The present invention relates to the identification of and use of biomarkers for disease and health conditions.
This invention is based on the observation that both normal and disease processes result in the covalent binding of small molecules to macromolecules and that these bound forms of small molecules constitute a new class of biomarkers for disease and therapeutic outcome and therapeutic leads that are accessible and measurable with a range of new technologies.
In one aspect of the invention there is provided a method for determining a disease state of an animal much as a human, which comprises determining levels of small molecules covalently bound to macromolecules (CBSM) in samples from that animal, and comparing said levels to standards.
In one embodiment the CBSM small molecules are sourced from gut microbiome derived, derived from metabolic processes, environmental chemical insult or abnormal chemical environment, endogenous or exogenous microorganism, or an interaction of processes between one or more of the above sources.
In another embodiment the macromolecule is selected from the group consisting of DNA, RNA, Protein, a complex carbohydrate and a Glycoprotein.
In yet another embodiment the disease state is selected from the group consisting of disease classification, disease sub categorization, disease progression, development of risk factors predictive of disease, specification of therapy, prediction of therapeutic outcome and development of therapeutic leads.
The present invention also provides a method for therapeutic intervention in disease in an animal much as a human comprising manipulating concentration levels of small molecules covalently hound to macromolecules (CBSM).
In one embodiment the macromolecule is selected from the group consisting of DNA, RNA, Protein, a complex carbohydrate and a Glycoprotein.
In another embodiment the small molecules are sourced from gut microbiome derived, derived from metabolic processes, environmental chemical insult or abnormal chemical environment, endogenous or exogenous microorganism, or an interaction of processes between and among one or more the above sources.
In one embodiment the disease is an affective disease selected from the group consisting of depression, schizophrenia and autism, a degenerative disease selected from the group consisting of Huntington's, Alzheimer's, Parkinson's, Mild Cognitive impairment, ALS, Freidrich Ataxia, cancer, diabetes, and cardiovascular disease, or from in-born errors of metabolism or genetic based disease.
The present invention also provides a method of intervention to prevent or ameliorate disease in an animal much as a human with disease risk which comprises manipulating the animal's small molecules covalently bound to macromolecules (CBSM).
In one embodiment the risk is for an affective disease selected from the group consisting of depression, schizophrenia and autism, a degenerative disease selected from the group consisting of Huntington's, Alzheimer's, Parkinson's, Mild Cognitive impairment, ALS or Freidrich Ataxia, cancer, diabetes, and cardiovascular disease, or from in-born errors of metabolism or genetic based disease.
The invention also provides a method for determining the nature of the source of small molecules covalently bound to macromolecules (CBSM) which comprises creating and analyzing synthetic combinations of small molecules and macromolecules with processes mimicking biochemical processes in an animal much as a human.
The invention further provides a method for modifying gene function in an animal comprising manipulating concentration levels of small molecules covalently bound to macromolecules (CBSM), whereby to increase or decrease expression of a target gene.
In one embodiment the macromolecule is selected from the group consisting of DNA, RNA, a Protein, a complex carbohydrate and a Glycoprotein.
In another embodiment the small molecule is sourced from gut microbiome derived, derived from metabolic processes, environmental chemical insult or abnormal chemical environment, endogenous or exogenous microorganism, or an interaction of processes between and among one or more the above sources.
In yet another embodiment the gene is associated with an affective disease selected from the group consisting of depression, schizophrenia or autism, a degenerative disease selected from the group consisting of Huntington's, Alzheimer's, Parkinson's, Mild Cognitive impairment, ALS, Freidrich Ataxia, cancer, diabetes, and cardiovascular disease, or from in-born errors of metabolism or genetic based.
The present invention also provides a method of therapeutic discovery comprising:
identifying a class of subjects with equivalent genetic risk factors;
identifying sub classes in this class who do and do not develop disease;
identifying differences in covalently bound molecules to DNA, RNA and protein that discriminate the class and subclasses and affect epigenetic differences in the system feedback control; isolating and determining chemical precursor sources and structures of the covalently bound discriminators; and providing or replacing such compounds as are missing or in reduced amount in the disease developing class and/or suppressing such compounds that are elevated or in excess in the disease developing class.
In one embodiment the disease is a neurodegenerative disease selected from the group consisting of Huntington's, Parkinson's, Mild Cognitive Impairment, Amyotrophic Lateral Sclerosis, Freidrich Ataxia, cancer, diabetes and cardiovascular disease, an affective disorder selected from the group consisting of depression, schizophrenia and autism, or from in-born errors of metabolism or genetic based disease.
Finally the invention provides small molecules covalently bound to macromolecules for determination of disease risk, diagnostic status, prediction of disease progression and development of therapy.
Further features and advantages of the present invention will be seen from the following detailed description, taken in conjunction with the accompanying drawings, wherein like numerals depict like parts, and wherein;
The network of biochemical interactions that define the functional operation of an individual is shown schematically in
Biomarkers, meaning those genes, proteins, RNA transcripts, or small molecules related to disease can generally be classified as: predictive biomarkers, i.e., those that show risk of disease; biomarkers of state, i.e., those that classify disease; biomarkers of progression, i.e.; those that progress with disease; and biomarkers of therapeutic outcome, i.e., biomarkers that change with therapeutic intervention. To these definitions we now add biomarkers that suggest therapeutic intervention strategies.
The search for biomarkers has almost universally been in specific “omic” compartments (A1-A4 in
Small molecule biomarkers are strongly coordinately bound to macro molecules in biological samples. Techniques for assessing small molecule biomarkers (Metabolomics) typically use extraction protocols to remove and concentrate such coordinately bound materials. However, biological/biochemical processes that are either enzyme driven or driven by normal/abnormal free radical production of, for instance, hydroxyl, oxy, or nitro free radical types or simple proximity reactions will cause covalent binding of these closely associated small molecules to macro molecules such as protein, DNA, or RNA. This binding can affect gene expression, the functionality of enzymes and the folding/aggregation of proteins. Since all of the above processes are implicated as risk factors, disease processes and disease progression, the levels and nature of the covalently bound and the distribution of free and coordinately bound small molecules in principal reflects the disease or risk factor processes better than single genes, transcripts, proteins or the totality of coordinately bound and free small molecules.
Referring again to
Process 1 involves covalently bound small molecule to protein biomarkers for blood-(plasma, leucocytes, platelets, RBC, lysed cells, lysed, whole blood) other bodily fluids and tissue.
In the simplest form protein pellets or other macromolecules (DNA, RNA, complex carbohydrates) derived from preparations using extraction and precipitation of plasma or other tissues for evaluating coordinately bound small molecules were further digested either chemically or enzymatically. The profiles of these preparations were then evaluated with metabolomic techniques such as liquid chromatography with electrochemical detection (LCECA), Mass spectrometry (MS), parallel or series combinations of LCEC/LCMS or nuclear magnetic resonance (NMR).
This is shown in the left branch of the sample preparation methodology flow chart in
Examples of potential biomarkers of HD vs. controls in this type of preparation are shown in
Samples were taken through stacked membrane filters in sequence from 1M-300K, 100K-50K-10K molecular weight cut off membranes. The below 10K fraction when processed or analyzed directly reflected the free Metabolome or that which is in equilibrium with coordinately bound fractions in the macro molecules. Sequential macromolecule fractions treated with standard extraction techniques such as precipitation with acetonitrile metha.11ol from which the supernatant was subsequently analyzed via the distribution of coordinately bound molecules as a function of molecular weight.
The analysis of this first set of distributional data provided greater insight into potential biomarkers than the total of all coordinately bound species. For instance the relationship of tryptophan to its primary metabolite kynurinine was partially descriptive of response to antidepressants. However, the relationship of tryptophan to kynurinine in the macromolecule fraction between 300 and 100K was more highly descriptive; the decrease in Indole propionate in AD plasma vs controls was more pronounced in the macromolecule fraction between 100 and 50K, and further pronounced in the ratio of free material to the material bound in the 100 to 50K fraction.
The second set of data was obtained from the macromolecule precipitates in the case of protein precipitates the protein is digested for instance with trypsin (TP) or proteinase k (PK) or beta peptidase or a combination thereof subsequently passing the digest from each fraction through a 10K membrane for PK digests or a 30 K membrane for TP digests and directly analyzing the filtrate.
Additional resolution of other potential markers could be had in the electrochemical array by introducing a boron doped diamond sensor as the last sensor in the series, and further resolution could be had by utilizing LCECA and liquid chromatography with Mass (LCMS) spectrometry in parallel following the teachings of my PCT Application Serial No. PCT/US13/33918, filed Mar. 26, 2013. Essentially any response that does not have the characteristic signature of a peptide in the EC array incorporating a Boron Doped diamond sensor or the extract mass of a peptide in the parallel LCEC/LCMS parallel configuration is a covalently bound small molecule to an amino acid moiety.
While standard preparative protocols for tissue and DNA/RNA extraction can be used, optimum preparative protocols seek to preserve the macromolecules in the least chemically compromised state. The preparative protocols for tissue involve solubilization of the macromolecules through such processes as grinding of the sample at liquid nitrogen temperatures or using a high speed “tissue mizer” grinder followed by processes such as repetitive freeze thawing in an acceptable matrix such as distilled water or normal saline, or by uses of cycled high pressure disruption again in a suitable matrix.
A second approach for clinical samples of whole blood was based on the ability of the LCECA and parallel LCMS platforms to resolve and compare multiple signals quantitatively. A process of isolating DNA from blood by serial filtration through sequentially small pore sizes provided a crude preparation containing DNA that can be sub aliquoted and analyzed with a sequence of extraction preparations for one fraction, and directly lysed with HCl for a second fraction to disrupt the DNA to the base purines and pyrimidines and release covalently bound materials as base adducts. Subsequently the profiles from the two fractions were compared to determine those moieties unique to the DNA.
For DNA, protocols which preserve the histone association with DNA are preferred for initial studies. The histones can be selectively removed by PK digestion and the digests analyzed as above for covalently bound small molecules.
RNA fractions can be evaluated either globally or as isolated using size fractionation protocols to evaluate binding to fractions from tRNA, mRNA exosomes etc. Macromolecule fractions from tissue were evaluated for distribution to various proteins of coordinately and covalently bound compounds from the metabolome are described above. DNA and RNA fractions were evaluated by precipitation/extraction of the coordinately bound metabolome followed by enzymatic disruption as with P 1 endonuclease or P 1 endonuclease followed by AP alkaline phosphatase or digestion with HCl or other weak acid. Purified DNA for instance showed under these protocols the base pairs as 5′ monophosphates (P 1), or the base pairs (P 11AP), or the bases guanine adenine cytidine, thymidine for HCL digests. As we reported in our prior paper, on reviewing entire profiles in our prior paper on 7 methyl guanine. (Anal Biochem. 2013 May 15; 436(2):112-20. doi: 10.1016/j.ab.2013.01.035. Epub 2013 Feb. 12, “A novel method for detecting 7-methyl guanine reveals aberrant methylation levels in Huntington disease”, Thomas B, Matson S, Chopra V, Sun L, Sharma S, Hersch S, Rosas H D, Scherzer C, Ferrante R, Matson W) that other peaks in the response profiles as well as direct modifications of base pairs such as 7 methyl guanine or 8 hydroxy guanine are directly related to other molecules covalently bound to the base pairs or base pair monophosphates or bases. These were made available for assay by the process of dissolution of DNA or RNA and represent species that either inherently respond to the sensors or respond as adducts to the base pairs showing different chromatographic separation.
In our prior paper we also reported on techniques of isolating RNA and DNA from brain tissue with the intent of developing a targeted method for guanine and 7 methyl guanine in DNA and RNA following the hypothesese that changes in the ratio of these would be indicative of epigenetic differences. The whole point of the targeted assay was to get a clean signal for the guanine and 7 methyl guanine which were indeed descriptive of epigenetic changes in the wild type and CAG 140 HD mouse model and in human postmortem HD and control brain. This involved removal of coordinately bound species and substantial manipulation of the LCECA. However when we recognized the potential significance of the “interferences” as covalently bound small molecules we re-analyzed the entire chromatographic output.
Shown below in
While not wishing to be bound by theory, it is believed that all of these species are likely involved in the functionality of the DNA and RNA and consequently both reflect and determine the operation of the network shown in
Strategies for identifying the source of a covalently bound material:
Many of the processes to covalently bind a small molecule to a macromolecule involve the creation of an intermediate small molecule radical by attack with for instance hydroxyl of nitro so radicals. We have made preparations of various proteins, RNA and DNA with coordinate sites saturated with small molecules such as kynurinine or indole propionic acid. These preparations were subjected to free radical attack using various variants of Fenton reactions, peroxide peroxide/nitrate and subsequently processed as above. This allowed the identification of the source of many of the responses as shown in
Our analysis showed that covalently bound small molecule biomarkers (CBSM) are strong discriminators of state and in the animal data predictors of progression. The distribution of CBSM in protein of other studies lead us to conclude that they also are predictive of outcome of therapy in depression and schizophrenia, time of pheno conversion in Huntington's disease and conversion of mild cognitive impairment to Alzheimer's disease. Thus they are believed to be applicable as biomarkers of state, risk, therapeutic monitoring and prediction in a range of disorders or potentials for disorders.
CBSM also can be used in therapeutic intervention and pharmaceutical development. The rationale for this is that many small molecules are relatively strongly co-ordinately bound to macro molecules. Early risk states whether genetic or induced by environmental factors or by the interaction of genetic and environmental factors as in the case of higher incidence of Amyotrophic Lateral Sclerosis in Gulf War veterans or Parkinson's in agricultural workers exposed to pesticides/herbicides can result in the binding of these small molecules to, for instance, DNA or critical proteins. This binding will in turn affect the operation of the genome (epigenetics) or the functionality of the enzymes. For instance a possibility of the latter effect would be the binding of small molecules to the enzymes in the kynurinine pathway affecting the onset of depression or the outcome of therapy. While not wishing to be bound by theory, it is believed this binding may be the reason that differences in the levels of compounds on this pathway are correlated with depression. Understanding which compounds are bound to the macro molecules provides a route to the design of compounds or strategies to displace them from their coordinate sites with compounds that are not subject to free radical or chemical processes that result in coordinate binding.
Alternatively understanding the compounds covalently associated with DNA allows strategies for design of compounds to specifically change the functionality of the genome-for instance shutting down the function of the breast cancer risk gene by epigenetic covalent binding.
This Application claims priority from U.S. Provisional Application Ser. No. 61/934,374, filed Jan. 31, 2014.
Number | Date | Country | |
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61934374 | Jan 2014 | US |