nucleic acids, either double-stranded DNA (dsDNA) or RNA, are amplified or reverse-transcribed (RT) and amplified, for DNA or RNA respectively. Following transcription, amplified target RNAs and fluorescently quenched cleavage reporters (grey star = fluorophore, blue circle = quencher) are incubated with Cas13a-crRNA complex. Detection and binding of the RNA target unleashes Cas13a’s collateral activity, resulting in reporter cleavage, fluorophore activation (green star), and signal amplification [114]. (C) DETECTR diagnostic test. Target dsDNA are amplified and, along with fluorescently quenched cleavage reporters, incubated with Cas12a-crRNA complex. Detection and binding of the dsDNA target unleashes Cas12a’s collateral activity, resulting in reporter cleavage, fluorophore activation, and signal amplification [115]. (D) CRISPR diagnostics achieve high specificity driven by crRNA guide-target base pairing (left), detection sensitivity from the highly efficient collateral cleavage of reporter molecules (middle), and diagnostic programmability due to facile guide RNA design for nucleic acid targets of interest (right).