BACKGROUD:Sebaceous carcinoma is a rare and aggressive cutaneous carcinoma. It is believed that this malignancy predominates in the periocular region and occurs more frequently in Asian populations and in women. The objective of the current study was to analyze demographic characteristics and outcomes for patients with this malignancy from a large United States‐based population registry.METHODS:An analysis of the National Cancer Institute's Surveillance, Epidemiology, and End Results database from 1973 through 2004 was performed.RESULTS:Of 1349 patients who were identified, 54% were men, 86.2% were white, and 5.5% were of Asian/Pacific Islander ancestry. The median age at diagnosis was 73 years. The most frequent site of disease was the eyelid (38.7%). The population‐matched 5‐ and 10‐year age‐matched relative survival rate was 91.9% (standard error [SE], 1.9%) and 79.2% (SE, 3.7%), respectively. Cause of death was attributable to cancer in 31% of patients. Orbital involvement did not predict for worsened survival compared with nonorbital involvement (5‐year overall survival, 75.2% vs 68%, respectively; P = .66). The overall population‐matched rate of sebaceous carcinoma was highest in whites (2.03 cases per 1000,000; SE, 0.08) versus Asian/Pacific Islanders (1.07 per 1000,000; SE, 0.18; P = .0001) versus blacks (0.48 per 1000,000; SE, 0.11; P < .0001).CONCLUSIONS:The current results support the finding of a predominance of men among patients with sebaceous carcinoma, and no difference was observed in the prognosis for orbital and periorbital involvement. This retrospective analysis also corroborated previous case reports of a higher incidence among patients with advanced age and the highest incidence for sites in the eyelid and skin of the face. The results also established that Asian/Pacific Islander ancestry is not a risk factor for developing sebaceous carcinoma. Cancer 2009. © 2008 American Cancer Society.

Plasmodium falciparum thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in folate biosynthesis, and a major malarial drug target. Point mutations in P. falciparum TS-DHFR have caused widespread global antifolate resistance, and yet the most effective antifolate known to overcome drug-resistance, WR99210, has poor oral bioavailability. More specific, less toxic therapies are urgently needed. Antifolates target the conversion of methylene tetrahydrofolate to dihydrofolate by TS, and that of dihydrofolate to tetrahydrofolate by DHFR. In humans, TS and DHFR are two discrete enzymes. In P. falciparum, however, TS-DHFR is a bifunctional enzyme, with TS and DHFR encoded within a single protein, and tethered together with by a ‘linker’ region. This linker is not homologous to any other known TS or DHFR enzymes, and is essential for enzyme activity. This bifunctional enzyme thus presents different design approaches for developing novel inhibitors against drug-resistant mutants: developing active-site inhibitors equally effective against wildtype and drug-resistant parasites, or targeting unique non-active site regions for parasite-specific inhibitors. As a first step in identifying unique inhibitors, we performed a high-throughput in silico screen of a database of diverse, drug-like molecules against a non-active site pocket within the linker region of TS-DHFR. The top compounds from the virtual screen were evaluated by enzymatic and cellular assays. In vitro enzymatic studies and cell culture studies of wildtype and drug-resistant P. falciparum parasites identified three compounds active to 20 μM IC50s in both wildtype and antifolate-resistant enzymatic studies, as well as in P. falciparum cell culture. Moreover no inhibition of human DHFR enzyme was observed indicating the inhibitory effects appeared to be parasite-specific. Notably, all three compounds had a biguanide scaffold. Further computational analysis was utilized to determine the relative free energy of binding and these calculations suggested that the compounds might preferentially interact with the active site over the screened ‘linker’ region. To resolve the two possible modes of binding, co-crystallization studies of the compounds complexed with TS-DHFR enzyme were performed to determine the three-dimensional structures. Surprisingly, the structural analysis revealed that these novel, biguanide compounds, distinct from WR99210, do indeed bind at the active site of DHFR, and additionally revealed the molecular basis by which they overcome drug-resistance. To our knowledge, these are the first co-crystal structures of novel, biguanide, non-WR99210 compounds that are active against folate-resistant malaria parasites in cell culture. These studies reveal how serendipity coupled with computational and structural analysis can identify unique compounds as a promising starting point for rational drug design to combat drug-resistant malaria.

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Plasmodium falciparum thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in nucleotide biosynthesis, and a validated molecular drug target in malaria. Because P. falciparum TS and DHFR are highly homologous to their human counterparts, existing active-site antifolate drugs can have dose-limiting toxicities. In humans, TS and DHFR are two separate proteins. In P. falciparum, however, TS-DHFR is bifunctional, with both TS and DHFR active sites on a single polypeptide chain of the enzyme. Consequently, P. falciparum TS-DHFR contains unique distant or 'non-active' regions which might modulate catalysis: 1) an N-terminal tail; and 2) a 'linker' region tethering DHFR to TS, and encoding a 'crossover helix' that forms critical electrostatic interactions with the DHFR active site. The role of these non-active sites in the bifunctional P. falciparum TS-DHFR is unknown. We report the first in-depth, pre-steady state, kinetic characterization of the fulllength, WT P. falciparum TS-DHFR enzyme, and probe the role of distant, non-active regions through mutational analysis. We show that the overall rate-limiting step in the WT P. falciparum TS-DHFR enzyme is TS catalysis. We further show that if TS is in an 'activated' (liganded) conformation, the DHFR rate is 2-fold activated, from 60 s −1 to 130 s −1 in the WT enzyme. The TS rate is also reciprocally activated by ~1.5-fold if DHFR is in an activated, ligand-bound conformation. Mutations to the linker region affect neither catalytic rate nor domain-domain communication. Deletion of the N-terminal tail -although in a location remote to the active site -decreases DHFR single and the bifunctional TS-DHFR rate by a factor of 2. The two-fold activation of the DHFR rate by TS ligands remains intact, although even the activated N-terminal mutant has just half the DHFR activity of the WT enzyme. However, the reciprocal communication -between TS active site and DHFR ligands -is impaired in N-terminal mutants. Surprisingly, deletion of the analogous N-terminal tail in Leishmania major TS-DHFR causes a 3-fold enhancement of the DHFR rate from ~14 s −1 tõ 40 s −1 . In summary, our results demonstrate a complex interplay of domain-domain communication and non-active site modulation of catalysis in P. falciparum TS-DHFR. Furthermore, each parasitic TS-DHFR is activated by unique mechanisms, modulated by their non-active site regions. Finally, our studies suggest the N-terminal tail of P. falciparum TS-DHFR is a highly selective, novel target for potential antifolate development in malaria. KeywordsDihydrofolate reductase-thymidylate synthase (DHFR-TS); Plasmodium falciparum; antifolates; presteady state kinetics; non-active site *CORRESPONDING AUTHOR FOOTNOTE, Karen S. Anderson, Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptMalaria is a parasitic disease that kills over 3,000 a day and infects some 300 million people per year. The ...