No abstract

Since 2011, with the approval of crizotinib and subsequent approval of four additional targeted therapies, ALK inhibitors have become important treatments for a subset of patients with lung cancer. Each generation of ALK inhibitor showed improvements in terms of CNS penetration and potency against wild-type ALK, yet a key continued limitation is their susceptibility to resistance from ALK active-site mutations. The solvent front mutation (G1202R) and gatekeeper mutation (L1196M) are major resistance mechanisms to the first two generations of inhibitors while patients treated with the third-generation ALK inhibitor lorlatinib often experience progressive disease with multiple mutations on the same allele (mutations in cis, compound mutations). TPX-0131 is a compact macrocyclic molecule designed to fit within the ATPbinding boundary to inhibit ALK fusion proteins. In cellular assays, TPX-0131 was more potent than all five approved ALK inhibitors against wild-type ALK and many types of ALK resistance mutations, e.g. G1202R, L1196M, and compound mutations. In biochemical assays, TPX-0131 potently inhibited (IC 50 <10 nmol/L) wild-type ALK and 26 ALK mutants (single and compound mutations). TPX-0131, but not lorlatinib, caused complete tumor regression in ALK (G1202R) and ALK compound mutation-dependent xenograft models. Following repeat oral administration of TPX-0131 to rats, brain levels of TPX-0131 were ~66% of those observed in plasma. Taken together, preclinical studies show that TPX-0131 is a CNS-penetrant, next-generation ALK inhibitor that has potency against wild-type ALK and a spectrum of acquired resistance mutations, especially the G1202R solvent front mutation and compound mutations, for which there are currently no effective therapies.

Anaplastic lymphoma kinase (ALK) gene rearrangements occur in up to 7% of patients with non-small cell lung cancer (NSCLC) with the majority as EML4-ALK fusions. Crizotinib (first generation ALK inhibitor) was the first approved ALK inhibitor for the treatment of ALK-positive metastatic non-small cell lung cancer. However, development of resistance to crizotinib caused by secondary kinase domain mutations, bypass signaling, or morphology changes occurs. Second generation ALK inhibitors alectinib, ceritinib, and brigatinib were able to overcome the majority of ALK resistant mutations (L1196M, G1269A and F1174L) acquired with crizotinib. The solvent front mutation (SFM) G1202R is a common resistant mutation to crizotinib and the second generation ALK inhibitors. Lorlatinib, a third generation ALK inhibitor, can overcome G1202R resistance with moderate IC50 values of 40 - 60 nM in cell-based assays. Although, compound mutations such as ones with both gatekeeper and solvent front mutations (L1196M/G1202R) are refractory to lorlatinib, representing an unmet medical need. TPX-0131 is a next generation ALK inhibitor designed with a compact macrocyclic structure that can bind completely within the ATP binding boundary to overcome a variety of ALK resistant mutations, especially SFM G1202R and compound mutations L1196M/G1202R. TPX-0131 potently inhibits wildtype (WT) ALK and over 20 different ALK mutations with IC50 values &lt;5 nM when tested in enzymatic kinase assays in the presence of 10 μM of ATP. In cell proliferation assays, TPX-0131 exhibited comparable antiproliferation activity to the most potent ALK inhibitor lorlatinib in Ba/F3 cells engineered with EML4-ALK WT. Importantly, TPX-0131 is more than 100-fold more potent against G1202R than lorlatinib in cell proliferation assays. Furthermore, TPX-0131 demonstrated antiproliferation IC50 values &lt;2 nM in Ba/F3 cell models engineered with compound mutations including L1196M/G1202R, L1198F/G1202R, L1196M/L1198F, and C1156Y/G1202R, while lorlatinib and other ALK inhibitors are not active (IC50s &gt;1 μM). Taken together, TPX-0131 is a next generation ALK inhibitor that can overcome a broad spectrum of acquired resistance mutations, especially the G1202R solvent front mutation and compound mutations (e.g. L1196M/G1202R). The nonclinical pharmacology profile of TPX-0131 warrants further preclinical investigation. Citation Format: J. Jean Cui, Evan Rogers, Dayong Zhai, Wei Deng, Jane Ung, Vivian Nguyen, Han Zhang, Xin Zhang, Ana Parra, Maria Barrera, Dong Lee, Brion Murray. TPX-0131: A next generation macrocyclic ALK inhibitor that overcomes ALK resistant mutations refractory to current approved ALK inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5226.

During the course of a large-scale HIV-1 vaccine field trial (VAX004), full-length gp120 sequences were determined for 349 new HIV-1 infections. The data collected represent the largest survey of full-length gp120 sequences from new HIV-1 infections ever assembled. Previous studies have shown that subtype B viruses typically possess 18 cysteine residues that are covalently linked to form 9 conserved disulfide bridges. However, in this study we found that approximately 20% of the trial participants possessed envelope proteins with an unusual number of cysteine residues that could very likely result in unusual protein structures. One class of variants included envelope proteins with two additional cysteine residues in close proximity, potentially yielding additional disulfide-bonded loops. Other classes of variants included envelope proteins where amino acid replacements increased or decreased the number of cysteine residues by one, resulting in molecules with either 19 or 17 cysteines, respectively. Initial functional analysis demonstrated that envelope proteins with 19 cysteine residues bind to CD4 and the CCR5 chemokine coreceptor, and are infectious. These results suggest that the protein structure of gp120 in newly transmitted viruses may be more heterogeneous than previously appreciated and potentially represent a new mechanism of virus variation. The disulfide variation that we report here may have important implications for HIV vaccine and drug development efforts.

The present study examines cortical neuronal morphology in the African lion (Panthera leo leo), African leopard (Panthera pardus pardus), and cheetah (Acinonyx jubatus jubatus). Tissue samples were removed from prefrontal, primary motor, and primary visual cortices and investigated with a Golgi stain and computer‐assisted morphometry to provide somatodendritic measures of 652 neurons. Although neurons in the African lion were insufficiently impregnated for accurate quantitative dendritic measurements, descriptions of neuronal morphologies were still possible. Qualitatively, the range of spiny and aspiny neurons across the three species was similar to those observed in other felids, with typical pyramidal neurons being the most prominent neuronal type. Quantitatively, somatodendritic measures of typical pyramidal neurons in the cheetah were generally larger than in the African leopard, despite similar brain sizes. A MARsplines analysis of dendritic measures correctly differentiated 87.4% of complete typical pyramidal neurons between the African leopard and cheetah. In addition, unbiased stereology was used to compare the soma size of typical pyramidal neurons (n = 2,238) across all three cortical regions and gigantopyramidal neurons (n = 1,189) in primary motor and primary visual cortices. Both morphological and stereological analyses indicated that primary motor gigantopyramidal neurons were exceptionally large across all three felids compared to other carnivores, possibly due to specializations related to the felid musculoskeletal systems. The large size of these neurons in the cheetah which, unlike lions and leopards, does not belong to the Panthera genus, suggests that exceptionally enlarged primary motor gigantopyramidal neurons evolved independently in these felid species.