key: cord-0855365-3zsqw5xv authors: O’Donnell, Henry R.; Tummino, Tia A.; Bardine, Conner; Craik, Charles S.; Shoichet, Brian K. title: Colloidal aggregators in biochemical SARS-CoV-2 repurposing screens date: 2021-08-31 journal: bioRxiv DOI: 10.1101/2021.08.31.458413 sha: 2c2eb09fdd73aea1fe77da84b84982f13684f629 doc_id: 855365 cord_uid: 3zsqw5xv To fight the SARS-CoV-2 pandemic, much effort has been directed toward drug repurposing, testing investigational and approved drugs against several viral or human proteins in vitro. Here we investigate the impact of colloidal aggregation, a common artifact in early drug discovery, in these repurposing screens. We selected 56 drugs reported to be active in biochemical assays and tested them for aggregation by both dynamic light scattering and by enzyme counter screening with and without detergent; seventeen of these drugs formed colloids at concentrations similar to their literature reported IC50s. To investigate the occurrence of colloidal aggregators more generally in repurposing libraries, we further selected 15 drugs that had physical properties resembling known aggregators from a common repurposing library, and found that 6 of these aggregated at micromolar concentrations. An attraction of repurposing is that drugs active on one target are considered de-risked on another. This study suggests not only that many of the drugs repurposed for SARS-CoV-2 in biochemical assays are artifacts, but that, more generally, when screened at relevant concentrations, drugs can act artifactually via colloidal aggregation. Understanding the role of aggregation, and detecting its effects rapidly, will allow the community to focus on those drugs and leads that genuinely have potential for treating COVID-19. Table of Contents Graphic Drug repurposing is an attractive idea in the face of a global pandemic, when rapid antiviral drug development is crucial. While the historical pragmatism of this approach has drawn scrutiny, 1, 2 drug repurposing has the potential to dramatically cut both the time and cost needed to develop a new therapeutic. 3 Repurposing campaigns typically screen curated libraries of thousands of approved drugs and investigational new drugs (INDs), and several assays have been developed to test these libraries for activity against SARS-CoV-2 4-6 . Most high throughput, biochemical screens were developed to detect activity against two proteins that are used in viral infection and maturation: the human ACE-2 (Angiotensin converting enzyme 2) and 3CL-Pro, 7 the major polypeptide processing protease of SARS-2-CoV-2. When testing molecules for biochemical activity at micromolar concentrations, it is important to control for artifacts [8] [9] [10] [11] [12] including colloidal aggregation, which is perhaps the single most common artifact in early drug discovery. 13, 14 Drugs, though in many ways de-risked, are not immune to aggregation and artifactual behavior when screened at relevant concentrations 15, 16 (though they are not expected to aggregate at on-target relevant concentrations). Knowing this, we wondered if colloidal aggregation was causing false positives in some COVID-19 drug repurposing studies, especially since several known aggregators, such as manidipine and methylene blue, were reported as apparently potent hits for Covid-19 targets. 17, 18 Aggregation is a is common source of false positives in early drug discovery, 19 arising from spontaneous formation of colloidal particles when organic, drug-like molecules are introduced into aqueous media. 15, 16, 19, 20 The resulting liquid particles are densely packed spheres 21 that promiscuously inhibit proteins by sequestering them on the colloid surface, 22 where they suffer partial unfolding. 23 The resulting inhibition is reversable by disruption of the colloid, and is characterized by an incubation effect on the order of several minutes due to enzyme crowding on the surface of the particle. 24 Colloids often can be disrupted by the addition of small amounts, often sub-critical micelle concentrations, of non-ionic detergent such as Triton-X 100. 25 Accordingly, addition of detergent is a common perturbation to rapidly detect aggregates in counter screens against model enzymes such as AmpC ß-lactamase or malate dehydrogenase (MDH). Aggregation can be physically detected by biophysical techniques such as nuclear magnetic resonance (NMR) 26 and by dynamic light scattering (DLS), as the colloids typically form particles in the 50 to 500 nm radius size range, which is wellsuited to measurement by this latter technique. Here we investigate the role of colloidal aggregation as a source of false positives in drug repurposing studies for SARS-CoV-2 targets. We focused on in vitro, ACE2 and 3CL-Pro screens since these are relevant for aggregation. We searched the literature and compiled the top hits from 12 studies 18, 27-37 and these were then visually inspected and ordered. How the results of this study may impact the design of future repurposing screens both for SARS-2 and for other indicators, will be considered. Colloidal aggregators are common hits in drug repurposing screens for SARS-CoV-2. We tested 56 drugs for colloidal aggregation that had been reported to be active in biochemical repurposing screens against SARS-CoV-2 18, 27-30, 32, 38 (SI Table 1 ). Five criteria were used to investigate whether reported hits formed colloidal aggregates: a. particle formation indicated by scattering intensity, b. clear autocorrelation curves, c. an MDH IC50 in the micromolar -high nanomolar range, d. restoration of MDH activity with the addition of detergent, and less stringently e. high Hill slopes in the inhibition concentration response curves (Fig 1) . Using the literature reported IC50 for the repurposed drugs as a starting point, we tested each drug for MDH inhibition and calculated the IC50 and Hill slope. We used IC50 values from the MDH concentration response curves and tested for detergent sensitivity at 3-fold the MDH IC50 (Fig 2) . Next, we calculated the critical aggregation concentration (CAC) by measuring normalized scattering intensity on the DLS, any point above 1x10 6 was considered from the aggregated form. By plotting a best fit line for aggregating concentrations and non-aggregating concentrations, the CAC was given by the point of intersection (Fig 3) . We also measured the DLS auto correlation curve as a criterion: if this was well-formed it gave further confidence (SI Fig 1) . Seventeen molecules formed well-behaved particles detectable by DLS with clean autocorrelation curves, and inhibited MDH in the absence of, but not the presence of 0.01% Triton X-100; these seem to be clear colloidal aggregators (Table 1, Fig 2, Fig 3) . Both DLS-based critical aggregation concentrations and MDH IC50 values were in the range of the primary IC50s reported in the literature against the two SARS-CoV-2 enzymes; indeed, molecules like Gossypol, manidipine, and TTNPB inhibited the counterscreening enzyme MDH even more potently than they did either ACE2 or 3CL-Pro. For most of the 17 drugs, the Hill slopes were high, though for several clear aggregators, such as Hemin and Shikonin, they were only in the 1.3 -1.4 range. Hill slope depends on the enzyme concentration to true KD ratio and so will vary from assay to assay 39 . While many consider it a harbinger of aggregation, we take it as only a soft criterion. reported to inhibit 3CL-Pro with an IC50 of 16.2 µM 18 . Lercanidipine satisfies our five criteria for aggregation: in aqueous buffer if forms particles that can be detected by a 10-fold increase in DLS scattering intensity (Cnts/sec); by a clearly defined autocorrelation curve in the DLS; it inhibits the counter-screening enzyme MDH with an IC50 of 2.2 µM, while MDH activity is restored on addition of 0.01% Triton-X 100 detergent (Fig 1) . In the absence of detergent, lercanidipine inhibits MDH with a Hill slope of 2.9. In addition to the 17 molecules that passed all five criteria for aggregation, another 19 molecules were more ambiguous, either forming particles by DLS but not inhibiting MDH, or inhibiting MDH in a detergent-dependent manner but not forming particles detectable by DLS (SI Table 1 ). These 19 drugs may also be acting artifactually, however further investigation is needed to determine their exact mechanisms. For this study, we focused only on clear colloidal aggregators. CAC is determined by finding the intersection of two best fit lines for points with scattering intensity above or below 1x10 6 . All measurements in triplicate. Figure 3 ). Only hemin continued to inhibit 3CL-Pro substantially, with an IC50 of 25 µM (but even this was 2.6-fold less potent than its literature value). As hemin's inhibition of MDH was disrupted by detergent ( Table 2) Taken together, these observations further support the aggregation-based activity of these 12 repurposed drugs. aggregators. We selected 15 of the latter for aggregation: six of these drugs satisfied our five criteria for aggregation, they inhibited MDH in the absence of, but not the presence of 0.01% Triton X-100 (Fig 4) and formed well-behaved particles detectable by DLS (Fig 5) with clean autocorrelation curves (SI Fig 2) , often with steep Hill slopes. In aggregate, these data suggest that these six drugs are prone to colloidal aggregation at screening-relevant concentrations ( Table 2 ). Two broad observations from this study merit emphasis. First, many drugs repurposed for Covid-19 aggregate and inhibit counter-screening enzymes promiscuously at concentrations relevant to their reported IC50s against the Covid-19 targets (ACE2 and 3CL-Pro). Of the 56 drugs tested, 17 fulfilled all five of our criteria for acting via colloidal aggregation: i. they formed particles that scattered strongly by DLS with ii. well-behaved autocorrelation curves, iii. they inhibited the counter-screening enzyme malate dehydrogenase-unrelated to either ACE2 or 3CL-Pro-at relevant concentrations in the absence but iv. not the presence of detergent and v. they typically inhibited with steep Hill slopes. Each of these criteria individually is a harbinger of colloidal aggregation; combined they strongly support its occurrence. Another 19 of the 56 drugs fulfilled only some of these criteria-for instance, forming particles at relevant concentrations but not inhibiting MDH in a detergent-dependent manner. Some of these 19 may also be aggregators, while others, like those that inhibit MDH but cannot be reversed by detergent, like tannic acid, may be acting via other PAINS-like artifacts. A second observation from this study is that these artifacts are not so much a feature of SARS-CoV-2 repurposing, but rather reflect the behavior of drugs at screening relevant concentrations. Thus, six of fifteen drugs investigated from a general purposing library were also aggregators at micromolar concentrations. An attraction of drug repurposing is that the molecules are thought to be derisked from the pathologies of early discovery. But at micromolar concentrations, drugs, which are often larger and more hydrophobic than the lead-like molecules found in most high- Literature search and chemoinformatic selection of potential aggregators. We used two approaches to identify drugs with the potential to form colloidal aggregates from µM oxaloacetic acid (324427, Sigma Aldrich) and the rate was monitored at 340 nm. A negative control was included in each run, in which 10 µL of DMSO without compound was added. The reactions were monitored for 90 seconds, and the initial rates were divided by the initial rate of the negative control to obtain the % inhibition and % enzyme activity. For dose response curves, 3 replicates were done for each concentration, the graphs were generated using GraphPad Prism version 9.1.1 (San Diego, CA). - What Are the Odds of Finding a COVID-19 Drug from a Lab Repurposing Screen? 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We thank Khanh Tang and John Irwin for help with Aggregation Advisor, and Isabella Glenn for help with aggregation assays.