Familial Clonal Hematopoiesis in a Long Telomere Syndrome.

BACKGROUND: Telomere shortening is a well-characterized cellular aging mechanism, and short telomere syndromes cause age-related disease. However, whether long telomere length is advantageous is poorly understood. METHODS: We examined the clinical and molecular features of aging and cancer in persons carrying heterozygous loss-of-function mutations in the telomere-related gene POT1 and noncarrier relatives. RESULTS: A total of 17 POT1 mutation carriers and 21 noncarrier relatives were initially included in the study, and a validation cohort of 6 additional mutation carriers was subsequently recruited. A majority of the POT1 mutation carriers with telomere length evaluated (9 of 13) had long telomeres (>99th percentile). POT1 mutation carriers had a range of benign and malignant neoplasms involving epithelial, mesenchymal, and neuronal tissues in addition to B- and T-cell lymphoma and myeloid cancers. Five of 18 POT1 mutation carriers (28%) had T-cell clonality, and 8 of 12 (67%) had clonal hematopoiesis of indeterminate potential. A predisposition to clonal hematopoiesis had an autosomal dominant pattern of inheritance, as well as penetrance that increased with age; somatic DNMT3A and JAK2 hotspot mutations were common. These and other somatic driver mutations probably arose in the first decades of life, and their lineages secondarily accumulated a higher mutation burden characterized by a clocklike signature. Successive generations showed genetic anticipation (i.e., an increasingly early onset of disease). In contrast to noncarrier relatives, who had the typical telomere shortening with age, POT1 mutation carriers maintained telomere length over the course of 2 years. CONCLUSIONS: POT1 mutations associated with long telomere length conferred a predisposition to a familial clonal hematopoiesis syndrome that was associated with a range of benign and malignant solid neoplasms. The risk of these phenotypes was mediated by extended cellular longevity and by the capacity to maintain telomeres over time. (Funded by the National Institutes of Health and others.).

T cell immune deficiency rather than chromosome instability predisposes patients with short telomere syndromes to squamous cancers.

Patients with short telomere syndromes (STS) are predisposed to developing cancer, believed to stem from chromosome instability in neoplastic cells. We tested this hypothesis in a large cohort assembled over the last 20 years. We found that the only solid cancers to which patients with STS are predisposed are squamous cell carcinomas of the head and neck, anus, or skin, a spectrum reminiscent of cancers seen in patients with immunodeficiency. Whole-genome sequencing showed no increase in chromosome instability, such as translocations or chromothripsis. Moreover, STS-associated cancers acquired telomere maintenance mechanisms, including telomerase reverse transcriptase (TERT) promoter mutations. A detailed study of the immune status of patients with STS revealed a striking T cell immunodeficiency at the time of cancer diagnosis. A similar immunodeficiency that impaired tumor surveillance was documented in mice with short telomeres. We conclude that STS patients' predisposition to solid cancers is due to T cell exhaustion rather than autonomous defects in the neoplastic cells themselves.

Somatic reversion impacts myelodysplastic syndromes and acute myeloid leukemia evolution in the short telomere disorders.

BACKGROUNDGermline mutations in telomerase and other telomere maintenance genes manifest in the premature aging short telomere syndromes. Myelodysplastic syndromes and acute myeloid leukemia (MDS/AML) account for 75% of associated malignancies, but how these cancers overcome the inherited telomere defect is unknown.METHODSWe used ultra-deep targeted sequencing to detect somatic reversion mutations in 17 candidate telomere lengthening genes among controls and patients with short telomere syndromes with and without MDS/AML, and we tested the functional significance of these mutations.RESULTSWhile no controls carried somatic mutations in telomere maintenance genes, 29% (16 of 56) of adults with germline telomere maintenance defects carried at least 1 (P < 0.001), and 13% (7 of 56) had 2 or more. In addition to TERT promoter mutations, which were present in 19%, another 13% of patients carried a mutation in POT1 or TERF2IP. POT1 mutations impaired telomere binding in vitro and some mutations were identical to ones seen in familial melanoma associated with longer telomere length. Exclusively in patients with germline defects in telomerase RNA (TR), we identified somatic mutations in nuclear RNA exosome genes RBM7, SKIV2L2, and DIS3, where loss-of-function upregulates mature TR levels. Somatic reversion events in 6 telomere-related genes were more prevalent in patients who were MDS/AML-free (P = 0.02, RR 4.4, 95% CI 1.2-16.7), and no patient with MDS/AML had more than 1 reversion mutation.CONCLUSIONOur data indicate that diverse adaptive somatic mutations arise in the short telomere syndromes. Their presence may alleviate the telomere crisis that promotes transformation to MDS/AML.FUNDINGThis work was supported by the NIH, the Commonwealth Foundation, the S&R Foundation Kuno Award, the Williams Foundation, the Vera and Joseph Dresner Foundation, the MacMillan Pathway to Independence Award, the American Society of Hematology Scholar Award, the Johns Hopkins Research Program for Medical Students, and the Turock Scholars Fund.

Ac-Trp-DPhe(p-I)-Arg-Trp-NH2, a 250-Fold Selective Melanocortin-4 Receptor (MC4R) Antagonist over the Melanocortin-3 Receptor (MC3R), Affects Energy Homeostasis in Male and Female Mice Differently.

The melanocortin-4 receptor (MC4R) has been indicated as a therapeutic target for metabolic disorders such as anorexia, cachexia, and obesity. The current study investigates the in vivo effects on energy homeostasis of a 15 nM MC4R antagonist SKY2-23-7, Ac-Trp-DPhe(p-I)-Arg-Trp-NH2, that is a 3700 nM melanocortin-3 receptor (MC3R) antagonist with minimal MC3R and MC4R agonist activity. When monitoring both male and female mice in TSE metabolic cages, sex-specific responses were observed in food intake, respiratory exchange ratio (RER), and energy expenditure. A 7.5 nmol dose of SKY2-23-7 increased food intake, increased RER, and trended toward decreasing energy expenditure in male mice. However, this compound had minimal effect on female mice's food intake and RER at the 7.5 nmol dose. A 2.5 nmol dose of SKY2-23-7 significantly increased female food intake, RER, and energy expenditure while having a minimal effect on male mice at this dose. The observed sex differences of SKY2-23-7 administration result in the discovery of a novel chemical probe for elucidating the molecular mechanisms of the sexual dimorphism present within the melanocortin pathway. To further explore the melanocortin sexual dimorphism, hypothalamic gene expression was examined. The mRNA expression of the MC3R and proopiomelanocortin (POMC) were not significantly different between sexes. However, the expression of agouti-related peptide (AGRP) was significantly higher in female mice which may be a possible mechanism for the sex-specific effects observed with SKY2-23-7.

Discovery of a ß-Hairpin Octapeptide, c[Pro-Arg-Phe-Phe-Dap-Ala-Phe-DPro], Mimetic of Agouti-Related Protein(87-132) [AGRP(87-132)] with Equipotent Mouse Melanocortin-4 Receptor (mMC4R) Antagonist Pharmacology.

Agouti-related protein (AGRP) is a potent orexigenic peptide that antagonizes the melanocortin-3 and -4 receptors (MC3R and MC4R). While the C-terminal domain of AGRP, AGRP(87-132), is equipotent to the full-length peptide, further truncation decreases potency at the MC3R and MC4R. Herein, we report AGRP-derived peptides designed to mimic the active ß-hairpin secondary structure that contains the hypothesized Arg-Phe-Phe pharmacophore. The most potent scaffold, c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro], comprised the hexa-peptide ß-hairpin loop from AGRP cyclized through a DPro-Pro motif. A 20 compound library was synthesized from this scaffold for further structure-activity relationship studies. The most potent peptide from this library was an asparagine to diaminopropionic acid substitution that possessed sub-nanomolar antagonist activity at the mMC4R and was greater than 160-fold selective for the mMC4R versus the mMC3R. The reported ligands may serve as probes to characterize the melanocortin receptors in vivo and leads in the development of novel therapeutics.

Semi-rigid tripeptide agonists of melanocortin receptors.

A series of 30 RCO-HfR-NH(2) derivatives show preference for the mouse MC1R vs MC3-5Rs. trans-4-HOC(6)H(4)CH=CHCO-HfR-NH(2) (13) [EC(50) (nM): MC1R 83, MC3R 20500, MC4R 18130 and MC5R 935; ratio 1:246:217:11] is 11 times more potent than the lead compound LK-394 Ph(CH(2))(3)CO-HfR-NH(2) (2) and only 11 times less potent than the native tridecapeptide alpha-MSH at mMC1R. Differences in conformations of 2 and 13 are discussed.

Peptide and small molecules rescue the functional activity and agonist potency of dysfunctional human melanocortin-4 receptor polymorphisms.

The melanocortin pathway, specifically the melanocortin-4 receptor and the cognate endogenous agonist and antagonist ligands, have been strongly implicated in the regulation of energy homeostasis and satiety. Genetic studies of morbidly obese human patients and normal weight control patients have resulted in the discovery of over 70 human melanocortin-4 receptor (MC4R) polymorphisms observed as both heterozygous and homozygous forms. A number of laboratories have been studying these hMC4R polymorphisms attempting to understand the molecular mechanism(s) that might explain the obese human phenotype. Herein, we have studied 13 polymorphic hMC4Rs that have been identified to possess statistically significant decreased endogenous agonist potency with synthetic peptides and small molecules attempting to identify ligands that can pharmacologically rescue the hMC4R polymorphic agonist response. The ligands examined in this study include NDP-MSH, MTII, Ac-His-DPhe-Arg-Trp-NH2 (JRH887-9), Ac-Anc-DPhe-Arg-Trp-NH2 (amino-2-naphtylcarboxylic acid, Anc, JRH420-12), Ac-His-(pI)DPhe-Arg-Trp-NH2 (JRH322-18), chimeric AGRP-melanocortin based ligands (Tyr-c[Cys-His-DPhe-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH2, AMW3-130 and Ac-mini-(His-DPhe-Arg-Trp)-hAGRP-NH2, AMW3-106), and the small molecules JB25 and THIQ. The hMC4R polymorphisms included in this study are S58C, N97D, I102S, L106P, S127L, T150I, R165Q, R165W, L250Q, G252S, C271Y, Y287Stop, and I301T. These studies resulted in the NDP-MSH, MTII, AMW3-130, THIQ, and AMW3-106 ligands possessing nanomolar to subnanomolar agonist potency at the hMC4R polymorphisms examined in this study. Thus, these ligands could generically rescue the potency and stimulatory response of the abnormally functioning hMC4Rs studied and may provide tools to further clarify the molecular mechanism(s) involving these receptor modifications.

Molecular mechanism of the constitutive activation of the L250Q human melanocortin-4 receptor polymorphism.

The Melanocortin-4 Receptor is a G-protein coupled receptor that has been physiologically linked to participate in the regulation of energy homeostasis. The Melanocortin-4 Receptor is stimulated by endogenous melanocortin agonists derived from the pro-opiomelanocortin gene transcript and antagonized by the endogenous antagonist agouti-related protein. Central administration of melanocortin agonists has been demonstrated to decrease food intake and conversely, treatment with antagonists resulted in increased food intake. Deletion of the Melanocortin-4 Receptor gene from the mouse genome results in an obese and hyperphagic phenotype. Polymorphisms of the human Melanocortin-4-Receptor have been found in severely obese individuals, suggesting that Melanocortin-4 Receptor malfunction might be involved in human obesity and obesity-associated diabetes. Herein, we have performed experiments to understand the molecular mechanisms associated with the L250Q human Melanocortin-4-Receptor polymorphism discovered in an extremely obese woman. This L250Q human Melanocortin-4-Receptor has been pharmacologically characterized to result in a constitutively active receptor. The fact that a constitutively active human Melanocortin-4-Receptor mutation was found in an obese person is a physiologic contradiction, as chronic activation of the human Melanocortin-4-Receptor and subsequently high cyclic adenosine monophosphate levels should theoretically result in a normal or lean phenotype. In this study, we demonstrated that agouti-related protein acts as an inverse agonist at this constitutively active receptor, and we propose a mechanism by which agouti-related protein might contribute to the obese phenotype in the L250Q patient. In addition, using receptor mutagenesis, pharmacology, and computer modeling approaches, we investigated the molecular mechanism by which modification of the L250 residue results in constitutive activation of the human Melanocortin-4-Receptor.

[Identification of triacylglycerols in coix oil by high performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry].

OBJECTIVE: To identify triacylglycerols in coix oil. METHOD: High performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry was used for identification. The experiment was operated under the conditions: spray voltage at 3 000 V, capillary temperature at 250 degrees C, APCI vaporizer temperature at 400 degrees C, and corona current of 4 microA. Sheath gas pressure (high purity liquid nitrogen) was 35 kPa. Mass spectra were obtained over the m/e range of 300 to 900 amu, scan duration of 1s and Q1 peak width at 0.7. The stationary phase was Zorbax Extend C18 column (4.6 mm x 250 mm, 5 microm). The mobile phase: dichloromethane-acetonitrile (35:65), flow rate: 1 mL x min(-1); column temperature: 25 degrees C. RESULT: 12 triacylglycerols were identified by HPLC-MS method. CONCLUSION: The result can be used to identify the components in a fingerprint chromatogram of coix oil and its related injection product.

Structural characterization and pharmacology of a potent (Cys101-Cys119, Cys110-Cys117) bicyclic agouti-related protein (AGRP) melanocortin receptor antagonist.

Agouti-related protein (AGRP) is one of two known naturally occurring antagonists of G-protein coupled receptors. AGRP is synthesized in the brain and is an antagonist of the melanocortin-3 and -4 receptors (MC3R, MC4R). These three proteins are involved in the regulation of energy homeostasis and obesity in both mice and humans. The human AGRP protein is 132 amino acids and contains five disulfide bridges in the C-terminal domain. Previous reports of the NMR structures of hAGRP(87-132) and a truncated 34 amino acid form consisting of four disulfide bridges identified that AGRP contains an inhibitor cystine knot (ICK) structural fold, and that is the first mammalian example. Herein, we report a bicyclic hAGRP analogue that, when compared to hAGRP(87-132), possesses equal binding affinity but is 80-fold less potent at the mouse MC4R. Using NMR, computer assisted molecular modeling (CAMM), and cluster analysis, we have identified five structural families, two of which are highly populated, of this bicyclic hAGRP analogue. Computational docking experiments of this bicyclic hAGRP derivative, using a three-dimensional homology molecular model of the mouse MC4R, identified that three of the five structural families could be docked into the MC4R without problems from steric hindrance. Those three docked mMC4R-bicyclic hAGRP family structures were compared with putative hAGRP(87-132) ligand-receptor interactions previously reported (Wilczynski et al. J. Med. Chem. 2004, 47, 2194) in attempts to identify a "bioactive" conformation of the bicyclic hAGRP peptide and account for the 80-fold decreased ligand potency compared to hAGRP(87-132).

Identification of putative agouti-related protein(87-132)-melanocortin-4 receptor interactions by homology molecular modeling and validation using chimeric peptide ligands.

Agouti-related protein (AGRP) is one of only two naturally known antagonists of G-protein-coupled receptors (GPCRs) identified to date. Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these melanocortin receptors. Insight into putative interactions between the antagonist AGRP amino acids with the melanocortin-4 receptor (MC4R) may be important for the design of unique ligands for the treatment of obesity related diseases and is currently lacking in the literature. A three-dimensional homology molecular model of the mouse MC4 receptor complex with the hAGRP(87-132) ligand docked into the receptor has been developed to identify putative antagonist ligand-receptor interactions. Key putative AGRP-MC4R interactions include the Arg111 of hAGRP(87-132) interacting in a negatively charged pocket located in a cavity formed by transmembrane spanning (TM) helices 1, 2, 3, and 7, capped by the acidic first extracellular loop (EL1) and specifically with the conserved melanocortin receptor residues mMC4R Glu92 (TM2), mMC4R Asp114 (TM3), and mMC4R Asp118 (TM3). Additionally, Phe112 and Phe113 of hAGRP(87-132) putatively interact with an aromatic hydrophobic pocket formed by the mMC4 receptor residues Phe176 (TM4), Phe193 (TM5), Phe253 (TM6), and Phe254 (TM6). To validate the AGRP-mMC4R model complex presented herein from a ligand perspective, we generated nine chimeric peptide ligands based on a modified antagonist template of the hAGRP(109-118) (Tyr-c[Asp-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2)). In these chimeric ligands, the antagonist AGRP Arg-Phe-Phe residues were replaced by the melanocortin agonist His/D-Phe-Arg-Trp amino acids. These peptides resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs). The most notable results include the identification of a novel subnanomolar melanocortin peptide template Tyr-c[Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) that is equipotent to alpha-MSH at the mMC1, mMC3, and mMC5 receptors but is 30-fold more potent than alpha-MSH at the mMC4R. Additionally, these studies identified a new and novel >200-fold MC4R versus MC3R selective peptide Tyr-c[Asp-D-Phe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) template. Furthermore, when the His-DPhe-Arg-Trp sequence is used to replace the hAGRP Arg-Phe-Phe residues in the "mini"-AGRP (hAGRP87-120, C105A) template, a potent nanomolar agonist resulted at the mMC1R and MC3-5Rs.

Design and pharmacology of peptoids and peptide-peptoid hybrids based on the melanocortin agonists core tetrapeptide sequence.

A series of N-substituted glycine oligomers (peptoids) and peptide-peptoid hybrids were synthesized based on the Ac-His-Phe-Arg-Trp-NH(2) tetrapeptide template. The compounds were pharmacologically characterized at the mouse melanocortin receptors (MC1R, MC3R-MC5R) for agonist activity.

Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity.

Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111-113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7-9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle4-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH2 peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.

A solid-phase approach to mouse melanocortin receptor agonists derived from a novel thioether cyclized peptidomimetic scaffold.

The solid-phase synthesis of a novel thioether cyclized peptidomimetic scaffold, displaying functionality at the i to i + 3 positions, is reported. The thioether bridge is formed on-bead by an intramolecular reaction between a chloroacetylated reduced peptide bond and the free thiol from a cysteine. The crude products were obtained in moderate to very high purity. A series of 19 compounds were prepared and tested for agonist activity at the mouse melanocortin receptors 1, 3, 4, and 5 (mMC1-5R). From these results, several compounds were identified as having low micromolar agonist activity at the mMC1R and mMC4R. The former is involved in skin pigmentation and animal coat coloration. The latter is involved in the regulation of appetite and food intake and is currently a drug target for potential treatment of obesity. The most potent compound 1n with the pharmacophore motif "His-DPhe-Arg-Trp" was identified as having an EC(50) value of 165 nM at mMC1R, 7600 nM at mMC3R, 650 nM at mMC4R, and 335 nM at mMC5R. In addition, some of the compounds showed moderate selectivity for the mMC1R.