Drug Resistance
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Engineering inducible morphotype switching control in Mycobacterium abscessus for investigating infection outcomes and discovering pathophysiological-targeted treatments
PROJECT SUMMARY Antibiotic-resistant nontuberculous mycobacteria (NTM) infections are rising at a rate of 8% each year and account for ~$1.7 billion in annual U.S. healthcare costs. Mycobacterium abscessus (Mabs), the most common rapidly growing NTM infection, is notoriously nicknamed the “antibiotic nightmare” for its extensive intrinsic and inducible broad-range multidrug resistance to antibiotic countermeasures. As part of its natural infection cycle, Mabs undergoes a morphotypical conversion from smooth to rough, characterized by irreversible genetic changes resulting in the loss of cell envelope glycopeptidolipids (GPLs). This morphotypic conversion is intimately associated with disease progression, ultimately leading to debilitating, refractory Mabs pulmonary disease. Specific stimuli triggering Mabs morphotypical conversion are unknown, thus preventing directed investigations into morphotype-specific immunological responses and the discovery of morphotype-specific therapeutic targets. This project leverages cutting-edge molecular genetic tools, including CRISPR (clustered regularly interspersed short palindromic repeats) interference (CRISPRi) and inducible knockdown control of CRISPRi via the anhydrotetracycline-inducible TetR-regulated promoter-operator system, to create six unique, reversible Mabs smooth to conditional rough morphotype strains. These molecular morphoswitchable strains allow precise investigator-mediated on-off control of Mabs surface GPLs, enabling investigations into Mabs morphological plasticity, unique pathophysiology traits associated with each morphotype, and the complex interplay between Mabs and morphotype-specific immunological responses. In Aim 1, we implement CRISPRi inducible knockdown tunable control of Mabs morphotype switching by targeting six, independent genetic targets directly involved in GPL biosynthesis (mps1, mps2) or transport (mmpS4, mmpL4a, mmpL4b, gap) and validate in vitro morphoswitching. In Aim 2, we establish and confirm Mabs morphoswitching and intracellular growth in infected THP-1 macrophages. Subsequently, we evaluate differential and distinct innate cellular immune responses elicited by Mabs smooth and Mabs conditional rough morphotypes during intracellular infection in human primary monocyte-derived macrophages. Collectively, these studies create a suite of characterized and reversible Mabs smooth and conditional rough morphoswitchable strains with controlled, regulated, and on- demand expression of Mabs surface GPLs. By enabling precisely timed and controlled induction of the Mabs conditional rough morphotype during intracellular growth, we can molecularly dissect and investigate fundamental Mabs host-pathogen interactions and immunological responses that so substantially influence negative clinical outcomes.
Implementing a New Paradigm for Antifungal Drug Development
About 30% of the drugs currently in clinical use function through covalent modification of their target. Yet, until recently, none of these covalent drugs were specifically designed to utilize this irreversible mode of action. It is our hypothesis that the production of a new class of covalent inactivators, designed to selectively modify new drug targets, will lead to novel agents with efficacy against both native and drug-resistant pathogenic fungal species. Because of their novelty these agents will also offer a greater opportunity to bypass the existing mechanisms of drug resistance. Pathogenic fungal infections remain among the leading causes of human mortality, and this threat is rising due to the increasing prevalence of drug- resistance strains and the paucity of effective antifungal drugs against the more virulent fungal species. Our proposed new drug target is an enzyme that plays a critical role in a uniquely microbial pathway that is essential for the survival of fungal organisms. To test our hypothesis and achieve the goals of this project we plan to complete the following specific aims during the initial R21 phase of this project: (1) Optimization of the potency of novel enzyme inactivators. Our goals here are to use our strong preliminary results to address critical barriers that must be overcome to convert potent enzyme inactivators into advanced drug candidates, thereby achieving higher target selectivity and increasing compound reactivity once bound to the target; (2) Enhance the antifungal capability of these enzyme inactivators. Our strategy for this aim is focused on the incorporation of conjugate partners into this new class of covalent inactivators, enabling them to potentially utilize the existing nutrient uptake systems to achieve toxic levels in Candida species; (3) Examine the target selectivity of our new antifungal agents. Results from fungal growth inhibition and fungal killing assays will be used to evaluate and rank the efficacy of our compounds against both wild-type and drug-resistant Candida strains. Specific milestones are presented to evaluate our achievement of these initial aims. Once accomplished we will immediately proceed to the R33 phase of this project, with the aims of: (4) Pharmacological evaluation of lead candidates, though ranking the drug candidates based on their ADME, pharmacokinetic and toxicity properties; and then (5) Evaluate the efficacy of our candidates against pathogenic fungal infections. A systematic infection animal model will be utilized for candidate screening to identify the best agents against disseminated fungal infections, followed by further efficacy screening in an oral infection model. Completion of these aims will produce, refine and evaluate a new class of antifungal agents with a novel mode of action against an unexplored but essential fungal target. The agents with the most promising drug profiles will then be moved into advanced preclinical trials used to select the most effective new antifungal agents.
Structure-Based Development of Nucleotide-Competing Inhibitors Against HIV-1 and LINE-1 Reverse Transcriptases
PROJECT SUMMARY Reverse transcriptases (RTs) from retroviruses and endogenous retroelements are essential polymerases that catalyze RNA- and DNA-dependent DNA synthesis. Nucleoside inhibitors (NIs) remain central to HIV-1 therapy and are also used against other viral infections and in cancer, but toxicity, limited selectivity, pharmacokinetic (PK) liabilities, and the emergence of drug resistance highlight the need for alternative RT inhibitor mechanisms. In contrast to NIs, nucleotide-competing inhibitors (NCIs) block the polymerase active site without requiring incorporation into nucleic acids. Structural studies by PI Ruiz have defined the NCI mechanism of action for HIV- 1 RT and revealed conserved binding modules shared across multiple polymerase families. These advances now enable rational discovery of improved NCIs. LINE-1 (L1) ORF2 RT is an emerging therapeutic target in cancer, autoimmunity, and aging, yet NIs are the only inhibitors known to act against L1 RT. Notably, the NCI-binding region is structurally similar between HIV-1 RT and L1 RT, suggesting that NCI recognition principles may extend across these two biologically distinct polymerases. This R21 seeks to establish proof-of-concept for NCI development against both enzymes. Aim 1 will discover and structurally optimize NCIs targeting HIV-1 RT by combining binding modules from known NCI chemotypes and determining their biochemical activity and co-crystal structures. Aim 2 will determine whether HIV-1 RT NCI principles translate to L1 RT by solving L1 RT/nucleic acid/NCI structures, evaluating enzymatic inhibition, and applying AI-based structure prediction and generative design to propose L1-specific NCI candidates. Cellular retrotransposition assays will test mechanism of action. Aim 3 will develop a fragment library tailored to protein–nucleic acid interfaces and perform fragment screening of HIV-1 and L1 RT/nucleic acid complexes to identify additional chemotypes that engage the NCI binding region. Successful completion will yield NCI scaffolds and mechanistic insights applicable to HIV-1 RT and L1 RT, define structural principles governing NCI recognition across two evolutionarily related polymerases, and establish new avenues for RT inhibitor development. The PI is highly qualified to lead this work, with extensive expertise in RT structural biology, drug design, and fragment-based discovery.
Blood-brain barrier dysfunction in epilepsy: Time for translation
The neurovascular unit (NVU) consists of cerebral blood vessels, neurons, astrocytes, microglia, and pericytes. It plays a vital role in regulating blood flow and ensuring the proper functioning of neural circuits. Among other, this is made possible by the blood-brain barrier (BBB), which acts as both a physical and functional barrier. Previous studies have shown that dysfunction of the BBB is common in most neurological disorders and is associated with neural dysfunction. Our studies have demonstrated that BBB dysfunction results in the transformation of astrocytes through transforming growth factor beta (TGFβ) signaling. This leads to activation of the innate neuroinflammatory system, changes in the extracellular matrix, and pathological plasticity. These changes ultimately result in dysfunction of the cortical circuit, lower seizure threshold, and spontaneous seizures. Blocking TGFβ signaling and its associated pro-inflammatory pathway can prevent this cascade of events, reduces neuroinflammation, repairs BBB dysfunction, and prevents post-injury epilepsy, as shown in experimental rodents. To further understand and assess BBB integrity in human epilepsy, we developed a novel imaging technique that quantitatively measures BBB permeability. Our findings have confirmed that BBB dysfunction is common in patients with drug-resistant epilepsy and can assist in identifying the ictal-onset zone prior to surgery. Current clinical studies are ongoing to explore the potential of targeting BBB dysfunction as a novel treatment approach and investigate its role in drug resistance, the spread of seizures, and comorbidities associated with epilepsy.
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