Racemic 2,4-diaminopyrimidine dihydrophthalazine derivatives BAL0030543, BAL0030544, and BAL0030545 exhibited low in vitro MICs toward small, selected panels of Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae, Moraxella catarrhalis, and Mycobacterium avium, though the compounds were less active against Haemophilus influenzae. The constellation of dihydrofolate reductases (DHFRs) present in 20 enterococci and 40 staphylococci was analyzed and correlated with the antibacterial activities of the dihydrophthalazines and trimethoprim. DHFRs encoded by dfrB, dfrA (S1 isozyme), dfrE, and folA were susceptible to the dihydrophthalazines, whereas DHFRs encoded by dfrG (S3 isozyme) and dfrF were not. Studies with the separated enantiomers of BAL0030543, BAL0030544, and BAL0030545 revealed preferential inhibition of susceptible DHFRs by the (R)-enantiomers. BAL0030543, BAL0030544, and BAL0030545 were well tolerated by mice during 5-and 10-day oral toxicity studies at doses of up to 400 mg/kg of body weight. Using a nonoptimized formulation, the dihydrophthalazines displayed acceptable oral bioavailabilities in mice, and efficacy studies with a septicemia model of mice infected with trimethoprim-resistant, methicillin-resistant Staphylococcus aureus gave 50% effective dose values in the range of 1.6 to 6.25 mg/kg.Despite sustained efforts to identify new drugs against antibiotic-resistant bacteria, treatment of infections caused by vancomycin-resistant enterococci and methicillin-resistant staphylococci continues to challenge clinicians (5). Enterococci are often associated with urinary tract infections, intra-abdominal abscesses, endocarditis, and bacteremia, whereas staphylococci are common causes of skin and skin structure infections, endocarditis, osteomyelitis, prosthetic joint infections, catheterrelated bacteremia, and nosocomial pneumonia. Methicillinresistant Staphylococcus aureus (MRSA) accounts for approximately 50% of all S. aureus infections in the United States (24). Once confined to healthcare settings, MRSA has emerged as a serious cause of community-acquired skin and skin structure infections (32), and an increased frequency of skin cultures positive for S. aureus is associated with nasal and intestinal colonization (2).Dihydrofolate reductase (DHFR; EC 1.5.1.3) is an essential enzyme in most pathogenic bacteria, and the clinical success of trimethoprim (28,33,41,43) confirms DHFR as an important chemotherapeutic target. A phenylalanine3tyrosine substitution at position 98 in the wild-type, nontransferable, chromosomally encoded DHFR renders S. aureus resistant to trimethoprim (15); a corresponding tyrosyl residue occurs in transferable plasmid-encoded wild-type staphylococcal DHFR isozymes S1, S2, and S3 (13,14,40) and in the enterococcal DHFRs encoded by dfrE and dfrF. The gene encoding DHFR S1 may also be associated with staphylococcal chromosomal cassette mec N1 (SCCmec-N1) (37).BAL0030543, BAL0030544, and BAL0030545 are novel dihydrophthalazine antifolates with potent activities a...

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Background: BAL0891 is a dual inhibitor of threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1). These kinases collaborate in activating the mitotic spindle assembly checkpoint (SAC) at the kinetochore (KT) to regulate chromosome alignment and segregation prior to mitotic exit. In vitro, BAL0891 has a combined prolonged effect on TTK and a transient effect on PLK1, leading to rapid disruption of the SAC that potentiates aberrant mitotic progression of tumor cells. In this work, efficacy of BAL0891 was investigated in mouse models of human triple negative breast cancer (TNBC) including evaluation of dose-dependency, drug exposure, target occupancy and a screen of activity across a panel of PDX models. Methods: The MDA-MB-231 cell line was grown sc in nude mice and treated with BAL0891, administered IV weekly (QW) or twice-weekly (2QW). Thirteen sc TNBC PDX models were screened for BAL0891 response using 2QW administration. Efficacy was quantified as deltaT/C (treated/control tumors). Plasma and tumor were analyzed for drug levels or TTK target occupancy by LC-MS/MS. The latter used a biotinylated TTK-specific probe and streptavidin-mediated isolation of unoccupied TTK, trypsin digestion and quantification of TTK-representative peptides. Results: BAL0891 efficacy was tested in the TNBC xenograft model MDA-MB-231 with QW or 2QW IV dosing schedules. All treatments were well tolerated, with no drug-related animal deaths. With MTD dosing, tumor regressions were observed, while different MTD fractions for both QW and 2QW schedules showed dose-dependent anti-tumor activity. The weekly MTD group was followed for an additional 20 days after treatment cessation on day 100. Strikingly, 3 of 8 tumors continued to shrink resulting in 2 (25%) pathologically confirmed cures. Consistent with the potent efficacy of intermittent MTD dosing, and prolonged tumor drug exposure, tumor TTK was fully drug-occupied for ≥ 6 days after the last administration; target occupancy was also dose-and drug exposure-dependent. To further evaluate BAL0891 anti-cancer activity in TNBC, a screen in 13 TNBC PDX models was conducted. Seven models exhibited deltaT/C < 50%, with regressions observed in 3. Of these, 2 models showed persistent regressions ≥ 70% vs. baseline. Interestingly, evaluation of TTK target occupancy in selected models showed high target occupancy independent of tumor response, indicating target dependency rather than drug availability is important for anti-cancer activity. Conclusion: BAL0891 is a novel dual TTK/PLK1 mitotic checkpoint inhibitor with potent anti-cancer activity in TNBC models. Intermittent IV administration is well tolerated and associated with prolonged tumor drug exposure, prolonged TTK inhibition and notable anti-tumor efficacy. These data support further investigation of BAL0891 for the treatment of cancer patients (incl. TNBC). Citation Format: Heidi A. Lane, Felix Bachmann, Elisa Zanini, Paul McSheehy, Karine Litherland, Nicole Forster-Gross, Luc Bury, Diep Vu-Pham, Jos de Man, Wilhelmina E. van Riel, Guido JR Zaman, Rogier C. Buijsman, Laurenz Kellenberger. BAL0891: A novel dual TTK/PLK1 mitotic checkpoint inhibitor (MCI) that drives aberrant tumor cell division resulting in potent anti-cancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5645.