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Solid organ transplant recipients are at an increased risk of developing skin cancers due to chronic immunosuppression, particularly with calcineurin inhibitors. Tacrolimus is the most prescribed calcineurin inhibitor in this patient cohort, and understanding tacrolimus concentrations in the skin will facilitate the development of anti-cancer preventive and therapeutic strategies. Here, we show that in mice, tacrolimus blood levels peaked rapidly ∼1 h post last oral dose while skin levels rose more slowly and remained high for at least 6 h. Subsequently, tacrolimus skin and blood concentrations were assessed in 15 kidney transplant recipients. The mean age was 61 years, the average time post-transplant was 7 years (range 0–21 years) and 87% were male. The average skin sampling time post tacrolimus dosing was 6 h 32 min. Skin tacrolimus concentrations ranged from 7.1 ng/g to 71.2 ng/g and correlated with blood concentrations (r = 0.6). Mouse and human mean skin concentrations were in a similar range. Our data suggests that tacrolimus measurements in the blood may be used to approximate tacrolimus concentrations in the skin of kidney transplant recipients, and further exploited for the delivery of anti-cancer therapies designed to antagonize the immunosuppressive effects of tacrolimus in the skin.

Slow and continuous release of H2S by GYY4137 has previously been demonstrated to kill cancer cells by increasing glycolysis and impairing anion exchanger and sodium/proton exchanger activity. This action is specific for cancer cells. The resulting lactate overproduction and defective pH homeostasis bring about intracellular acidification-induced cancer cell death. The present study investigated the potency of H2S released by GYY4137 against invasive and radio- as well as chemo-resistant cancers, known to be glycolytically active. We characterized and utilized cancer cell line pairs of various organ origins, based on their aggressive behaviors, and assessed their response to GYY4137. We compared glycolytic activity, via lactate production, and intracellular pH of each cancer cell line pair after exposure to H2S. Invasive and therapy resistant cancers, collectively termed aggressive cancers, are receptive to H2S-mediated cytotoxicity, albeit at a higher concentration of GYY4137 donor. While lactate production was enhanced, intracellular pH of aggressive cancers was only modestly decreased. Inherently, the magnitude of intracellular pH decrease is a key determinant for cancer cell sensitivity to H2S. We demonstrated the utility of coupling GYY4137 with either simvastatin, known to inhibit monocarboxylate transporter 4 (MCT4), or metformin, to further boost glycolysis, in bringing about cell death for aggressive cancers. Simvastatin inhibiting lactate extrusion thence contained excess lactate induced by GYY4137 within intracellular compartment. In contrast, the combined exposure to both GYY4137 and metformin overwhelms cancer cells with lactate over-production exceeding its expulsion rate. Together, GYY4137 and simvastatin or metformin synergize to induce intracellular hyper-acidification-mediated cancer cell death.

Mycobacteria harbor two main degradative proteolytic machineries, the caseinolytic protease ClpP1P2 and a proteasome. We recently showed that Bortezomib inhibits ClpP1P2 and exhibits whole cell activity against Mycobacterium tuberculosis. Bortezomib, a dipeptide with a boronic acid warhead, is a human proteasome inhibitor approved for cancer therapy. The boronic acid warhead of the compound has been shown to drive potency against both the human proteasome and ClpP1P2 protease. Selectivity for the bacterial ClpP1P2 protease over the human proteasome is lacking but needs to be achieved to move this new anti-tuberculosis lead forward. In this study we explored whether an alternative warhead could influence Bortezomib's selectivity. We synthesized an analog containing a chloromethyl ketone instead of the boronic acid warhead and determined potencies against the bacterial and human enzymes. Surprisingly, the analog retained activity against mycobacterial ClpP1P2 and was active against the mycobacterial proteasome, but was devoid of activity against the human proteasome. Interrogation of a set of chloromethyl ketone peptides identified three additional compounds similarly inhibiting both ClpP1P2 and the proteasome in the bacteria while leaving the human proteasome untouched. Finally, we showed that these compounds display bactericidal activity against M. tuberculosis with cytotoxicity ranging from acceptable to undetectable. These results suggest that selectivity over the human proteasome is achievable. Selectivity, together with dual-targeting of mycobacterial ClpP1P2 and proteasome makes this new scaffold an attractive starting point for optimization.