Chemical tools for epichaperome-mediated interactome dysfunctions of the central nervous system

Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer’s disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administered by intravenous injection. In sum, we introduce a discovery platform for brain-directed chemical probes that specifically modulate epichaperomes and provide proof-of-principle applications in their use in the detection, quantification, and modulation of the target in complex biological systems.


Statistics
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Software and code
Policy information about availability of computer code Data collection Gabriela Chiosis and Fumiko Shimizu May 17, 2021 As described in the Methods, the identity and purity of each product was characterized by MS, HPLC, TLC, and NMR. 1H/13C NMR spectra were recorded on either a Bruker 400, 500 or 600 MHz instrument. High resolution mass spectra were recorded on a Waters LCT Premier system. Low resolution mass spectra were obtained on Waters Acquity Ultra Performance LC with electrospray ionization and SQ detector. HPLC analysis was done on Waters Autopurification system with PDA, MicroMass ZQ and ELSD detector and a reversed phase column (Waters X-Bridge C18, 4.6 x 150 mm, 5 µm) eluted with water/acetonitrile gradients, containing 0.1% TFA. FP measurements were performed on an Analyst GT instrument (Molecular Devices, Sunnyvale, CA). General SEP II was performed by Caliper Life Sciences (now PerkinElmer). Radioactivity metabolite analysis was performed using Shimadzu (Columbia, Maryland USA) HPLC system equipped with binary pumps LC-20AB, UV detector and sodium iodide radioactivity detector connected to flow-ram (Lab Logic, Tampa, FL) and 250 x 4.6 mm Phenomenex Luna (Torrance, CA) C-18 HPLC column (10 µM, 100 A). For metabolite characterization, samples were analyzed by LC-MS using positive-ion electrospray ionization and reverse-phase chromatography in full-scan mode with MS/MS product-ion spectra generated for detected metabolite masses. This was performed using the MS2 scan mode of the G6410B mass spectrometer to produce MS/MS product-ion spectra for masses detected at sufficient intensities. Chromatography was performed using a ZORBAX Eclipse XDB-C18 4.6 x 50 mm, 5 µm, column, with gradient elution at 1mL /min-1. Absorbance was measured by UV spectroscopy on the GT Analyst (Molecular Devices) plate reader at 260 nm. Radiation (10 Gy) was delivered via X-RAD 225C irradiator (Precision X-Ray Inc., North Branford, CT). CD105+ tumor endothelial cells were isolated by a MoFlo cell sorter (Beckman Coulter, Pasadena, CA). I-131 was measured in a scintillation g-counter (Perkin Elmer 1480 Wizard 3 Auto Gamma counter, Waltham, MA) using a 260-340 keV energy window. For mouse PET imaging, small-animal PET scanner (Model # Focus 120 microPET; Siemens Medical Solutions, USA, formerly Concorde Microsystems, Knoxville, TN)) was used. LC-MS/MS (6410, Agilent Technologies) was used to measure concentrations of PU-HZ151 in mouse plasma and tissues. PET-CT in human patients was performed using an integrated PET-CT scanner (Discovery DSTE, GE Healthcare) and MRI was obtained using a 1.5 T magnet (Signa HDxt, GE Healthcare, Milwaukee, WI). For quantification of radioprobe clearance and metabolism, plasma samples were radioassayed by well counter and radioHPLC using standard techniques. Activity in whole blood and plasma specimens was radioassayed by well counter (1480 WIZARD® 3" Automatic Gamma Counter, PerkinElmer, Shelton, Connecticut USA).

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October 2018

Data analysis
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Data exclusions
Replication Randomization Blinding Prism v7and9 was used for statistical testings (t-tests and ANOVA). ImageJ (versions 1.4 and 1.52) was used for western blot quantification. SoftMaxPro6 was used for fluorescence polarization data analyses. Calculated physicochemical properties were obtained using standard commercial and freeware packages: Schrödinger software suite release 2018-1 (QikProp and Epik), ChemAxon Marvin suite (MarvinSketch 20.19) and VCCLAB (ALOGPS 2.1). The 2D structures of all the title compounds were generated by using ChemBioDraw Ultra 12.0. LigPrep module implemented in Schrödinger was used to generate energy minimized 3D structures. Acquired flow cytometry data was analysed using FlowJo v10.7 (FlowJo LLC.). Quantification and %ID g-1 values were calculated by manually drawing regions of interests in three different frames and determining the average values using ASI Pro VM™ MicroPET Analysis software (Siemens Medical Solutions, Knoxville, TX). For precise localization of tracer uptake to specific neuroanatomical structures, PET brain images were fused to MRI brain images using the Integrated Registration application of the AW Suite software package (GE Healthcare, Milwaukee, Wisconsin, USA). The software used for the fits and to derive the PK parameters was EXCEL 365 (Microsoft Corp, Redmon, WA). Relative count intensity of the autoradiography sections in each image was quantified using ImageJ 1.47u processing software.
The source data underlying all main and supplementary figures are provided as a Source Data file. All data generated or analyzed during this study are included in this published article (and its supplementary information files).
No statistical methods were used to predetermine sample size for in vitro but these are similar to those generally employed in the field. For mouse studies, sample sizes were determined by magnitude and consistency of measurable differences in a pilot study we conducted. Sample sizes for in vitro and in vivo experiments are fully disclosed in the manuscript. For the clinical study, this was an exploratory pilot study. Sample was not estimated according to statistical power calculation. In vitro and in vivo experiments were performed in at least 3 biological replicates to sufficiently detect statistical significance, and the number of technical replicates for in vitro studies were determined according to manufacturers' instructions for data acquisitions.
No data were excluded from the analyses. For the clinical study (NCT03371420), the first enrolled patient consented but withdrew prior to participating in the study due to disease worsening. For the third enrolled patient we had a failure in 124I-PU-HZ151 production and the patient could not complete the study. Exclusion Criteria for participation in NCT03371420: Subject has unacceptable pre-study organ function during screening defined as: Bilirubin > 1.5 x institutional upper limit of normal (ULN) AST/ALT >2.5 x ULN Albumin < 2 g/dl GGT > 2.5 x ULN (IF Alkaline phosphatase > 2.5 x ULN) Creatinine >1.5 x ULN or creatinine clearance < 60 mL/min. Subject has history of acute major illness (i.e., unstable cardiovascular condition.) Subject has concurrent participation in any interventional studies within 30 days of first dose of study drug.
All in vitro and in vivo experiments were performed, in at least 3 biological replicates, with biological and technical replicates which are fully disclosed in the manuscript. Several alternative methods were used to validate observations. Experiments were also replicated through multiple cohort analyses. Results shown are representative of several independently performed experiments (see figure legends, at least 3). There were no findings that could not be replicated or reproduced. The data in the clinical analyses were derived from a clinical trial; therefore, experimental replication was not feasible.
Mice were randomized prior to treatment with vehicle control or compound. For in vitro experiments, samples were allocated into experimental groups by randomization. For the clinical PET imaging study randomization does not apply. This is a single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420).
Investigators were not blinded to group allocation during data collection and/or analysis for the preclinical studies. All values were determined by methods that are independent of operator bias. Mice treatments were not blinded since most of the experiments required daily treatments and treatment groups and mice cage numbers had to be known for investigators. For the clinical study, outcome assessment for nature research | reporting summary

October 2018
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Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals the primary endpoint (blood PK parameters) was performed by strictly blinded scientists. Blinding was not relevant to the remaining experiments described in the study due to primary investigators performing experiments from start to finish due to technicality required. Wild animals

Field-collected samples
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Recruitment
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Study protocol
Data collection acclimatize at the MSKCC vivarium for 1 week prior to experiments. Mice were housed in ventilated cage enclosures in an environment maintained at 50% humidity with ambient temperatures range between 66°F and 78°F and 12h day/light cycles. Mice were provided with food and water ad libitum. All mice in all studies were observed for clinical signs at least once daily.
The study did not involve wild animals none All procedures were approved by the MSKCC Institutional Animal Care and Use Committee.
Patients with solid malignancy, myeloproliferative neoplasm, myeloma, and/or lymphoma (histology confirmed by MSKCC Department of Pathology). Disease is measurable or evaluable as defined by RECIST (1.1 or original version) or other tumor response criteria from an MSKCC IRB-approved clinical research protocol.
Patients have established diagnosis of mild-moderate Alzheimer's Disease by board-certified neurologist (MSKCC or non-MSKCC) based upon neurological and neuropsychological evaluation following the National Institute on Aging-Alzheimer's Disease Association criteria that recently revisited the NINCDS-ADRDA criteria Ages Eligible for Study: 18 Years to 90 Years (Adult, Older Adult) Sexes Eligible for Study: All Given the diverse population of patients at MSKCC, minorities and women patients had full access to this study and were fairly represented in the accrual.
Patients were screened by trained personnel, and the principal investigator was responsible for evaluating and confirming eligibility. Informed consent and HIPAA compliance forms were obtained from all subjects prior to their enrollment. Any patient who met the inclusion/exclusion criteria as detailed in the study protocol was considered for enrollment, therefore, to the best of our knowledge, there was no selection bias during recruitment. 124I-PU-HZ151 and 124I-PU-H71 were synthesized in-house by the institutional cyclotron core facility at high specific activity. Analyses of the epichaperome by positron emission tomography (PET-CT) were performed as previously reported. In brief, research PET-CT was performed using an integrated PET-CT scanner (Discovery DSTE, GE Healthcare). CT scans for attenuation correction and anatomic coregistration were performed before tracer injection. Patients received 185 megabecquerel (MBq) of 124I-PU-HZ151 or 124I-PU-H71 by peripheral vein over two minutes. PET data were reconstructed using a standard ordered subset expected maximization iterative algorithm. Emission data were corrected for scatter, attenuation, and decay. PET scans were performed 0.5, 3 and 24 h after tracer administration. For blood PK, all patient blood samples had a valid chain of custody from venipuncture through onsite specimen analysis and disposal. Safety of 124I-PU-AD in subjects as assessed by evaluation of the incidence, nature, and severity of adverse events and serious adverse events.
Single cell suspension was prepared from a freshly resected GBM tumor as described in Online methods and CD105+ tumor endothelial cells (ECs) were isolated by a MoFlo cell sorter (Beckman Coulter, Pasadena, CA) using a FITC-conjugated CD105 antibody (1:20, BD Biosciences).

FowJo version 10
An aliquot of the CD105+ post-sort fraction was analyzed by a fluorescence microscope. The purity of CD105+ cells was 98%.
FSC/SSC gates were set to exclude debris in the sample. CD105-FITC signal (FL1: green channel) was plotted against cell autofluorescence (FL2: orange channel) to allow a clear separation of CD105+ vs CD105-cells on a two-dimensional plot. Standard brain MRI obtained as part of routine patient care. MRI brain were performed per institutional standard of care according to the standardized brain tumor imaging protocol (https://pubmed.ncbi.nlm.nih.gov/26250565/) Experimental design specifications are not applicable as the brain MRI was obtained as standard-of-care (ie, routine brain MRI is a common, non-experimental procedure in the hospital setting). Standard brain MRI can vary in technical details; the technical details of the MRI procedure (ie, image acquisition protocol) employed for the MRI scans used in this research project are therefore provided below (under Acquisition, Preprocessing, Statistical Modeling).
N/A. The brain MRI was performed for tumor imaging as part of cancer patient care. Any quantitative analysis to correlate behavioral performance was not part of the study scale. Therefore, behavioral performance was not measured.