Novel small molecules inhibit proteotoxicity and inflammation: Mechanistic and therapeutic implications for Alzheimer's Disease, healthspan and lifespan- Aging as a consequence of glycolysis.

Inflammation drives many age-related, especially neurological, diseases, and likely mediates age-related proteotoxicity. For example, dementia due to Alzheimer's Disease (AD), cerebral vascular disease, many other neurodegenerative conditions is increasingly among the most devastating burdens on the American (and world) health system and threatens to bankrupt the American health system as the population ages unless effective treatments are developed. Dementia due to either AD or cerebral vascular disease, and plausibly many other neurodegenerative and even psychiatric conditions, is driven by increased age-related inflammation, which in turn appears to mediate Abeta and related proteotoxic processes. The functional significance of inflammation during aging is also supported by the fact that Humira, which is simply an antibody to the pro-inflammatory cytokine TNF-a, is the best-selling drug in the world by revenue. These observations led us to develop parallel high-throughput screens to discover small molecules which inhibit age-related Abeta proteotoxicity in a C. elegans model of AD AND LPS-induced microglial TNF-a. In the initial screen of 2560 compounds (Microsource Spectrum library) to delay Abeta proteotoxicity, the most protective compounds were, in order, phenylbutyrate, methicillin, and quetiapine, which belong to drug classes (HDAC inhibitors, beta lactam antibiotics, and tricyclic antipsychotics, respectably) already robustly implicated as promising to protect in neurodegenerative diseases, especially AD. RNAi and chemical screens indicated that the protective effects of HDAC inhibitors to reduce Abeta proteotoxicity are mediated by inhibition of HDAC2, also implicated in human AD, dependent on the HAT Creb binding protein (Cbp), which is also required for the protective effects of both dietary restriction and the daf-2 mutation (inactivation of IGF-1 signaling) during aging. In addition to methicillin, several other beta lactam antibiotics also delayed Abeta proteotoxicity and reduced microglial TNF-a. In addition to quetiapine, several other tricyclic antipsychotic drugs also delayed age-related Abeta proteotoxicity and increased microglial TNF-a, leading to the synthesis of a novel congener, GM310, which delays Abeta as well as Huntingtin proteotoxicity, inhibits LPS-induced mouse and human microglial and monocyte TNF-a, is highly concentrated in brain after oral delivery with no apparent toxicity, increases lifespan, and produces molecular responses highly similar to those produced by dietary restriction, including induction of Cbp inhibition of inhibitors of Cbp, and genes promoting a shift away from glycolysis and toward metabolism of alternate (e.g., lipid) substrates. GM310, as well as FDA-approved tricyclic congeners, prevented functional impairments and associated increase in TNF-a in a mouse model of stroke. Robust reduction of glycolysis by GM310 was functionally corroborated by flux analysis, and the glycolytic inhibitor 2-DG inhibited microglial TNF-a and other markers of inflammation, delayed Abeta proteotoxicity, and increased lifespan. These results support the value of phenotypic screens to discover drugs to treat age-related, especially neurological and even psychiatric diseases, including AD and stroke, and to clarify novel mechanisms driving neurodegeneration (e.g., increased microglial glycolysis drives neuroinflammation and subsequent neurotoxicity) suggesting novel treatments (selective inhibitors of microglial glycolysis).

Diets, genes, and drugs that increase lifespan and delay age-related diseases: Role of nutrient-sensing neurons and Creb-binding protein.

Discovery of interventions that delay or minimize age-related diseases is arguably the major goal of aging research. Conversely discovery of interventions based on phenotypic screens have often led to further elucidation of pathophysiological mechanisms. Although most hypotheses to explain lifespan focus on cell-autonomous processes, increasing evidence suggests that in multicellular organisms, neurons, particularly nutrient-sensing neurons, play a determinative role in lifespan and age-related diseases. For example, protective effects of dietary restriction and inactivation of insulin-like signaling increase lifespan and delay age-related diseases dependent on Creb-binding protein in GABA neurons, and Nrf2/Skn1 in just 2 nutrient-sensing neurons in C. elegans. Screens for drugs that increase lifespan also indicate that such drugs are predominantly active through neuronal signaling. Our own screens also indicate that neuroactive drugs also delay pathology in an animal model of Alzheimer's Disease, as well as inhibit cytokine production implicated in driving many age-related diseases. The most likely mechanism by which nutrient-sensing neurons influence lifespan and the onset of age-related diseases is by regulating metabolic architecture, particularly the relative rate of glycolysis vs. alternative metabolic pathways such as ketone and lipid metabolism. These results suggest that neuroactive compounds are a most promising class of drugs to delay or minimize age-related diseases.

Mitomycin C and decarbamoyl mitomycin C induce p53-independent p21WAF1/CIP1 activation.

Mitomycin C (MC), a commonly used anticancer drug, induces DNA damage via DNA alkylation. Decarbamoyl mitomycin C (DMC), another mitomycin lacking the carbamate at C10, generates similar lesions as MC. Interstrand cross-links (ICLs) are believed to be the lesions primarily responsible for the cytotoxicity of MC and DMC. The major ICL generated by MC (α-ICL) has a trans stereochemistry at the guanine-drug linkage whereas the major ICL from DMC (ß-ICL) has the opposite, cis, stereochemistry. In addition, DMC can provoke strong p53-independent cell death. Our hypothesis is that the stereochemistry of the major unique ß-ICL generated by DMC is responsible for this p53-independent cell death signaling. p53 gene is inactively mutated in more than half of human cancers. p21WAF1/CIP1 known as a major effector of p53 is involved in p53-dependent and -independent control of cell proliferation and death. This study revealed the role of p21WAF1/CIP1 on MC and DMC triggered cell damage. MCF-7 (p53-proficient) and K562 (p53-deficient) cells were used. Cell cycle distributions were shifted to the G1/S phase in MCF-7 treated with MC and DMC, but were shifted to the S phase in K562. p21WAF1/CIP1 activation was observed in both cells treated with MC and DMC, and DMC triggered more significant activation. Knocking down p53 in MCF-7 did not attenuate MC and DMC induced p21WAF1/CIP1 activation. The α-ICL itself was enough to cause p21WAF1/CIP1 activation.

Synthesis of Mitomycin C and Decarbamoylmitomycin C N(2) deoxyguanosine-adducts.

Mitomycin C (MC) and Decarbamoylmitomycin C (DMC) - a derivative of MC lacking the carbamate on C10 - are DNA alkylating agents. Their cytotoxicity is attributed to their ability to generate DNA monoadducts as well as intrastrand and interstrand cross-links (ICLs). The major monoadducts generated by MC and DMC in tumor cells have opposite stereochemistry at carbon one of the guanine-mitosene bond: trans (or alpha) for MC and cis (or beta) for DMC. We hypothesize that local disruptions of DNA structure from trans or cis adducts are responsible for the different biochemical responses produced by MC and DMC. Access to DNA substrates bearing cis and trans MC/DMC lesions is essential to verify this hypothesis. Synthetic oligonucleotides bearing trans lesions can be obtained by bio-mimetic methods. However, this approach does not yield cis adducts. This report presents the first chemical synthesis of a cis mitosene DNA adduct. We also examined the stereopreference exhibited by the two drugs at the mononucleotide level by analyzing the formation of cis and trans adducts in the reaction of deoxyguanosine with MC or DMC using a variety of activation conditions. In addition, we performed Density Functional Theory calculations to evaluate the energies of these reactions. Direct alkylation under autocatalytic or bifunctional conditions yielded preferentially alpha adducts with both MC and DMC. DFT calculations showed that under bifunctional activation, the thermodynamically favored adducts are alpha, trans, for MC and beta, cis, for DMC. This suggests that the duplex DNA structure may stabilize/oriente the activated pro-drugs so that, with DMC, formation of the thermodynamically favored beta products are possible in a cellular environment.